Specific inhibitors of NFAT activation by calcineurin and their use in treating immune-related diseases

Info

Publication number: 20040002117
Type: Application
Filed: Feb 4, 2003
Publication Date: Jan 1, 2004
Inventors: Patrick G. Hogan (Cambridge, MA), Anjana Rao (Cambridge, MA), Jose Aramburu (Barcelona), Michael H. A. Roehrl (Brookline, MA), Gerhard Wagner (Chestnut Hill, MA), Sunghyun Kang (Brookline, MA)
Application Number: 10358052

Abstract

Isolated peptide fragments of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof are described. Isolated polynucleotides and gene therapy vectors encoding such peptide fragments are also described. In addition, methods for treating immune-related diseases or conditions and methods for high throughput screening of candidate agents are described. Pharmaceutical compositions are also provided.

Description

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patent application Ser. No. 10/066,151, filed on Jan. 31, 2002, which is a continuation of U.S. patent application Ser. No. 09/248,620, filed Feb. 11, 1999, which claims the benefit of U.S. Provisional Application No. 60/074,467, filed Feb. 12, 1998, all of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0003] This invention relates generally to NFAT peptide fragments, NFAT polynucleotides, NFAT gene therapy vectors, treatments for immune system related diseases, and methods for identifying immunosuppressive agents.

BACKGROUND OF THE INVENTION

[0004] Hyperactivity or inappropriate activity of the immune system is a serious and widespread medical problem. It contributes to acute and chronic immune diseases, e.g., allergic and atopic diseases, e.g., asthma, allergic rhinitis, allergic conjunctivitis and atopic dermatitis, and to autoimmune diseases, e.g., rheumatoid arthritis, insulin-dependent diabetes, inflammatory bowel disease, autoimmune thyroiditis, hemolytic anemia and multiple sclerosis. Hyperactivity or inappropriate activity of the immune system is also involved in transplant graft rejections and graft-versus-host disease.

[0005] A certain family of transcription factors, the NFAT proteins, are expressed in immune cells and play a key role in eliciting immune responses. The NFAT proteins are activated by calcineurin, and the activated NFAT proteins, in turn, induce transcription of cytokine genes which are required for an immune response. The immunosuppressive drugs cyclosporin A and FK506 are potent inhibitors of cytokine gene transcription in activated immune cells, and have been reported to act by inhibiting calcineurin such that calcineurin is not able to activate NFAT. These drugs, however, can display nephrotoxic and neurotoxic effects after long term usage. Since calcineurin is ubiquitously expressed in many tissues, the drugs' inhibition of calcineurin activity toward substrates other than NFAT may contribute to the observed toxicity.

[0006] There is a need for immunosuppressive agents which selectively inhibit the calcineurin-NFAT interactions and which do not inhibit the enzymatic activity of calcineurin for its other substrates.

SUMMARY OF THE INVENTION

[0007] It is an object of the invention to provide an immunosuppressive agent with reduced toxic effects.

[0008] It is another object of the invention to provide an immunosuppressive agent that inhibits interaction between calcineurin and NFAT.

[0009] It is yet another object of the invention to provide an immunosuppressive agent that selectively inhibits interaction between calcineurin and NFAT, and which does not inhibit enzymatic activity of calcineurin for its other substrates.

[0010] It is yet another object of the invention to provide a gene therapy vector encoding an immunosuppressive agent that selectively inhibits interaction between calcineurin and NFAT.

[0011] It is yet another object of the invention to provide a method for inhibiting an immune response using an immunosuppressive agent that selectively inhibits interaction between calcineurin and NFAT.

[0012] It is yet another object of the invention to provide methods for high-throughput screening of candidate agents to identify agents that inhibit one or more aspects of calcineurin-mediated NFAT activation.

[0013] According to the invention, an isolated fragment of the conserved regulatory domain of NFAT protein, e.g., NFAT1, NFAT2, NFAT3 or NFAT4, capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof, is provided. Preferably, the peptide fragment or biologically active analog thereof does not inhibit or does not substantially inhibit the activity of calcineurin toward non-NFAT calcineurin substrates.

[0014] In certain embodiments, the peptide fragment comprises the amino acid sequence IX2X3T (SEQ ID NO:104), wherein X2 is E, R or Q, and X3 is I or F. Preferred amino acid sequences are, e.g., IEIT (SEQ ID NO:105), IRIT (SEQ ID NO:106), IQIT (SEQ ID NO:107), and IQFT (SEQ ID NO:108).

[0015] In certain embodiments, the peptide fragment comprises the amino acid sequence X1IX2X3T (SEQ ID NO:73), wherein X1 is R or S, X2 is E, R or Q, and X3 is I or F. Preferred amino acid sequences are, e.g., X1IX2IT (SEQ ID NO:74), RIX2IT (SEQ ID NO:75), X1IEIT (SEQ ID NO:76), RIEIT (SEQ ID NO:1), SIRIT (SEQ ID NO:2), SIQIT (SEQ ID NO:3), and SIQFT (SEQ ID NO:4).

[0016] In certain embodiments, the peptide fragment comprises the amino acid sequence PX1IX2X3T (SEQ ID NO:77), wherein X1 is R or S, X2 is E, R or Q, and X3 is I or F. Preferred amino acid sequences are, e.g., PRIEIT (SEQ ID NO:5), PSIRIT (SEQ ID NO:6), PSIQIT (SEQ ID NO:71) and PSIQFT (SEQ ID NO:7).

[0017] In certain embodiments, the peptide fragment comprises the amino acid sequence X5PX1IX2X3T (SEQ ID NO:78), wherein X1 is R or S, X2 is E, R or Q, X3 is I or F and X5 is S or C. Preferred amino acid sequences are, e.g., SPRIEIT (SEQ ID NO:8), CPSIRIT (SEQ ID NO:9), CPSIQIT (SEQ ID NO:10) and CPSIQFT (SEQ ID NO:11).

[0018] In certain embodiments, the peptide fragment comprises the amino acid sequence X5PX1IX2X3TX6 (SEQ ID NO:79), wherein X1 is R or S, X2 is E, R or Q, X3 is I or F, X5 is S or C, and X6 is P or S. Preferred amino acid sequences are, e.g., SPRIEITP (SEQ ID NO:12), SPRIEITS (SEQ ID NO:13), CPSIRITS (SEQ ID NO:14), CPSIQITS (SEQ ID NO:15) and CPSIQFTS (SEQ ID NO:16).

[0019] In certain embodiments, the peptide fragment comprises the amino acid sequence X5PX1IX2X3TX6X7 (SEQ ID NO:80), wherein X1 is R or S, X2 is E, R or Q, X3 is I or F, X5 is S or C, X6 is P or S, and X7 is S, C or I. Preferred amino acid sequences are 1 SPRIEITPS, (SEQ ID NO:17) SPRIEITSC, (SEQ ID NO:18) CPSIRITSI, SEQ ID NO:19) CPSIQITSI, and (SEQ ID NO:20) CPSIQFTSI. (SEQ ID NO:21)

[0020] In certain embodiments, the peptide fragment comprises the amino acid sequence X11X10X9X5PX1IX2X3TX6X7X8 (SEQ ID NO:81), wherein X1 is R or S, X2 is E, R or Q, X3 is I or F, X5 is S or C, X6 is P or S, X7 is S, C or I, X8 is H, L or S, X9 is P, L or E, X10, is G, L or F, and X11 is S, A, V or P. Preferred amino acid sequences are, e.g., SGPSPRIEITPSH (SEQ ID NO:22), SGLSPRIEITPSH (SEQ ID NO:23), ALESPRIEITSCL (SEQ ID NO:24), VLECPSIRITSIS (SEQ ID NO:25), PFECPSIQITSIS (SEQ ID NO:26), PFECPSIQITSIS (SEQ ID NO:27) and PFECPSIQFTSIS (SEQ ID NO:28). Other preferred amino acid sequences are, e.g., 2 KPAGASGPSPRIEITPSHELMQAGG, (SEQ ID NO:29) KPAGASGLSPRIEITPSHELIQAVG, (SEQ ID NO:30) PDGAPALESPRIEITSCLGLYHNNN, (SEQ ID NO:31) AGGGRVLECPSIRITSISPTPEPPA, (SED ID NO:32) LGGPKPFECPSIQITSISPNCHQEL, (SEQ ID NO:33) LGGPKPFECPSIQITSISPNCHQGT and (SEQ ID NO:34) LGGPKPFECPSIQFTSISPNCQQEL. (SEQ ID NO:35)

[0021] Another aspect of the invention is an isolated polynucleotide encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, or biologically active analogs thereof. Preferred polynucleotide sequences are, e.g., the sequences as set forth in SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114 and SEQ ID NO:115.

[0022] Another aspect of the invention is an isolated polynucleotide encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or biologically active analogs thereof. Preferred polynucleotide sequences are, e.g., the sequences as set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:83 and SEQ ID NO:84.

[0023] Another aspect of the invention is an isolated polynucleotide encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:71, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or biologically active analogs thereof. Preferred polynucleotide sequences are, e.g., the sequences as set forth in SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:72, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92.

[0024] Another aspect of the invention is an isolated polynucleotide encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or biologically active analogs thereof. Preferred polynucleotide sequences are, e.g., the sequences as set forth in SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:63.

[0025] Another aspect of the invention is an isolated polynucleotide encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or biologically active analogs thereof. Preferred polynucleotide sequences are, e.g., sequences as set forth in SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:70.

[0026] Another aspect of the invention is a gene therapy vector comprising a nucleotide sequence encoding a peptide fragment of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog of the peptide fragment.

[0027] In preferred embodiments, the gene therapy vector comprises a nucleotide sequence encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, or biologically active analogs thereof. In certain embodiments, the gene therapy vector comprises the nucleotide sequences as set forth in SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114 or SEQ ID NO:115.

[0028] In preferred embodiments, the gene therapy vector comprises a nucleotide sequence encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or biologically active analogs thereof. In certain embodiments, the gene therapy vector comprises the nucleotide sequences as set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:83 or SEQ ID NO:84.

[0029] In preferred embodiments, the gene therapy vector comprises a nucleotide sequence encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:71, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or biologically active analogs thereof. In certain embodiments, the gene therapy vector comprises the nucleotide sequence as set forth in SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:72, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92.

[0030] In preferred embodiments, the gene therapy vector comprises a nucleotide sequence encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or biologically active analogs thereof. In certain embodiments, the gene therapy vector comprises the nucleotide sequence as set forth in SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 or SEQ ID NO:63.

[0031] In preferred embodiments, the gene therapy vector comprises a nucleotide sequence encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, or biologically active analogs thereof. In certain embodiments, the gene therapy vector comprises the nucleotide sequence as set forth in SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 or SEQ ID NO:70.

[0032] Another aspect of the invention is a cell having a gene therapy vector described herein.

[0033] Another aspect of the invention is a method for producing a peptide capable of inhibiting protein-protein interaction between calcineurin and NFAT, comprising culturing a cell having a gene therapy vector described herein under conditions that permit expression of the peptide.

[0034] Another aspect of the invention is a method for treating an immune-related disease or condition, e.g., acute immune diseases, chronic immune diseases or autoimmune diseases, in an animal. It is also meant to include treatment of tissue or organ transplant graft rejections or graft-versus-host disease. A gene therapy vector described herein is administered to the animal.

[0035] Another aspect of the invention is a method for providing an animal having an immune-related disease or condition with a therapeutically effective level of a peptide capable of inhibiting protein-protein interaction between calcineurin and NFAT. A gene therapy vector described herein is administered to the animal.

[0036] Another aspect of the invention is a method for inhibiting an immune response in an animal. An animal in need of inhibition of an immune response is provided. A therapeutically effective amount of a peptide fragment of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof, is provided. The peptide fragment or biologically active analog thereof is administered to the animal so as to inhibit the immune-response in the animal.

[0037] In certain embodiments, the therapeutically effective amount of the peptide fragment is provided by providing to the animal a recombinant nucleic acid having a nucleotide sequence encoding the peptide fragment or a biologically active analog thereof, and which is capable of expressing the peptide fragment or biologically active analog thereof in vivo. The peptide fragment is administered to the animal by administering the recombinant nucleic acid. The nucleic acid can be, e.g., any of the polynucleotides described herein.

[0038] In certain embodiments, the therapeutically effective amount of the peptide fragment is provided by providing to the animal a composition comprising animal cells wherein a recombinant nucleic acid having a nucleotide sequence encoding the peptide fragment has been introduced ex vivo into the animal cells so as to express the peptide fragment in the animal cells. The peptide fragment is administered to the animal by administering the animal cells having the recombinant nucleic acid. Preferably, the recombinant nucleic acid is a gene therapy vector, e.g., as described herein. Preferably, the animal cells are derived from the animal to be treated or allogeneic cells.

[0039] Another aspect of the invention is a method for treating a disease involving hyperactivity or inappropriate activity of the immune system, a transplant graft rejection or graft-versus-host disease, in an animal. An animal in need of treatment for a disease involving hyperactivity or inappropriate activity of the immune system, a transplant graft rejection or graft-versus-host disease, is provided. A therapeutically effective amount of a peptide fragment of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof, is provided. The peptide fragment or biologically active analog thereof is administered to the animal in a therapeutically effective amount such that treatment of the disease involving hyperactivity or inappropriate activity of the immune system, transplant graft rejection or graft-versus-host disease, occurs.

[0040] Another aspect of the invention is a method for treating an animal at risk for a disease involving hyperactivity or inappropriate activity of the immune system, a transplant graft rejection or graft-versus-host disease. An animal at risk for a disease involving hyperactivity or inappropriate activity of the immune system, a transplant graft rejection or graft-versus-host disease, is provided. A therapeutically effective amount of a peptide fragment of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof, is provided. The peptide fragment or biologically active analog thereof is administered in a therapeutically effective amount such that treatment occurs.

[0041] Another aspect of the invention is a method for gene therapy. An animal cell is genetically modified such that it is able to express a peptide fragment or biologically active analog thereof of the conserved regulatory domain of NFAT protein, the peptide fragment being capable of inhibiting calcineurin-mediated NFAT activation, so as to effect gene therapy. In certain embodiments, the animal cells are genetically modified by introducing into the cells a recombinant nucleic acid having a nucleotide sequence encoding the peptide fragment and which is capable of expressing the peptide fragment in vivo. Preferably, the recombinant nucleic acid is a gene therapy vector, e.g., as described herein.

[0042] Another aspect of the invention is a pharmaceutical composition for treating an immune-related disease or condition in an animal comprising a therapeutically effective amount of a peptide fragment of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof, and a pharmaceutically acceptable carrier. The peptide fragment can be, e.g., any of the peptide fragments described herein.

[0043] Another aspect of the invention is a pharmaceutical composition for treating an immune-related disease or condition in an animal, comprising a therapeutically effective amount of a recombinant nucleic acid having a nucleotide sequence encoding a peptide fragment of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof, and a pharmaceutically acceptable carrier. The nucleic acid can be, e.g., any of the polynucleotides described herein.

[0044] Another aspect of the invention is a pharmaceutical composition for treating an immune-related disease or condition in an animal, comprising a therapeutically effective amount of animal cells wherein a recombinant nucleic acid having a nucleotide sequence encoding a peptide fragment of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof, has been introduced into the animal cells so as to express the peptide fragment; and a pharmaceutically acceptable carrier. Preferably, the animal cells are cells derived from the animal to be treated or allogeneic cells.

[0045] Another aspect of the invention is a method for inhibiting protein-protein interaction between calcineurin and NFAT in vivo. A cell having calcineurin and NFAT is provided. A peptide fragment or a biologically active analog thereof of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT is provided. The calcineurin and peptide fragment or biologically active analog thereof are contacted in vivo such that protein-protein interaction between the calcineurin and the NFAT is inhibited.

[0046] Another aspect of the invention is a method for inhibiting protein-protein interaction between calcineurin and NFAT in vitro. Calcineurin and NFAT are provided. A peptide fragment or a biologically active analog thereof of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT is provided. The calcineurin and peptide fragment or biologically active analog thereof are contacted in vitro such that protein-protein interaction between the calcineurin and the NFAT is inhibited.

[0047] Another aspect of the invention is a method for evaluating an agent for use in modulating an immune response. A cell is provided. An agent, e.g., a peptide fragment of the conserved regulatory domain of NFAT protein or biologically active analogs thereof, is provided. The effect of the agent on an aspect of calcineurin-mediated NFAT activation is evaluated, e.g., protein-protein interaction between calcineurin and NFAT, dephosphorylation of NFAT by calcineurin, recruitment of NFAT to the nucleus in a cell, conformational change in NFAT, or activation of NFAT-dependent gene transcription. A change in the aspect of calcineurin-mediated NFAT activation is indicative of the usefulness of the agent in modulating an immune response.

[0048] Another aspect of the invention is a method for high throughput screening of candidate agents to identify an agent that inhibits protein-protein interaction between calcineurin and NFAT. A first compound is provided. The first compound is calcineurin or a biologically active derivative thereof, or the first compound is NFAT or a biologically active derivative thereof. A second compound is provided which is different from the first compound and which is labeled. The second compound is calcineurin or a biologically active derivative thereof, or the second compound is NFAT or a biologically active derivative thereof. A candidate agent is provided. The first compound, second compound and candidate agent are contacted with each other. The amount of label bound to the first compound is determined. A reduction in protein-protein interaction between the first compound and the second compound as assessed by label bound is indicative of the usefulness of the agent in inhibiting protein-protein interaction between calcineurin and NFAT. In certain embodiments, the method includes a washing step after the contacting step, so as to separate bound and unbound label.

[0049] Another aspect of the invention is a method for high throughput screening of candidate agents to identify an agent that inhibits protein-protein interaction between calcineurin and NFAT. A first compound is provided. The first compound is calcineurin or a biologically active active derivative thereof, or the first compound is an organic molecule capable of binding to calcineurin and inhibiting protein-protein interaction between calcineurin and NFAT. A second compound is provided which is different from the first compound and which is labeled. The second compound is calcineurin or a biologically active active derivative thereof, or the second compound is an organic molecule capable of binding to calcineurin and inhibiting protein-protein interaction between calcineurin and NFAT. A candidate agent is provided. The first compound, second compound and candidate agent are contacted with each other. The amount of label bound to the first compound is determined. A reduction in protein-protein interaction between the first compound and the second compound as assessed by label bound is indicative of the usefulness of the agent in inhibiting protein-protein interaction between calcineurin and NFAT. In certain embodiments, the method includes a washing step after the contacting step, so as to separate bound and unbound label.

[0050] In another aspect, the invention provides a method for high throughput screening of candidate agents to identify an agent that inhibits protein-protein interaction between calcineurin and NFAT. A first compound is provided. The first compound is NFAT or a biologically active active derivative thereof, or the first compound is an organic molecule capable of binding to NFAT and inhibiting protein-protein interaction between calcineurin and NFAT. A second compound is provided which is different from the first compound and which is labeled. The second compound is NFAT or a biologically active active derivative thereof, or the second compound is an organic molecule capable of binding to NFAT and inhibiting protein-protein interaction between calcineurin and NFAT. A candidate agent is provided. The first compound, second compound and candidate agent are contacted with each other. The amount of label bound to the first compound is determined. A reduction in protein-protein interaction between the first compound and the second compound as assessed by label bound is indicative of the usefulness of the agent in inhibiting protein-protein interaction between calcineurin and NFAT. In certain embodiments, the method includes a washing step after the contacting step, so as to separate bound and unbound label.

[0051] Another aspect of the invention is a method for high-throughput screening of candidate agents to identify an agent that inhibits dephosphorylation of NFAT by calcineurin. Phosphorylated NFAT is provided. Calcineurin or a biologically active derivative thereof having enzymatic activity is provided. A candidate agent is provided. The phosphorylated NFAT, the calcineurin or biologically active derivative thereof, and the candidate agent are contacted with each other in reaction media, e.g., buffer, under conditions that allow enzymatic activity of calcineurin. In certain embodiment, the NFAT is separated from the reaction media. It is determined whether phosphate remains associated with the NFAT. If phosphate remains associated with NFAT, it is indicative of the usefulness of the agent in inhibiting dephosphorylation of NFAT by calcineurin.

[0052] Another aspect of the invention is a method for high-throughput screening of candidate agents to identify an agent that inhibits conformational change in NFAT from dephosphorylation by calcineurin. Phosphorylated NFAT is provided. Calcineurin or a biologically active derivative thereof having enzymatic activity is provided. A candidate agent is provided. An oligonucleotide having an NFAT site is provided. The phosphorylated NFAT, calcineurin or biologically active derivative thereof, and the candidate agent are contacted with each other in reaction media under conditions that allow enzymatic activity of calcineurin. Specific binding of NFAT to the oligonucletide having the NFAT site is determined. A reduction of binding is indicative of the usefulness of the agent in inhibiting conformational change in NFAT from dephosphorylation by calcineurin.

[0053] Another aspect of the invention is a method for high-throughput screening of candidate agents to identify an agent that inhibits calcineurin-dependent import of NFAT into the nucleus of a cell. Cells expressing NFAT are provided. A stimulant that activates NFAT through the calcium/calcineurin pathway is provided. A candidate agent is provided. The cells, stimulant and candidate agent are contacted with each other. The presence or absence of nuclear NFAT in the cells is determined. A reduction in nuclear NFAT is indicative of the agent inhibiting calcineurin-dependent import of NFAT into the nucleus of a cell.

[0054] Another aspect of the invention is a method for assessing the state of NFAT activation of immune system cells from an animal. Immune system cells isolated from an animal are provided. The presence or absence of nuclear NFAT in the cells is determined. The presence of nuclear NFAT in the cells is indicative of activation of NFAT in the cells.

[0055] Another aspect of the invention is a method for assessing the ability of immune system cells isolated from an animal to respond to an NFAT activating signal. Immune system cells from an animal are provided, the cells being unactivated for NFAT. A stimulant that activates NFAT is provided. The cells are contacted with the stimulant. The presence or absence of nuclear NFAT in the cells is determined. A reduction in nuclear NFAT is indicative of impairment of the ability of the cells to respond to an NFAT activating signal.

[0056] Another aspect of the invention is a method for identifying a stimulant that can activate NFAT in immune system cells isolated from an animal. Immune system cells isolated from an animal are provided. A candidate stimulant, e.g., allergen, is provided. The cells are contacted with the candidate stimulant. The presence or absence of nuclear NFAT in the cells is determined. The presence of nuclear NFAT is indicative of the stimulant activating NFAT in the cells.

[0057] Another aspect of the invention is a method for identifying an allergen. An animal cell line expressing NFAT is provided. IgE from an animal, e.g., a human, is provided. A candidate allergen is provided. The cell line is contacted with the IgE. The cell line is contacted with the candidate allergen. The presence or absence of nuclear NFAT in cells of the cell line is determined. The presence of nuclear NFAT is indicative of the candidate allergen being an allergen.

[0058] In another aspect, the invention features a method for treating or preventing an NFAT-related disease or disorder, e.g., a disease or disorder caused by excessive or inappropriate activation of NFAT or a molecular target of NFAT, e.g., an immune disease, a cardiovascular disease, a skeletal muscle disease, a neurological disorder, or a weight disorder, in an animal. The method includes administering to an animal an agent, e.g., an organic molecule (e.g., an an organic molecule described herein), that modulates, e.g., inhibits, an interaction (e.g., a protein-protein interaction, e.g., binding) between calcineurin and NFAT, in an amount sufficient to treat the NFAT-related disease or disorder in the animal.

[0059] In another aspect, the invention features a pharmaceutical composition comprising a therapeutically effective amount of an organic molecule capable of inhibiting protein-protein interaction between calcineurin and NFAT, and a pharmaceutically acceptable carrier. The agent can be, e.g., an agent that inhibits dephosphorylation of NFAT by calcineurin. The molecular weight of the organic molecule can be less than 2500 Da, e.g., about 100 to 2000 Da, about 200 to 1500 Da, or about 300 and 1000 Da. The organic molecule can bind to calcineurin with an affinity constant of at least about 2×104 M−1, e.g., with an affinity constant of at least about 106 M−1, at least about 107 M−1, or at least about 108 M−1. The organic molecule can be a compound generally represented by:

[0060] formula (I): 1

[0061] wherein R1 is hydrogen, C1-C20 alkyl optionally substituted with 1-20 R6, C3-C8 cycloalkyl optionally substituted with 1-3 R6, aryl optionally substituted with 1-4 R6, heterocyclyl optionally substituted with 1-3 R6; heteroaryl optionally substituted with 1-4 R6; C2-C8 alkenyl, or C2-C8 alkynyl, cyano, nitro, carboxy, carbo(C1-C6)alkoxy, trihalomethyl, halogen, C1-C6 alkoxy, hydroxy, aryloxy, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, or ureido; R2 is C1-C20 alkyl optionally substituted with 1-20 R6, C3-C8 cycloalkyl optionally substituted with 1-3 R6, aryl optionally substituted with 1-4 R6, heterocyclyl optionally substituted with 1-3 R6, heteroaryl optionally substituted with 1-4 R6, C1-C6 alkoxy, or hydroxy; R3 is hydrogen or halogen; R4 is hydrogen, C1-C20 alkyl optionally substituted with 1-20 R6, C3-C8 cycloalkyl optionally substituted with 1-3 R6, aryl optionally substituted with 1-4 R6, heterocyclyl optionally substituted with 1-3 R6, heteroaryl optionally substituted with 1-4 R6, or halogen; R5 is NR7, O or S; R6 is halogen, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido; and R7 is C1-C6 alkyl;

[0062] formula (II): 2

[0063] wherein: R1 and R2 are each independently hydrogen, halogen, amino, C1-C6alkylamino, di(C1-C6)alkylamino, arylamino, diarylamino, or 4,4-dimethyl-2,6-dioxocyclohexyl; R3 is NR11 or O; R4, R5 and R8 are each independently hydrogen, C1-C6 alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido; R6 is hydrogen, halogen, or when taken together with R7 forms a double bond between the carbon atoms to which they are attached, R7 is hydrogen, halogen, or when taken together with R6 forms a double bond between the carbon atoms to which they are attached, R9 is OR3, or when taken together with R10 forms a double bond between the carbon and nitrogen atoms to which they are attached, R10 is hydrogen, or when taken together with R9 forms a double bond between the carbon and nitrogen atoms to which they are attached;

[0064] R11 is SO2R12; and R12 is aryl optionally substituted with alkyl, R13 is alkyl or aryl; and

[0065] formula (III): 3

[0066] wherein R1 and R4 are each independently O or NR8; R2 and R3 are each independently hydrogen, halogen, or R2 and R3 together combine to form aryl optionally substituted with 1-4 R9; R5 is hydrogen, halogen, carboxy, acylamino, alkoxycarbonyl, carboxy, alkylcarbonyl, acyloxy, or cyano; R6, R7 and R9 are each independently hydrogen, C1-C6 alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido; R8 is SO2R10; and R10 is aryl optionally substituted with alkyl.

[0067] In another aspect, the invention features a method for inhibiting protein-protein interaction between calcineurin and NFAT. The method includes providing calcineurin and NFAT, providing a pharmaceutical composition, e.g., a pharmaceutical composition described above, and contacting the calcineurin, NFAT, and pharmaceutical composition together, such that the protein-protein interaction between calcineurin and NFAT is inhibited.

[0068] In another aspect, the invention features a method of inhibiting an immune response in an animal, which includes administering to the animal an organic molecule, e.g., an organic molecule described herein, capable of inhibiting protein-protein interaction between calcineurin and NFAT. The organic molecule can be administered to the animal as a pharmaceutical composition as described herein.

[0069] In another aspect, the invention features a method for treating a disease involving hyperactivity or inappropriate activity of the immune system. The method includes identifying an animal suffering from a disease involving hyperactivity or inappropriate activity of the immune system, and administering to the animal a therapeutically effective amount of a pharmaceutical composition, e.g., a pharmaceutical composition comprising a therapeutically effective amount of an organic molecule capable of inhibiting protein-protein interaction between calcineurin and NFAT, and a pharmaceutically acceptable carrier, e.g., a pharmaceutical composition as described above, to thereby treat the disease involving hyperactivity or inappropriate activity of the immune system. The disease involving hyperactivity or inappropriate activity of the immune system can be, e.g., an acute immune disease or disorder, a chronic immune disease or disorder, or an autoimmune disease or disorder.

[0070] In another aspect, the invention features a method for treating a disease involving excessive or inappropriate activation of NFAT, or a molecular target thereof. Molecular targets include, but are not limited to genes involved in immune activation and inflammation, e.g., genes encoding cytokines and chemokines, e.g,. IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, GM-CSF, IFN-gamma, TNF-alpha, MIP-1alpha, MIP-1beta, RANTES, lymphotactin, IL-16, and IL-18, and cell surface proteins, e.g., CD25, CD40L, CD69, CTLA-4, FasL, CD5, CD21, CD3-gamma, CD23, and LIGHT; transcription factors, e.g., NFkappaB p50; c-Rel; Oct2; NFAT2 (also called NFATc, NFATc1); Nur77; NF-IL3/E4BP4; EGR1; EGR2; and EGR3; and effectors and other targets, e.g., granzyme B; perforin; COX-2; CDK4; and p21/CIP1. Molecular targets also include genes involved in immune anergy or tolerance, e.g., transcriptional activators and repressors, e.g., Jumonji, Ikaros, Groucho-related protein-4, SATB1, NF-IL3/E4BP4, RNF19, HIF-1, Elf-1, and LAD/TSAd; genes encoding receptor and non-receptor tyrosine phosphatases, e.g., RPTP-sigma, RPTP-kappa, and PTP-1B; genes encoding G-proteins or other signalling proteins, enzymes, and cell surface receptors (e.g., Rab10; GBP-3; RGS-2; diacylglycerol kinase alpha; Mlp (MARCKS-like protein); LAD/TSAd; ZAP70; phosphoglycerate mutase; glutamate dehydrogenase; LDH-alpha; CD3 epsilon; 4-IBB ligand; CD98 heavy chain; FasL; cation-dependent mannose-6-phosphate receptor; gamma-aminobutyric acid receptor-associated protein 1); and genes and proteins involved in proteolytic pathways, endocytosis, lysosomal degradation (e.g., SOCS-2; TRAF5; caspase 3; serpin 1b; and cystatin C); and others (e.g., tetracycline transporter-like protein; M-CSF; osmotic stress protein 94; heme oxygenase 2a; calcyclin; CDK4. Molecular targets further include genes involved in osteoclast differentiation and function, e.g., tartrate-resistant acid phosphatase, osteoclast stimulating factor, and calcitonin receptor; genes involved in cardiac hypertrophy, e.g., atrial natriuretic factor, adenylosuccinate synthetase-1; and B-type natriuretic peptide; and genes involved in viral replication and activation, e.g., HIV-1 LTR, and gene(s) involved in calcium-dependent re-activation of latent KSHV; and other genes, e.g., IP3R; COX-2; eNOS; iNOS; CDK4; p21/CIP1; BMP2; Myf5; myosin heavy chain IIa; VEGF; and gene(s) involved in growth of vascular smooth muscle cells in response to PDGF. The method includes identifying an animal suffering from a disease involving excessive or inappropriate activation of NFAT or a molecular target thereof; and administering to the animal a therapeutically effective amount of a pharmaceutical composition described herein, to thereby treat the disease involving excessive or inappropriate activation of NFAT or molecular target thereof.

[0071] In another aspect, the invention features a method of manufacturing an agent that inhibits protein-protein interaction between calcineurin and NFAT. The method includes providing an organic compound capable of inhibiting protein-protein interaction between calcineurin and NFAT, e.g., an organic compound as described herein, providing at least one pharmaceutically acceptable carrier; and combining the organic compound with the pharmaceutically acceptable carrier, to thereby manufacture an agent that inhibits protein-protein interaction between calcineurin and NFAT

[0072] The invention also features a method of manufacturing an agent that inhibits protein-protein interaction between calcineurin and NFAT. The process includes carrying out a method to identify an agent that inhibits protein-protein interaction between calcineurin and NFAT, e.g., a method as described herein. In one embodiment, the method includes providing a first compound selected from the group consisting of calcineurin or a biologically active derivative thereof, and NFAT or a biologically active derivative thereof; providing a second compound selected from the group consisting of calcineurin or a biologically active derivative thereof, and NFAT or a biologically active derivative thereof, wherein the second compound is different from the first compound, and wherein the second compound is labeled; providing a candidate agent; contacting the first compound, the second compound, and the candidate agent with each other; and determining the amount of label bound to the first compound, wherein a reduction in interaction between the first compound and the second compound as assessed by label bound is indicative of usefulness of the candidate agent in inhibiting protein-protein intereaction between calcineurin and NFAT; and manufacturing the agent, to thereby make an agent that inhibits protein-protein interaction between calcineurin and NFAT. In one embodiment, the first compound is calcineurin and the second compound is a biologically active derivative of NFAT. In another embodiment, the biologically active derivative of NFAT comprises the amino acid sequence GPHPVIVITGPHEE.

[0073] In some embodiments, the methods of manufacturing further comprise the step of manufacturing the agent into a form suitable for administration to an animal via a particular route, e.g., an oral, parenteral, topical, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, or intrasternal route.

[0074] In another aspect, the invention features a method for inhibiting protein-protein interaction between calcineurin and NFAT. The method includes providing calcineurin and NFAT, providing an organic molecule capable of inhibiting protein-protein interaction between calcineurin and NFAT, e.g., an organic compound described herein, and contacting the calcineurin and the organic compound such that protein-protein interaction between the calcineurin and the NFAT is inhibited.

[0075] In another aspect, the invention features a method of transplanting an organ. The method includes: (a) providing an organ from a donor, (b) transplanting the organ into a recipient; and (c) before, during, and/or after (b), administering to the recipient an amount of an organic molecule described herein capable of inhibiting protein-protein interaction between calcineurin and NFAT, to thereby transplant an organ. The organic molecule can be administered to the recipient in the form of a pharmaceutical composition as described herein. The term “donor” as used herein refers to an animal (human or non-human) from whom an organ or tissue can be obtained for the purposes of transplantation to a recipient. The term “recipient” refers to an animal (human or non-human) into which an organ or tissue can be transferred. The term “organ(s)” is used throughout the specification as a general term to describe any anatomical part or member having a specific function in the animal. Further included within the meaning of this term are substantial portions of organs, e.g., cohesive tissues obtained from an organ. Such organs include but are not limited to kidney, liver, heart, intestine, e.g., large or small intestine, pancreas, and lungs. Further included in this definition are bones and blood vessels, e.g. aortic transplants.

[0076] The invention also features a method of treating an animal to prevent transplant rejection. The method includes (a) transplanting an organ into an animal; and (b) before, during, or after (a), administering to the animal an organic compound, e.g., an organic molecule desribed herein, in an amount sufficient to inhibit the protein-protein interaction between calcineurin and NFAT, to thereby prevent transplant rejection in the animal. In an embodiment, the organic compound can be administered as a pharmaceutical composition.

[0077] The above and other features, objects and advantages of the present invention will be better understood by a reading of the following specification in conjunction with the drawings. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although suitable methods and materials for the practice or testing of the present invention are described below, other methods and materials similar or equivalent to those described herein, which are well known in the art, can also be used. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0078] FIGS. 1A-1C depict amino acid sequences of certain immunosuppressive agents of this invention, including 5-mers, 6-mers, 7-mers, 8-mers, 9-mers, 13-mers and 25-mers.

[0079] FIGS. 2A-2E depict cDNA sequences from murine NFAT1, human NFAT1, human NFAT2, human NFAT3, human NFAT4 and murine NFAT4, encoding certain immunosuppressive agents of this invention, and the corresponding amino acid sequences.

[0080] FIGS. 3A-3B depict the nucleotide and amino acid sequences of a truncated human calcineurin A&agr;, respectively.

[0081] FIG. 4A is a sequence alignment comparing the calcineurin-docking sequences present in the NFAT family of proteins. Consensus=consensus sequence of the four NFAT sequences shown.

[0082] FIG. 4B depicts an amino acid sequence and a first round of selection for an optimized peptide inhibitor. Particular amino acid sequences selected in the degenerate positions are shown with preference values indicated in parenthesis. Residues showing strong selection are underlined.

[0083] FIG. 4C depicts an amino acid sequence and a second round of selection for an optimized peptide inhibitor. An alternative set of residues was chosen based on the initial screen shown in FIG. 4B to orient a secondary library, and the second library was selected on GST-calcineurin (amino acids 2 to 347) to derive high affinity peptides. Residues locked in from the screening in FIG. B are boxed. “Z” indicates a set of non-natural amino acids.

[0084] FIG. 4D depicts the amino acid sequence of the VIVIT peptide.

[0085] FIG. 5A is a Western blot that illustrates that SPRIEIT and VIVIT peptide inhibit the NFAT-calcineurin interaction. Cn=calcineurin, CaM=calmodulin, Ca2+=CaCl2, Cn A=calcineurin A chain.

[0086] FIG. 5B is a Western blot that illustrates that that SPRIEIT and VIVIT peptide inhibit calcineurin-mediated dephosphorylation of NFAT. The positions of phospho-and dephosphoNFAT1 are indicated by arrows. Cn=calcineurin, CaM=calmodulin, Ca2+=CaCl2, NaPPi=sodium pyrophosphate.

[0087] FIG. 5C is a bar graph illustrating that SPRIEIT and VIVIT peptides do not block dephosphorylation of RII phosphopeptide by calcineurin. The numbers next to each peptide label indicate peptide concentrations in &mgr;M. CsA/CypA complexes were used at 10 &mgr;M. CsA/Cyp=cyclosporin A/cyclophilin A.

[0088] FIG. 6A is a bar graph illustrating that VIVIT and SPRIEIT peptide, delivered intracellularly as a GFP-VIVIT and GFP-SPRIEIT fusion proteins, respectively, block expression of an NFAT-dependent reporter gene. Endog.=endogenous NFAT; NFAT1=overexpressed human NFAT1; NFAT2=overexpressed human NFAT2; NFAT4=overexpressed human NFAT4; PMA+iono=PMA and ionomycin stimulated cells; unstimulated=cells not stimulated with PMA and ionomycin.

[0089] FIG. 6B is a set of bar graphs illustrating that VIVIT peptide, delivered intracellularly as a GFP-VIVIT fusion protein, blocks expression of an NFAT-dependent reporter gene, but not of an NF-&kgr;B-dependent reporter gene. 3×NFAT-Luc=reporter plasmid containing three tandem NFAT sites in a promoter controlling a luciferase gene; 2×NF-&kgr;B-Luc=reporter plasmid containing two NF-&kgr;B sites in a promoter controlling a luciferase gene; PMA+iono=PMA and ionomycin stimulated cells; Unstim.=cells not stimulated with PMA and ionomycin.

[0090] FIG. 6C is a set of bar graphs illustrating that cyclosporin A blocks expression of both reporter genes used in FIG. 6B. 3×NFAT-Luc=reporter plasmid containing three tandem NFAT sites in a promoter controlling a luciferase gene; 2×NF-&kgr;B-Luc=reporter plasmid containing two NF-&kgr;B sites in a promoter controlling a luciferase gene; CsA=cyclosporin A; P+I=PMA plus ionomycin.

[0091] FIG. 6D is a set of bar graphs that illustrate that the VIVIT peptide, delivered intracellularly as a GFP-VIVIT fusion protein, inhibits reporter activity driven by IL-2 and TNF-&agr; promoters. IL-2 promoter=reporter plasmid containing human IL-2 promoter controlling a luciferase gene; TNF-&agr; promoter=reporter plasmid containing human TNF-&agr; promoter controlling a luciferase gene; PMA+iono=PMA and ionomycin stimulated cells; Unstim.=cells neither stimulated with PMA and ionomycin nor stimulated with antibodies; CD3+CD28=cells stimulated with anti-CD3 and anti-CD28 antibodies.

[0092] FIG. 7A is a picture of an RNA gel autoradiogram illustrating that T cells expressing GFP-VIVIT displayed attenuated induction of IL-2 and IL-13 mRNAs.

[0093] FIG. 7B is a picture of an RNA gel autoradiogram illustrating that T cells expressing GFP-VIVIT display attenuated induction of IL-3, TNF&agr;, GM-CSF, and MIP-1&agr; mRNAs.

[0094] FIG. 7C is a picture of an RNA gel autoradiogram illustrating that T cells expressing GFP-VIVIT display attenuated induction TNF&agr;.

[0095] FIG. 8A is a line graph illustrating saturable binding of a fluorescent VIVIT peptide to calcineurin.

[0096] FIG. 8B is a line graph illustrating competition for binding to calcineurin by unlabelled VIVIT peptide.

[0097] FIG. 9A is a line graph illustrating that the organic compounds INCA1 and INCA2 compete with a VIVIT peptide for binding to calcineurin.

[0098] FIG. 9B is a line graph illustrating that the organic compound INCA6 competes with a VIVIT peptide for binding to calcineurin.

[0099] FIG. 10A is an NMR spectrum illustrating that INCA1 binds to calcineurin.

[0100] FIG. 10B is an NMR spectrum illustrating that INCA2 binds to calcineurin.

[0101] FIG. 10C is an NMR spectrum illustrating that INCA6 binds to calcineurin.

[0102] FIG. 11A is a picture of a Western Blot illustrating that INCA2 blocks dephosphorylation of NFAT by calcineurin in vitro. Iono=ionomycin. PPi=sodium pyrophosphate; CN=calcineurin in the absence of inhibitor; DMSO=Dimethyl Sulfoxide.

[0103] FIG. 11B is a bar graph illustrating that INCA compounds do not block dephosphorylation of RII phosphopeptide. DMSO=Dimethyl Sulfoxide; CsA/Cyp=cyclosporin A/cyclophilin A.

[0104] FIG. 12A is a picture of a Western blot illustrating that INCA6 blocks dephosphorylation of NFAT by calcineurin in cells. Iono=ionomycin.

[0105] FIG. 12B is a picture of a Western blot illustrating that INCA6 does not block phosphorylation of MAP kinase. PMA=phorbol 12-myristate 13-acetate.

[0106] FIG. 13 is a set of micrographs illustrating that INCA6 blocks the nuclear import of NFAT in cells. Iono=ionomycin; CsA=cyclosporin A.

[0107] FIG. 14A is a picture of an RNA gel autoradiogram illustrating that INCA6 attenuated induction of GM-CSF in T cells. CsA/FK506=cyclosporin A and FK506; Iono=ionomycin; PMA=phorbol 12-myristate 13-acetate.

[0108] FIG. 14B is a picture of an RNA gel autoradiogram illustrating that INCA6 attenuated induction of TNF&agr; and IFN&ggr; in T cells. CsA/FK506=cyclosporin A and FK506; Iono=ionomycin; PMA=phorbol 12-myristate 13-acetate.

[0109] FIG. 14C is a picture of an RNA gel autoradiogram illustrating that INCA6 attenuated induction of lymphotactin (Ltn), MIP-1&bgr;, and MIP-1&agr; in T cells. CsA/FK506=cyclosporin A and FK506; Iono=ionomycin; PMA=phorbol 12-myristate 13-acetate.

[0110] FIG. 15A illustrates the general chemical structure of INCA1, typical chemical modifications that can be made thereto, and the inhibitory activity exhibited by the modified compounds.

[0111] FIG. 15B illustrates the general chemical structure of INCA2, typical chemical modifications that can be made thereto, and the inhibitory activity exhibited by the modified compounds.

[0112] FIG. 15C illustrates the general chemical structure of INCA6, typical chemical modifications that can be made thereto, and the inhibitory activity exhibited by the modified compounds.

DETAILED DESCRIPTION

[0113] This invention provides an isolated fragment of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof.

[0114] By NFAT protein (nuclear factor of activated T cells) is meant a member of a family of transcription factors comprising the members NFAT1, NFAT2, NFAT3 and NFAT4, with several isoforms. Any other NFAT protein whose activation is calcineurin dependent is also meant to be included. NFAT proteins can be, e.g., mammalian proteins, e.g., human or murine. NFAT1, NFAT2 and NFAT4 are expressed in immune cells, e.g., T lymphocytes, and play a role in eliciting immune responses. NFAT proteins are involved in the transcriptional regulation of cytokine genes, e.g., IL-2, IL-3, IL-4, TNF-&agr; and IFN-&ggr;, during the immune response.

[0115] cDNA sequences for NFAT have been previously reported. See McCaffrey et al., Science 262:750-754 (1993) and Luo et al., Mol. Cell Biol. 16:3955-3966 (1996) for murine NFAT1. See Luo et al., Mol. Cell Biol. 16:3955-3966 (1996) for human NFAT1. See Northrop et al., Nature 369:497-502 (1994) for human NFAT2, and Park et al., J. Biol. Chem. 271:20914-20921 (1996) for human NFAT2b. The published sequences for human NFAT2 represent two isoforms differing by alternative splicing at the N and C termini, but having the same regulatory domain and DNA-binding domain. See Hoey et al., Immunity 2:461-472 (1995) for human NFAT3. See Masuda et al., Mol. Cell Biol. 15:2697-2706 (1995) and Hoey et al., Immunity 2:461-472 (1995) for human NFAT4. See Ho et al., J. Biol. Chem. 270:19898-19907 (1995) and Liu et al., Mol. Cell Biol. 8:157-170 (1997) for murine NFAT4. The two published sequences for murine NFAT4 are not identical.

[0116] NFAT proteins have been shown to be direct substrates of calcineurin. Calcineurin is a calmodulin-dependent, cyclosporin A/FK506-sensitive, phosphatase. Calcineurin is activated through its interaction with Ca+2 activated calmodulin when intracellular calcium levels are elevated as a result of receptor crosslinking and phospholipase C activation. The activated calcineurin in turn activates NFAT from an inactive cytoplasmic pool. NFAT activation involves protein-protein interaction between calcineurin and NFAT, dephosphorylation of NFAT by calcineurin, conformational change in NFAT resulting from the interaction between calcineurin and NFAT or the dephosphorylation of NFAT and translocation of NFAT to the nucleus. NFAT activation results in induction of NFAT-dependent gene expression of, e.g., cytokine genes.

[0117] The conserved regulatory domain of NFAT is an N-terminal region of NFAT which is about 300 amino acids in length. The conserved regulatory domain of murine NFAT1 is a region extending from amino acid residue 100 through amino acid residue 397, of human NFAT1 is a region extending from amino acid residue 100 through 395, of human NFAT2 is a region extending from amino acid residue 106 through 413, of human NFAT2b is a region extending from amino acid residue 93 through 400, of human NFAT3 is a region extending from amino acid residue 102 through 404, and of human NFAT4 is a region extending from amino acid residue 97 through 418. The conserved regulatory domain is moderately conserved among the members of the NFAT family, NFAT1, NFAT2, NFAT3 and NFAT4. The conserved regulatory region binds directly to calcineurin. The conserved regulatory region is located immediately N-terminal to the DNA-binding domain (amino acid residues 398 through 680 in murine NFAT1, amino acid residues 396 through 678 in human NFAT1, amino acid residues 414 through 696 in human NFAT2, amino acid residues 401 through 683 in human NFAT2b, amino acid residues 405 through 686 in human NFAT3, and amino acid residues 419 through 700 in human NFAT4).

[0118] In certain embodiments of the invention, the peptide fragment or biologically active analog thereof is further capable of inhibiting dephosphorylation of NFAT by calcineurin. In certain embodiments, the peptide fragment or biologically active analog thereof is further capable of inhibiting recruitment of NFAT to the nucleus in a cell. In certain embodiments, the peptide fragment or biologically active analog thereof is further capable of inhibiting conformational change in NFAT that results from the protein-protein interaction between NFAT and calcineurin or from the dephosphorylation of NFAT by calcineurin. In certain embodiments, the peptide fragment or biologically active analog thereof is further capable of inhibiting NFAT-dependent gene transcription.

[0119] Preferably, the peptide fragment or biologically active analog thereof does not inhibit or does not substantially inhibit the activity of calcineurin toward non-NFAT calcineurin substrates. Calcineurin normally is capable of interacting with many different substrates, e.g., NFAT and the microtubule-associated protein tau (Fleming and Johnson, Biochem J 309:41-47 (1995); Yamamoto et al, J Biochem 118:1224-1231 (1995)), the regulatory subunit RII of cAMP-dependent protein kinase (Blumenthal and Krebs, Biophys J 41:409a (1983)), inhibitor-1 (Hemmings et al, Nature 310:503-505 (1984); Mulkey et al, Nature 369:486-488 (1994)), dopamine-and cAMP-regulated phosphoprotein DARPP-32 (Hemmings et al, Nature 310:503-505 (1984)), a dihydropyridine-sensitive voltage-dependent Ca2+ channel (Hosey et al, Proc Natl Acad Sci USA 83:3733-3737 (1986)), nitric oxide synthase (Dawson et al, Proc Natl Acad Sci USA 90:9808-9812 (1993)), dynamin (Liu et al, Science 265:970-973 (1994); Nichols et al, J Biol Chem 269:23817-23823 (1994)), the inositol 1, 4, 5-trisphosphate receptor-FKBP12 complex (Cameron et al, Cell 83:463-472 (1995)), and the ryanodine receptor-FKBP12 complex (Cameron et al, Cell 83:463-472 (1995)). A key advantage of this invention is that it includes peptide fragments and analogs thereof which are specific for the interaction between calcineurin and NFAT. Such specific inhibitors can be used for therapeutic purposes with reduced toxic effects, as compared to general immunosuppressants.

[0120] By fragment of the conserved regulatory domain of NFAT protein is meant some portion of the naturally occurring conserved regulatory domain of NFAT protein. Preferably, the fragment is less than about 150 amino acid residues, more preferably is less than about 100 amino acid residues, more preferably yet is less than about 50 amino acid residues, more preferably yet is less than about 30 amino acid residues, more preferably yet is less than about 20 amino acid residues, more preferably yet is less than about 10 amino acid residues, and most preferably is less than about 6 amino acid residues in length. Preferably, the fragment is greater than about 3 amino acid residues in length. Fragments include, e.g., truncated secreted forms, cleaved fragments, proteolytic fragments, splicing fragments, other fragments, and chimeric constructs between at least a portion of the relevant gene and another molecule. Fragments of the conserved regulatory domain of NFAT protein can be generated by methods known to those skilled in the art. In preferred embodiments, the fragment is biologically active. The ability of a candidate fragment to exhibit a biological activity of the conserved regulatory domain of NFAT can be assessed by, e.g., its ability to form a protein-protein interaction with calcineurin, or its ability to inhibit the binding of NFAT to calcineurin, by methods as described herein. Also included are fragments containing residues that are not required for biological activity of the fragment or that result from alternative mRNA splicing or alternative protein processing events.

[0121] Fragments of a protein can be produced by any of a variety of methods known to those skilled in the art, e.g., recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Expression of the mutagenized DNA produces polypeptide fragments. Digestion with “end-nibbling” endonucleases can thus generate DNAs which encode an array of fragments. DNAs which encode fragments of a protein can also be generated, e.g., by random shearing, restriction digestion, chemical synthesis of oligonucleotides, amplification of DNA using the polymerase chain reaction, or a combination of the above-discussed methods.

[0122] Fragments can also be chemically synthesized using techniques known in the art, e.g., conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, peptides of the present invention can be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length.

[0123] An NFAT protein used for generating analogs or fragments can be obtained, e.g., from purification or secretion of a naturally occurring NFAT protein, from recombinant NFAT protein, or from synthesized NFAT protein.

[0124] In certain embodiments, the peptide fragment comprises the amino acid sequence IX2X3T (SEQ ID NO:104), wherein X2 is E, R or Q, and X3 is I or F. Preferred amino acid sequences are, e.g., IEIT (SEQ ID NO:105), IRIT (SEQ ID NO:106), IQIT (SEQ ID NO:107), and IQFT (SEQ ID NO:108).

[0125] In certain embodiments, the peptide fragment comprises the amino acid sequence X1IX2X3T (SEQ ID NO:73), wherein X1 is R or S, X2 is E, R or Q, and X3 is I or F. Preferred amino acid sequences are, e.g., X1IX2IT (SEQ ID NO:74), RIX2IT (SEQ ID NO:75), X1IEIT (SEQ ID NO:76), RIEIT (SEQ ID NO:1), SIRIT (SEQ ID NO:2), SIQIT (SEQ ID NO:3), and SIQFT (SEQ. ID NO:4).

[0126] In certain embodiments, the peptide fragment comprises the amino acid sequence PX1IX2X3T (SEQ ID NO:77), wherein X1 is R or S, X2 is E, R or Q, and X3 is I or F. Preferred amino acid sequences are, e.g., PRIEIT (SEQ ID NO:5), PSIRIT (SEQ ID NO:6), PSIQIT (SEQ ID NO:71) and PSIQFT (SEQ ID NO:7).

[0127] In certain embodiments, the peptide fragment comprises the amino acid sequence X5PX1IX2X3T (SEQ ID NO:78), wherein X1 is R or S, X2 is E, R or Q, X3 is I or F and X5 is S or C. Preferred amino acid sequences are, e.g., SPRIEIT (SEQ ID NO:8), CPSIRIT (SEQ ID NO:9), CPSIQIT (SEQ ID NO:10) and CPSIQFT (SEQ ID NO:11).

[0128] In certain embodiments, the peptide fragment comprises the amino acid sequence X5PX1IX2X3TX6 (SEQ ID NO:79), wherein X1 is R or S, X2 is E, R or Q, X3 is I or F, X5 is S or C, and X6 is P or S. Preferred amino acid sequences are, e.g., SPRIEITP (SEQ ID NO:12), SPRIEITS (SEQ ID NO:13), CPSIRITS (SEQ ID NO:14), CPSIQITS (SEQ ID NO:15) and CPSIQFTS (SEQ ID NO:16).

[0129] In certain embodiments, the peptide fragment comprises the amino acid sequence X5PX1IX2X3TX6X7 (SEQ ID NO:80), wherein X1 is R or S, X2 is E, R or Q, X3 is I or F, X5 is S or C, X6 is P or S, and X7 is S, C or I. Preferred amino acid sequences are 3 SPRIEITPS, (SEQ ID NO:17) SPRIEITSC, (SEQ ID NO:18) CPSTRITSI, SEQ ID NO:19) CPSIQITSI and (SEQ ID NO:20) CPSIQFTSI. (SEQ ID NO:21)

[0130] In certain embodiments, the peptide fragment comprises the amino acid sequence X11IX10X9X5PX1IX2X3TX6X7X8 (SEQ ID NO:81), wherein X1 is R or S, X2 is E, R or Q, X3 is I or F, X5 is S or C, X6 is P or S, X7 is S, C or I, X8 is H, L or S, X9 is P, L or E, X10, is G, L or F, and X11 is S, A, V or P. Preferred amino acid sequences are, e.g., SGPSPRIEITPSH (SEQ ID NO:22), SGLSPRIEITPSH (SEQ ID NO:23), ALESPRIEITSCL (SEQ ID NO:24), VLECPSIRITSIS (SEQ ID NO:25), PFECPSIQITSIS (SEQ ID NO:26), PFECPSIQITSIS (SEQ ID NO:27) and PFECPSIQFTSIS (SEQ ID NO:28). Other preferred amino acid sequences are, e.g., 4 KPAGASGPSPRIEITPSHELMQAGG, (SEQ ID NO:29) KPAGASGLSPRIEITPSHELIQAVG, (SEQ ID NO:30) PDGAPALESPRIEITSCLGLYHNNN, (SEQ ID NO:31) AGGGRVLECPSIRITSISPTPEPPA, (SEQ ID NO:32) LGGPKPFECPSIQITSISPNCHQEL, (SEQ ID NO:33) LGGPKPFECPSIQITSISPNCHQGT and (SEQ ID NO:34) LGGPKPFECPSIQFTSISPNCQQEL. (SEQ ID NO:35)

[0131] By a biologically active analog of the NFAT fragment is meant an analog that is capable of inhibiting protein-protein interaction between calcineurin and NFAT.

[0132] By analog is meant a compound that differs from the naturally occurring NFAT fragment in amino acid sequence or in ways that do not involve sequence, or both. Peptide analogs of the invention generally exhibit at least about 70% homology, preferably at least about 80% homology, more preferably at least about 90% homology, more preferably yet at least about 95% homology, more preferably yet at least about 97% homology, and most preferably at least about 98% homology, with substantially the entire sequence of a naturally occurring NFAT fragment, preferably with a segment of about 150 amino acid residues, more preferably with a segment of about 100 amino acid residues, more preferably yet with a segment of about 50 amino acid residues, more preferably yet with a segment of about 30 amino acid residues, more preferably yet with a segment of about 20 amino acid residues, more preferably yet with a segment of about 10 amino acid residues, more preferably yet with a segment of about 5 amino acid residues, and most preferably yet with a segment of about 4 amino acid residues. Non-sequence modifications include, e.g., in vivo or in vitro chemical derivatizations of the NFAT fragment. Non-sequence modifications include, e.g., changes in phosphorylation, acetylation, methylation, carboxylation, or glycosylation. Methods for making such modifications are known to those skilled in the art. For example, phosphorylation can be modified by exposing the peptide to phosphorylation-altering enzymes, e.g., kinases or phosphatases.

[0133] Preferred analogs include an NFAT fragment whose sequence differs from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions, which do not abolish biological activity of the peptide. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other examples of conservative substitutions are shown in Table 1. 5 TABLE 1 CONSERVATIVE AMINO ACID SUBSTITUTIONS For Amino Acid Code Replace with any of Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Acid Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Acid Glycine G Ala, D-Ala, Pro, D-Pro, P-Ala Acp Histidine H D-His Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenyl- F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His; Trp, alanine D-Trp, Trans-3, 4, or 5-phenylproline, cis-3, 4, or 5-phenylproline Proline P D-Pro, L-I-thioazolidine-4-carboxylic acid, D-or L-I-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val Tryptophan W D-Trp, Phe, D-Phe, Tyr, D-Tyr Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

[0134] Amino acid sequence variants of a protein can be prepared by any of a variety of methods known to those skilled in the art. For example, random mutagenesis of DNA which encodes a protein or a particular domain or region of a protein can be used, e.g., PCR mutagenesis (using, e.g., reduced Taq polymerase fidelity to introduce random mutations into a cloned fragment of DNA; Leung et al., BioTechnique 1:11-15 (1989)), or saturation mutagenesis (by, e.g., chemical treatment or irradiation of single-stranded DNA in vitro, and synthesis of a complementary DNA strand; Mayers et al., Science 229:242 (1985)). Random mutagenesis can also be accomplished by, e.g., degenerate oligonucleotide generation (using, e.g., an automatic DNA synthesizer to chemically synthesize degenerate sequences; Narang, Tetrahedron 39:3 (1983); Itakura et al., Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. A. G. Walton, Amsterdam: Elsevier, pp. 273-289 (1981)). Non-random or directed mutagenesis can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, e.g., deletions, insertions, or substitutions, of residues of the known amino acid sequence of a protein. The sites for mutation can be modified individually or in series, e.g., by (i) substituting first with conserved amino acids and then with more radical choices depending upon results achieved, (ii) deleting the target residue, (iii) inserting residues of the same or a different class adjacent to the located site, or (iv) combinations of the above.

[0135] Methods for identifying desirable mutations include, e.g., alanine scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)), oligonucleotide-mediated mutagenesis (Adelman et al., DNA 2:183 (1983)), cassette mutagenesis (Wells et al., Gene 34:315 (1985)), combinatorial mutagenesis, and phage display libraries (Ladner et al., PCT International Appln. No. WO88/06630). The NFAT fragment analogs can be tested in physical and/or functional assays, e.g., in their ability to inhibit protein-protein interaction between calcineurin and NFAT, as described herein.

[0136] Other analogs within the invention include, e.g., those with modifications which increase peptide stability. Such analogs can contain, e.g., one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are, e.g., analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., &bgr; or &ggr; amino acids and cyclic analogs.

[0137] Analogs are also meant to include peptides in which structural modifications have been introduced into the peptide backbone so as to make the peptide non-hydrolyzable. Such peptides are particularly useful for oral administration, as they are not digested. Peptide backbone modifications include, e.g., modifications of the amide nitrogen, the &agr;-carbon, the amide carbonyl, or the amide bond, and modifications involving extensions, deletions or backbone crosslinks. For example, the backbone can be modified by substitution of a sulfoxide for the carbonyl, by reversing the peptide bond, or by substituting a methylene for the carbonyl group. Such modifications can be made by standard procedures known to those skilled in the art. See, e.g., Spatola, A. F., “Peptide Backbone Modifications: A Structure-Activity Analysis of Peptides Containing Amide Bond Surrogates, Conformational Constraints, and Related Backbone Replacements,” in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp. 267-357, B. Weinstein (ed.), Marcel Dekker, Inc., New York (1983).

[0138] An analog is also meant to include polypeptides in which one or more of the amino acid residues include a substituent group, or polypeptides which are fused with another compound, e.g., a compound to increase the half-life of the polypeptide, e.g., polyethylene glycol.

[0139] Analogs are also meant to include those produced by introduction of amino acid substitutions, or the design of constrained peptides, cyclic peptides, and other modified peptides or analogs, where the modifications or constraints are introduced on the basis of knowledge of the conformation of the peptide bound to calcineurin, or on the basis of knowledge of the structure of a protein-protein complex formed by NFAT and calcineurin or by a fragment of NFAT (including, e.g., the 13-mer peptide described herein) and a fragment of calcineurin. The conformation of the bound peptide can be determined by techniques known to those skilled in the art, e.g., NMR, e.g., transferred nuclear Overhauser effect spectroscopy (transferred NOESY) of a rapidly dissociating peptide to determine distance constraints (Campbell and Sykes, J. Magn. Reson. 93:77-92 (1991); Lian et al., Methods Enzymol. 239:657-700 (1994)), with or without additional NMR techniques, followed by the use of the distance constraints and of constrained molecular dynamics simulations and energy minimization with available computer software (e.g., the NMR_Refine module of the InsightII™ suite of programs (Biosym/MSI, San Diego, Calif.), and the Discover™ or Discover 3.0™ molecular simulation programs (Biosym/MSI, San Diego, Calif.)) to arrive at a structural model. Alternatively, the structure of the specified complexes, including the conformation assumed by the claimed peptides in the complex, can be determined by x-ray crystallography.

[0140] Other analogs within the scope of the invention include compounds in which the peptide fragment or biologically active analog thereof is covalently linked to a ligand that binds to a site adjacent to that recognized by the 13-mer peptide described herein (See Shuker et al., Science 274:1531-1534 (1996)), in order to produce a biologically active compound with increased affinity or specificity for the calcineurin-NFAT interaction.

[0141] The invention also includes an isolated polynucleotide encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, or biologically active analogs thereof. The invention also includes an isolated polynucleotide encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, or biologically active analogs thereof. Preferred polynucleotide sequences are, e.g., the sequences as set forth in SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114 and SEQ ID NO:115.

[0142] The invention also includes an isolated polynucleotide encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or biologically active analogs thereof. Preferred polynucleotide sequences are, e.g., the sequences as set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:83 and SEQ ID NO:84.

[0143] The invention also includes an isolated polynucleotide encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, or biologically active analogs thereof.

[0144] The invention also includes an isolated polynucleotide encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:71, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or biologically active analogs thereof. Preferred polynucleotide sequences are, e.g., the sequences as set forth in SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:72, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92.

[0145] The invention also includes an isolated polynucleotide encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or biologically active analogs thereof. Preferred polynucleotide sequences are, e.g., the sequences as set forth in SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:63.

[0146] The invention also includes an isolated polynucleotide encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or biologically active analogs thereof. Preferred polynucleotide sequences are, e.g., sequences as set forth in SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:70.

[0147] The invention also includes nucleotide sequences which are capable of hybridizing to and which are at least about 70%, preferably at least about 80%, more preferably yet at least about 85%, more preferably yet at least about 90%, more preferably yet at least about 95%, more preferably yet at least about 97%, and most preferably at least about 98% identical to the polynucleotides described herein, and which encode a peptide having biological activity. By percent identity is meant the maximal percent identity obtained by aligning the first base of the oligonucleotide with any base in the nucleotide sequence and then scoring the identity of aligned bases for each base in the oligonucleotide without introduction of any gaps.

[0148] The nucleotide sequences of the present invention can be in the form of, e.g., RNA, DNA or PNA, e.g., cRNA, cDNA, genomic DNA, or e.g., synthetic RNA, DNA or PNA. The nucleotide sequence can be double-stranded or single stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.

[0149] The coding sequence which encodes the peptide fragments can be identical to the coding sequences as set forth in SEQ ID NOS:36-70, 72, 83-92 or 110-115, or can be a different coding sequence, which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same peptide fragments as the nucleic acid as set forth in SEQ ID NOS:36-70, 72, 83-92 or 110-115.

[0150] The invention also includes a gene therapy vector comprising a nucleotide sequence encoding a peptide fragment of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog of the peptide fragment.

[0151] By a gene therapy vector is meant a vector useful for gene therapy. Gene therapy vectors carry a gene of interest that is useful for gene therapy. The gene therapy vectors are able to be transferred to the cells of an animal, e.g., a human, and are able to express the gene of interest in such cells so as to effect gene therapy. The vector can be, e.g., chromosomal, nonchromosomal or synthetic. It can be, e.g., RNA or DNA. The vector can be, e.g., a plasmid, a virus or a phage. Preferred vectors include, e.g., retroviral vectors, adenoviral vectors, adeno-associated vectors, herpes virus vectors and Semliki Forest virus vector. A preferred retroviral vector is Murine Stem Cell Virus (MSCV), which is a variant of Moloney Murine Leukemia Virus (MoMLV).

[0152] In preferred embodiments, the gene therapy vector comprises a nucleotide sequence encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, or biologically active analogs thereof. In certain embodiments, the gene therapy vector comprises the nucleotide sequences as set forth in SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114 or SEQ ID NO:115. The invention also includes nucleotide sequences which are capable of hybridizing to and which are at least about 70%, preferably at least about 80%, more preferably yet at least about 85%, more preferably yet at least about 90%, more preferably yet at least about 95%, more preferably yet at least about 97%, and most preferably at least about 98% identical to these nucleotide sequences, and which encode a peptide having biological activity.

[0153] In preferred embodiments, the gene therapy vector comprises a nucleotide sequence encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or biologically active analogs thereof. In certain embodiments, the gene therapy vector comprises the nucleotide sequences as set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:83 or SEQ ID NO:84. The invention also includes nucleotide sequences which are capable of hybridizing to and which are at least about 70%, preferably at least about 80%, more preferably yet at least about 85%, more preferably yet at least about 90%, more preferably yet at least about 95%, more preferably yet at least about 97%, and most preferably at least about 98% identical to these nucleotide sequences, and which encode a peptide having biological activity. In preferred embodiments, the gene therapy vector comprises a nucleotide sequence encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:71, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or biologically active analogs thereof. In certain embodiments, the gene therapy vector comprises the nucleotide sequence as set forth in SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:72, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 or SEQ ID NO:92. The invention also includes nucleotide sequences which are capable of hybridizing to and which are at least about 70%, preferably at least about 80%, more preferably yet at least about 85%, more preferably yet at least about 90%, more preferably yet at least about 95%, more preferably yet at least about 97%, and most preferably at least about 98% identical to these nucleotide sequences, and which encode a peptide having biological activity. In preferred embodiments, the gene therapy vector comprises a nucleotide sequence encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or biologically active analogs thereof. In certain embodiments, the gene therapy vector comprises the nucleotide sequence as set forth in SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 or SEQ ID NO:63. The invention also includes nucleotide sequences which are capable of hybridizing to and which are at least about 70%, preferably at least about 80%, more preferably yet at least about 85%, more preferably yet at least about 90%, more preferably yet at least about 95%, more preferably yet at least about 97%, and most preferably at least about 98% identical to these nucleotide sequences, and which encode a peptide having biological activity.

[0154] In preferred embodiments, the gene therapy vector comprises a nucleotide sequence encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:29, SEQ ID NO:30, SEQ NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID SEQ ID NO:34 and SEQ ID NO:35, or biologically active analogs thereof. In certain embodiments, the gene therapy vector comprises the nucleotide sequence as set forth in SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 or SEQ ID NO:70. The invention also includes nucleotide sequences which are capable of hybridizing to and which are at least about 70%, preferably at least about 80%, more preferably yet at least about 85%, more preferably yet at least about 90%, more preferably yet at least about 95%, more preferably yet at least about 97%, and most preferably at least about 98% identical to these nucleotide sequences, and which encode a peptide having biological activity.

[0155] In certain embodiments, the gene therapy vector also has a nucleotide sequence encoding a signal peptide for effecting secretion of the NFAT peptide fragment. Examples of signal peptides include those of (pre)prolactin (Walter and Blobel, J Cell Biol 91:557-561(1981)), apolipoprotein AI (Stoffel et al, Eur J Biochem 120:519-522 (1981)), and &bgr;-lactamase (Muller et al, J Biol Chem 257:11860-11863 (1982)). Preferably, the signal peptide is encoded 5′ of the nucleotide sequence encoding the NFAT peptide fragment.

[0156] In certain embodiments, the gene therapy vector has a nucleotide sequence encoding a tag for identification of the NFAT peptide fragment. Examples of tags that can be detected with commercially available antibodies (Shiio et al, Methods Enzymol 254:497-502 (1995)) are the FLAG peptide, the peptide YPYDVPDYA (SEQ ID NO:93) from influenza virus haemagglutinin, and other peptides from T7 gene 10 protein, Myc, and bovine papillomavirus L1 protein. The sequence encoding the tag can be 5′ or 3′ of the nucleotide sequence encoding the NFAT peptide fragment.

[0157] In certain embodiments the gene therapy vector has a selectable marker. Examples of selectable markers include a Neomycin phosphotransferase gene, a humanized red-shifted green fluorescent protein, hygromycin resistance, puromycin resistance, luciferase, or a cell-surface protein that is recognized by a specific monoclonal antibody.

[0158] In certain embodiments, the gene therapy vector has an inducible promoter, e.g., a promoter that will allow expression of the therapeutic peptide at a specific time or in a graded manner. Such a construct is valuable, e.g., for the purpose of treating graft-versus-host disease after a transplant of bone marrow cells or stem cells genetically engineered to carry the gene therapy vector with a promoter inducible by a compound that can be administered orally; or for cell therapy of multiple sclerosis with glial cells genetically engineered to express an immunosuppressive protein or peptide in response to a cytokine or other molecule produced at a site of autoimmune demyelination.

[0159] In certain embodiments, the gene therapy vector has a cell-specific promoter to allow inhibition of the calcineurin-NFAT interaction in one cell type, without disturbing normal NFAT function in other types of cells.

[0160] The invention also includes a cell having a gene therapy vector described herein. Preferably, the cell is an animal cell. The gene therapy vectors described herein can be introduced into a cell, e.g., by transformation, transfection, transduction, infection, or ex vivo injection. Preferably, they are targeted to a particular cell type or cell.

[0161] The invention also includes a method for producing a peptide capable of inhibiting protein-protein interaction between calcineurin and NFAT, comprising culturing a cell having a gene therapy vector described herein under conditions that permit expression of the peptide.

[0162] The invention also includes a method for treating an immune related disease or condition in an animal. Immune-related diseases or conditions include, e.g., acute immune diseases, chronic immune diseases and autoimmune diseases. It is also meant to include treatment of tissue or organ transplant graft rejections or graft-versus-host disease. A gene therapy vector described herein is administered to the animal.

[0163] The invention also includes a method for providing an animal having an immune-related disease or condition with a therapeutically effective level of a peptide capable of inhibiting protein-protein interaction between calcineurin and NFAT. A gene therapy vector described herein is administered to the animal.

[0164] The invention also includes a method for inhibiting an immune response in an animal. An animal in need of inhibition of an immune response is provided. A therapeutically effective amount of a peptide fragment of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof, is provided. The peptide fragment or biologically active analog thereof is administered to the animal so as to inhibit the immune response in the animal.

[0165] The peptide fragment can be any of the peptide fragments of the conserved regulatory domain of NFAT protein of this invention described herein. Certain preferred peptide fragments comprise the amino acid sequence X1IX2X3T (SEQ ID NO:73) or a biologically active analog thereof, wherein X1 is R or S, X2 is E, R or Q, and X3 is I or F. In certain embodiments, the peptide fragment comprises the amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or biologically active analogs thereof.

[0166] Other preferred peptide fragments comprise the amino acid sequence IX2X3T (SEQ ID NO:104) or a biologically active analog thereof, wherein X2 is E, R or Q, and X3 is I or F. In certain embodiments, the peptide fragment comprises the amino acid sequence as set forth in SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, or biologically active analogs thereof.

[0167] In certain embodiments, the therapeutically effective amount of the peptide fragment is provided by providing to the animal a recombinant nucleic acid having a nucleotide sequence encoding the peptide fragment or a biologically active analog thereof, and which is capable of expressing the peptide fragment or biologically active analog thereof in vivo. The peptide fragment is administered to the animal by administering the recombinant nucleic acid. The nucleic acid can be, e.g., any of the polynucleotides described herein. Certain preferred nucleic acids are polynucleotides encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or biologically active analogs thereof, e.g., the sequences as set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39. Other preferred nucleic acids are polynucleotides encoding the peptide comprising the amino acid sequence as set forth in SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, or biologically active analogs thereof, e.g., the sequences as set forth in SEQ ID NO:110, SEQ ID NO:11, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114 and SEQ ID NO:115. In certain embodiments, the recombinant nucleic acid is a gene therapy vector, e.g., as described herein.

[0168] In certain embodiments, the therapeutically effective amount of the peptide fragment is provided by providing to the animal a composition comprising animal cells wherein a recombinant nucleic acid having a nucleotide sequence encoding the peptide fragment has been introduced ex vivo into the animal cells so as to express the peptide fragment in the animal cells. The peptide fragment is administered to the animal by administering the animal cells having the recombinant nucleic acid. Preferably, the recombinant nucleic acid is a gene therapy vector, e.g., as described herein. Preferably, the animal cells are derived from the animal to be treated or allogeneic cells.

[0169] Administration of an agent, e.g., a peptide or nucleic acid can be accomplished by any method which allows the agent to reach the target cells. These methods include, e.g., injection, deposition, implantation, suppositories, oral ingestion, inhalation, topical administration, or any other method of administration where access to the target cells by the agent is obtained. Injections can be, e.g., intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal. Implantation includes inserting implantable drug delivery systems, e.g., microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, polymeric systems, e.g., matrix erosion and/or diffusion systems and non-polymeric systems, e.g., compressed, fused or partially fused pellets. Suppositories include glycerin suppositories. Oral ingestion doses can be enterically coated. Inhalation includes administering the agent with an aerosol in an inhalator, either alone or attached to a carrier that can be absorbed. The agent can be suspended in liquid, e.g., in dissolved or colloidal form. The liquid can be a solvent, partial solvent or non-solvent. In many cases, water or an organic liquid can be used.

[0170] In certain embodiments of the invention, the administration can be designed so as to result in sequential exposures to the agent over some time period, e.g., hours, days, weeks, months or years. This can be accomplished by repeated administrations of the agent, e.g., by one of the methods described above, or alternatively, by a controlled release delivery system in which the agent is delivered to the animal over a prolonged period without repeated administrations. By a controlled release delivery system is meant that total release of the agent does not occur immediately upon administration, but rather is delayed for some time. Release can occur in bursts or it can occur gradually and continuously. Administration of such a system can be, e.g., by long acting oral dosage forms, bolus injections, transdermal patches or subcutaneous implants. Examples of systems in which release occurs in bursts include, e.g., systems in which the agent is entrapped in liposomes which are encapsulated in a polymer matrix, the liposomes being sensitive to a specific stimulus, e.g., temperature, pH, light, magnetic field, or a degrading enzyme, and systems in which the agent is encapsulated by an ionically-coated microcapsule with a microcapsule core-degrading enzyme. Examples of systems in which release of the agent is gradual and continuous include, e.g., erosional systems in which the agent is contained in a form within a matrix, and diffusional systems in which the agent permeates at a controlled rate, e.g., through a polymer. Such sustained release systems can be, e.g., in the form of pellets or capsules.

[0171] The agent can be administered prior to or subsequent to the appearance of disease symptoms. In certain embodiments, the agent is administered to patients with familial histories of the disease, or who have phenotypes that may indicate a predisposition to the disease, or who have been diagnosed as having a genotype which predisposes the patient to the disease, or who have other risk factors.

[0172] The agent is administered to the animal in a therapeutically effective amount. By therapeutically effective amount is meant that amount which is capable of at least partially preventing or reversing the disease. A therapeutically effective amount can be determined on an individual basis and will be based, at least in part, on consideration of the species of animal, the animal's size, the animal's age, the efficacy of the particular agent used, the longevity of the particular agent used, the type of delivery system used, the time of administration relative to the onset of disease symptoms, and whether a single, multiple, or controlled release dose regimen is employed. A therapeutically effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.

[0173] In certain preferred embodiments, the concentration of the agent if it is a peptide is at a dose of about 0.1 to about 1000 mg/kg body weight/day, more preferably at about 0.1 to about 500 mg/kg/day, more preferably yet at about 0.1 to about 100 mg/kg/day, and most preferably at about 0.1 to about 5 mg/kg/day. Preferably, the dosage form is such that it does not substantially deleteriously affect the animal.

[0174] In certain embodiments, a therapeutically effective amount of an agent which is a peptide can be administered by providing to the animal a nucleic acid encoding the peptide and expressing the peptide in vivo. Preferably, the dosage form is such that it does not substantially deleteriously affect the animal. Nucleic acids encoding the peptide can be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the nucleotide sequence for the peptide to cells in vivo. Approaches include, e.g., insertion of the nucleic acid into viral vectors, including, e.g., retrovirus, adenovirus, adeno-associated virus, herpes virus and Semliki Forest virus vectors. Viral vectors can be delivered to the cells, e.g., by infection or transduction using the virus. Viral vectors can also be delivered to the cells, e.g., by physical means, e.g., by electroporation, lipids, cationic lipids, liposomes, DNA gun, Ca3(PO4)2 precipitation, or delivery of naked DNA. In certain preferred embodiments, the virus is administered by injection, e.g., intramuscular injection, in a dose range of about 103 to about 1010 infectious particles per injection per treatment, more preferably in a dose range of about 105 to about 108 infectious particles per injection per treatment. Single or multiple doses can be administered over a given period of time, depending, e.g., upon the disease. An alternative is insertion of the nucleic acid encoding the peptide into a bacterial or eukaryotic plasmid. Plasmid DNA can be delivered to cells with the help of, e.g., cationic liposomes (lipofectin™; Life Technologies, Inc., Gaithersburg, Md.) or derivatized (e.g., antibody conjugated) polylysine conjugates, gramicidin S, streptolysin O artificial viral envelopes or other such carriers or delivery aids, as well as direct injection of the gene construct or Ca3(PO4)2 precipitation carried out in vivo, or by use of a gene gun. The above-described methods are known to those skilled in the art and can be performed without undue experimentation.

[0175] In certain embodiments, the nucleic acid is administered to the animal by introducing ex vivo the nucleic acid into cells of the animal or allogeneic cells, and administering the cells having the nucleic acid to the animal. Any cell type can be used. In certain embodiments, the cells having the introduced nucleic acid are expanded and/or selected after the nucleic acid transfer. The cells having the transferred nucleic acid are subsequently administered to the animal. Preferably, the cells are administered to the animal in a dose range of about 1×106 to about 1×109 cells/dosage/treatment, and most preferably at about 1×107 to about 1×108 cells/dosage/treatment. The cells can be administered by any method which results in delivering the transferred nucleic acid in the cells to the desired target. For example, the cells can be implanted directly into a specific tissue of the animal, or implanted after encapsulation within an artificial polymer matrix. Examples of sites of implantation include the lungs or airways, skin, conjunctiva, central nervous system, peripheral nerve, a grafted kidney, or an inflamed joint.

[0176] Choice of the particular delivery system will depend on such factors as the intended target and the route of administration, e.g., locally or systemically. Targets for delivery of the agent can be, e.g., specific target cells which are causing or contributing to disease. For example, the target can be resident or infiltrating cells in the lungs or airways that are contributing to an asthmatic illness; resident or infiltrating cells in the nervous system that are contributing to demyelinating disease; resident or infiltrating cells responsible for rejection of a kidney graft; grafted cells whose activation produces graft-versus-host disease; or resident or infiltrating cells whose activation underlies inflammation or arthritic degeneration of a joint. Administration can be directed to one or more cell types, and to one or more subsets of cells within a cell type, so as to be therapeutically effective, by methods known to those skilled in the art. For example, the agent can be coupled to an antibody, to a ligand to a cell surface receptor, or to a toxin component, or can be contained in a particle which is selectively internalized into cells, e.g., liposomes, or a virus where the viral receptor binds specifically to a certain cell type, or a viral particle lacking the viral nucleic acid, or can be administered by local injection. In certain embodiments, administration is done in a prenatal animal or embryonic cell.

[0177] In other embodiments, the agent is a non-peptide molecule, e.g., an organic or inorganic compound, e.g., as isolated from a library of organic or inorganic compounds as described herein. Exemplary doses of such agents include milligram or microgram amounts of the compound per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). It is furthermore understood that appropriate doses of such compounds depend upon the potency of the compound with respect to the activity to be modulated. When one or more of these compounds is to be administered to an animal (e.g., a human) in order to inhibit the protein-protein interaction between calcineurin and NFAT, e.g., to modulate an immune response or to treat a disease or condition described herein, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[0178] In certain embodiments, other therapy is additionally administered. For example, another therapeutic agent, chemotherapy, radiation or surgery, is additionally administered to the animal, either simultaneously or at different times.

[0179] The invention also includes a method for treating a disease involving hyperactivity or inappropriate activity of the immune system, a transplant graft rejection, or graft-versus-host disease in an animal. An animal in need of treatment for a disease involving hyperactivity or inappropriate activity of the immune system, a transplant graft rejection, or graft-versus-host disease, is provided. A therapeutically effective amount of an agent, e.g., a peptide fragment of the conserved regulatory domain of NFAT protein or a biologically active analog thereof, or a non-peptide agent, e.g., an organic or inorganic compound, capable of inhibiting protein-protein interaction between calcineurin and NFAT, is provided. The agent, e.g., peptide fragment or biologically active analog thereof, or non-peptide agent is administered to the animal in a therapeutically effective amount such that treatment of the disease involving hyperactivity or inappropriate activity of the immune system, transplant graft rejection, or graft-versus-host disease, occurs.

[0180] Immune system diseases involving hyperactivity or inappropriate activity of the immune system include, e.g., acute immune diseases, chronic immune diseases and autoimmune diseases. Examples of such diseases include rheumatoid arthritis, inflammatory bowel disease, allogeneic or xenogeneic transplantation rejection (organ, bone marrow, stem cells, other cells and tissues), graft-versus-host disease, aplastic anemia, psoriasis, lupus erytematosus, inflammatory disease, type I diabetes, asthma, pulmonary fibrosis, scleroderma, dermatomyositis, Sjogren's syndrome, postpericardiotomy syndrome, Kawasaki disease, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pemphigus vulgaris, autoimmune hemolytic anemia, idiopathic thrombopenia, chronic glomerulonephritis, Goodpasture's syndrome, Wegner's granulomatosis, multiple sclerosis, cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis, uveitis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, Crohn's disease, ulcerative colitis, Guilllain-Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, eczema and autoimmune thyroiditis. Transplant graft rejections can result from tissue or organ transplants. Graft-versus-host disease can result from bone marrow or stem cell transplantation.

[0181] The methods of the present invention can also be utilized to treat conditions and diseases that are not immune mediated, but which nevertheless involve the protein-protein interaction between calcineurin and NFAT. Examples include myocardial hypertrophy, dilated cardiomyopathy, excessive or pathological bone resorption, excessive adipocyte differentiation, obesity, and reactivation of latent human herpesvirus-8 or other viruses.

[0182] Treating is meant to include, e.g., preventing, treating, reducing the symptoms of, or curing the disease or condition. In certain embodiments, the therapeutically effective amount of the peptide fragment or biologically active analog thereof is administered by providing to the animal a nucleic acid encoding the peptide fragment or biologically active analog thereof, and expressing the peptide fragment or biologically active analog thereof in vivo.

[0183] The invention also includes a method for treating an animal at risk for a disease involving hyperactivity or inappropriate activity of the immune system, a transplant graft rejection, or graft-versus-host disease. An animal at risk for a disease involving hyperactivity or inappropriate activity of the immune system, a transplant graft rejection, or graft-versus-host disease, is provided. A therapeutically effective amount of a peptide fragment of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof, is provided. The peptide fragment or biologically active analog thereof is administered in a therapeutically effective amount such that treatment occurs.

[0184] Being at risk for the disease can result from, e.g., a family history of the disease, a genotype which predisposes to the disease, or phenotypic symptoms which predispose to the disease.

[0185] The invention also includes a method for gene therapy. An animal cell is genetically modified such that it is able to express a peptide fragment or biologically active analog thereof of the conserved regulatory domain of NFAT protein, the peptide fragment being capable of inhibiting calcineurin-mediated NFAT activation, so as to effect gene therapy. In certain embodiments, the animal cells are genetically modified by introducing into the cells a recombinant nucleic acid having a nucleotide sequence encoding the peptide fragment and which is capable of expressing the peptide fragment in vivo. Preferably, the recombinant nucleic acid is a gene therapy vector, e.g., as described herein.

[0186] The invention also includes a pharmaceutical composition for treating an immune-related disease or condition in an animal comprising a therapeutically effective amount of a peptide fragment of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof, and a pharmaceutically acceptable carrier. The peptide fragment can be, e.g., any of the peptide fragments described herein. Pharmaceutically acceptable carriers include, e.g., water, saline, dextrose, glycerol, ethanol, liposomes and lipid emulsions.

[0187] The invention also includes a pharmaceutical composition for treating an immune-related disease or condition in an animal, comprising a therapeutically effective amount of a recombinant nucleic acid having a nucleotide sequence encoding a peptide fragment of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof, and a pharmaceutically acceptable carrier. The nucleic acid can be, e.g., any of the polynucleotides described herein.

[0188] The invention also includes a pharmaceutical composition for treating an immune-related disease or condition in an animal, comprising a therapeutically effective amount of animal cells wherein a recombinant nucleic acid having a nucleotide sequence encoding a peptide fragment of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof, has been introduced into the animal cells so as to express the peptide fragment; and a pharmaceutically acceptable carrier. Preferably, the animal cells are cells derived from the animal to be treated or allogeneic cells.

[0189] The invention also includes a method for inhibiting protein-protein interaction between calcineurin and NFAT in vivo. A cell having calcineurin and NFAT is provided. A peptide fragment or a biologically active analog thereof of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT is provided. The calcineurin and peptide fragment or biologically active analog thereof are contacted in vivo such that protein-protein interaction between the calcineurin and the NFAT is inhibited.

[0190] The invention also includes a method for inhibiting protein-protein interaction between calcineurin and NFAT in vitro. Calcineurin and NFAT are provided. A peptide fragment or a biologically active analog thereof of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT is provided. The calcineurin and peptide fragment or biologically active analog thereof are contacted b vitro such that protein-protein interaction between the calcineurin and the NFAT is inhibited.

[0191] The invention also includes a method for evaluating an agent for use in modulating an immune response. A cell is provided. An agent, e.g., a peptide fragment of the conserved regulatory domain of NFAT protein or biologically active analogs thereof, is provided. The effect of the agent on an aspect of calcineurinmediated NFAT activation is evaluated. A change in the aspect of calcineurin-mediated NFAT activation is indicative of the usefulness of the agent in modulating an immune response.

[0192] Any aspect of calcineurin-mediated NFAT activation can be evaluated, e.g., protein-protein interaction between calcineurin and NFAT, dephosphorylation of NFAT by calcineurin, recruitment of NFAT to the nucleus in a cell, conformational change in NFAT, or activation of NFAT-dependent gene transcription.

[0193] The invention also includes a method for high throughput screening of candidate agents to identify an agent that inhibits protein-protein interaction between calcineurin and NFAT. A first compound is provided. The first compound is calcineurin or a biologically active derivative thereof, or the first compound is NFAT or a biologically active derivative thereof. A second compound is provided which is different from the first compound and which is labeled. The second compound is calcineurin or a biologically active derivative thereof, or the second compound is NFAT or a biologically active derivative thereof. A candidate agent is provided. The first compound, second compound and candidate agent are contacted with each other. The amount of label bound to the first compound is determined. A reduction in protein-protein interaction between the first compound and the second compound as assessed by label bound is indicative of the usefulness of the agent in inhibiting protein-protein interaction between calcineurin and NFAT. Preferably, the reduction is assessed relative to the same reaction without addition of the candidate agent.

[0194] In certain embodiments, the first compound which is provided is attached to a solid support. Solid supports include, e.g., resins, e.g., agarose and a multiwell plate. In certain embodiments, the method includes a washing step after the contacting step, so as to separate bound and unbound label.

[0195] By high-throughput screening is meant that the method can be used to screen a large number of candidate agents easily and quickly. In preferred embodiments, a plurality of candidate agents are contacted with the first compound and second compound. The different candidate agents can be contacted with the other compounds in groups or separately. Preferably, each of the candidate agents is contacted with both the first compound and the second compound in separate wells. For example, the method can screen libraries of potential agents. Libraries are meant to include, e.g., natural product libraries, organic chemical libraries, combinatorial chemical libraries, peptide libraries, and modified peptide libraries, including, e.g., D-amino acids, unconventional amino acids, or N-substituted amino acids. Preferably, the libraries are in a form compatible with screening in multiwell plates, e.g., 96-well plates. The assay is particularly useful for automated execution in a multiwell format in which many of the steps are controlled by computer and carried out by robotic equipment. The libraries can also be used in other formats, e.g., synthetic chemical libraries affixed to a solid support and available for release into microdroplets.

[0196] Calcineurin and biologically active derivatives thereof is meant to include, e.g., intact calcineurin; calcineurin A chain; fragments of calcineurin that are biologically active in binding NFAT, e.g., a catalytic domain fragment of the calcineurin A chain that binds to NFAT; analogs of calcineurin or a calcineurin fragment that are biologically active in binding NFAT; and chimeric recombinant proteins, e.g., calcineurin or a biologically active fragment of calcineurin fused to another peptide or protein such that calcineurin retains its NFAT-binding activity. The calcineurin and its biologically active derivatives can be natural, recombinant or synthesized. In certain preferred embodiments, the calcineurin can be from, e.g., a mammal, e.g., a human, or yeast. Calcineurin can be obtained, e.g., in cell extracts of cells that normally express calcineurin, or by expressing recombinant calcineurin protein in eukaryotic or prokaryotic cells. In certain embodiments, calmodulin is included in the assay so as to confer calcium responsiveness on calcineurin.

[0197] NFAT and biologically active derivatives thereof is meant to include intact NFAT, e.g., NFAT1, NFAT2, NFAT3 or NFAT4; fragments of NFAT that are biologically active, e.g., that retain the ability to form a protein-protein interaction with calcineurin or the ability of inhibiting the binding of NFAT to calcineurin, e.g., a peptide fragment of the conserved regulatory domain of NFAT, as described herein; analogs of NFAT or an NFAT fragment that are biologically active; and chimeric recombinant proteins, e.g., NFAT or a biologically active fragment of NFAT fused to another peptide or protein such that NFAT retains its activity. Examples of such chimeric recombinant proteins include: (i) NFAT fused to maltose-binding protein or glutathione S-transferase (GST) so as to immobilize the NFAT on a solid support, e.g., a resin; (ii) NFAT fused to green fluorescent protein or one of its variants for use in a fluorescence assay or a fluorescence energy transfer assay; and (iii) NFAT fused to a peptide tag so as to allow its recognition by a specific antibody or its labeling by a specific protein kinase. The NFAT and its biologically active derivatives can be natural, recombinant or synthesized. NFAT can be, e.g., a mammalian protein, e.g., human or murine. NFAT can be obtained, e.g., in cell extracts of cells that normally express NFAT, or by expressing a recombinant NFAT protein in eukaryotic or prokaryotic cells.

[0198] In certain embodiments, the NFAT derivative is a mutated NFAT that has increased affinity for calcineurin. Such mutants are obtained, e.g., by applying a two-hybrid screen to mutagenized NFAT (see, e.g., Mendelsohn and Brent, Curr. Opin. Biotech. 5:482-486 (1994); Goldfarb et al, J Biol Chem 271:2683-2688 (1996); Colas et al, Nature 380:548-550 (1996)) or using any other selection or screening method known to those skilled in the art, or produced by introducing into NFAT amino acid substitutions identified through screening peptide libraries or phage display libraries (see, e.g., Kast and Hilvert, Curr. Opin. Struct. Biol. 7:470-479 (1997)). Advantages of using mutant NFAT proteins that bind calcineurin with higher affinity are reducing the amount of radiolabeled calcineurin required for an assay, permitting more stringent washing, and expanding the range of assays that produce a detectable signal. A compelling example is Example 17, wherein the high sensitivity and high signal-to-noise ratio of the screening assay is directly attributable to utilization of the optimized NFAT derivative, VIVIT peptide.

[0199] In certain embodiments, the first compound is calcineurin or a biologically active derivative thereof, and the second compound is NFAT or a biologically active derivative thereof. In other embodiments, the first compound is NFAT or a biologically active derivative thereof, and the second compound is calcineurin or a biologically active derivative thereof. The solid support to which the first compound is attached includes, e.g., Sepharose beads, SPA beads and a multiwell plate. Preferably, SPA beads (microspheres that incorporate a scintillant) are used when the assay is performed without a washing step, e.g., in a scintillation proximity assay. Preferably, Sepharose beads are used when the assay is performed with a washing step. The second compound can be labeled with any label that will allow its detection, e.g., a radiolabel, a fluorescent agent, biotin, a peptide tag, or an enzyme fragment. Preferably, the second compound is radiolabeled, e.g., with 125I or 3H.

[0200] In certain embodiments, the enzymatic activity of an enzyme chemically conjugated to, or expressed as a fusion protein with, the first or second compound, is used to detect bound protein. A binding assay in which a standard immunological method is used to detect bound protein is also included. Methods based on surface plasmon resonance, as, e.g., in the BIAcore biosensor (Pharmacia Biosensor, Uppsala, Sweden) or evanescent wave excitation of fluorescence are particularly suited to measure recruitment of, e.g., NFAT (or fluorescently labeled NFAT) to a surface on which calcineurin is immobilized. In certain other embodiments, the interaction of NFAT and calcineurin is detected by fluorescence resonance energy transfer (FRET) between a donor fluorophore covalently linked to NFAT (e.g., a fluorescent group chemically conjugated to NFAT, or a variant of green fluorescent protein (GFP) expressed as an NFAT-GFP chimeric protein) and an acceptor fluorophore covalently linked to calcineurin, where there is suitable overlap of the donor emission spectrum and the acceptor excitation spectrum to give efficient nonradiative energy transfer when the fluorophores are brought into close proximity through the protein-protein interaction of NFAT and calcineurin.

[0201] In certain embodiments, the protein-protein interaction is detected by reconstituting domains of an enzyme, e.g., pgalactosidase (see Rossi et al, Proc. Natl. Acad. Sci. USA 94:8405-8410 (1997)). The detection method used is appropriate for the particular enzymatic reaction, e.g., by chemiluminescence with Galacton Plus substrate from the Galacto-Light Plus assay kit (Tropix, Bedford, Mass.) or by fluorescence with fluorescein di-&bgr;-D-galactopyranoside (Molecular Probes, Eugene, Oreg.) for &bgr;-galactosidase. Competition of the protein-protein interaction by the candidate agents or by the 13-mer or 26-mer inhibitory peptides described herein, is evident in a reduction of the measured enzyme activity. This assay can be performed with proteins in vitro or in vivo. An advantage of this embodiment in vivo is that only compounds sufficiently permeable through the membrane of living cells will be scored positive, and thus agents most likely to reach effective concentrations within cells when administered therapeutically can be identified. Measurement of reconstituted S-galactosidase activity in living cells has been demonstrated with fluorescein di-&bgr;-D-galactopyranoside (Molecular Probes, Eugene, Oreg.) as substrate. See Rossi et al., Proc. Natl. Acad. Sci. USA 94:8405-8410 (1997).

[0202] In certain embodiments, the protein-protein interaction is assessed by fluorescence ratio imaging (Bacskai et al, Science 260:222-226 (1993)) of suitable chimeric constructs of NFAT and calcineurin in cells, or by variants of the two-hybrid assay (Fearon et al, Proc Natl Acad Sci USA 89:7958-7962 (1992); Takacs et al, Proc Natl Acad Sci USA 90:10375-10379 (1993); Vidal et al, Proc Natl Acad Sci USA 93:10315-10320 (1996); Vidal et al, Proc Natl Acad Sci USA 93:10321-10326 (1996)) employing suitable constructs of NFAT and calcineurin and tailored for a highthroughput assay to detect compounds that inhibit the NFAT calcineurin interaction. These embodiments have the advantage that the cell permeability of compounds that act as specific inhibitors in the assay is assured.

[0203] Any false positives identified in these assays, such as protein denaturants or natural product samples contaminated with a protease activity, can be detected and eliminated through secondary assays that demonstrate that their inhibitory action is nonspecific, e.g., that such compounds interfere with known protein-protein interactions between pairs of proteins unrelated to NFAT and calcineurin.

[0204] The invention also includes a method for high-throughput screening of candidate agents to identify an agent that inhibits dephosphorylation of NFAT by calcineurin. Phosphorylated NFAT is provided. Calcineurin or a biologically active derivative thereof having enzymatic activity is provided. A candidate agent is provided. The phosphorylated NFAT, the calcineurin or biologically active derivative thereof, and the candidate agent are contacted with each other in reaction media, e.g., buffer, under conditions that allow enzymatic activity of calcineurin. In certain embodiments, the NFAT is separated from the reaction media. It is determined whether phosphate remains associated with the NFAT. If phosphate remains associated is indicative of the usefulness of the agent in dephosphorylation of NFAT by calcineurin.

[0205] In certain embodiments, the phosphorylated NFAT is labeled. The phosphate can be labeled with any label that will allow its detection. Preferably, the phosphate is radiolabeled, e.g., with 32P or 33P. In certain embodiments, determination of whether phosphate remains associated with the NFAT is accomplished by determining the release of labeled phosphate in the reaction media, or the retention of labeled phosphate on the NFAT. A reduction in release of labeled phosphate from the NFAT by the calcineurin, or an increase in retention of labeled phosphate on the NFAT, is indicative of the usefulness of the agent in inhibiting dephosphorylation of NFAT by calcineurin. Preferably, the reduction is assessed relative to the same reaction without addition of the candidate agent.

[0206] In certain embodiments, the phosphorylated NFAT that is provided is attached to a solid support.

[0207] In preferred embodiments, a plurality of candidate agents are contacted with the phosphorylated NFAT which optionally is attached to a solid support, and the calcineurin or biologically active derivative thereof. The different candidate agents can be contacted with the NFAT and calcineurin in groups or separately. Preferably, each of the candidate agents is contacted with the NFAT and the calcineurin in separate wells.

[0208] NFAT in its phosphorylated form can be obtained by any method known to those skilled in the art. Methods that involve enzymatic labeling using a protein kinase in vitro are preferred where 32P is incorporated, since high specific activities can be achieved. ERK2 phosphorylates GST-NFAT1 fusion protein on sites accessible to calcineurin. A combination of protein kinase A (protein kinase, catalytic subunit; Sigma Chemical Co., St. Louis, Mo.) and glycogen synthase kinase 3&bgr; (New England Biolabs, Beverly, Mass.) phosphorylates GST-NFAT2 on a set of sites that correspond to those phosphorylated b vivo (Beals et al, Science 275:1930-1933 (1997)). For assays that measure 32P-phosphate remaining covalently associated with the protein after the incubation with calcineurin, the background signal due to phosphate incorporated at calcineurin-insensitive sites may be lowered by preblocking all substrate sites for the kinase in a reaction with unlabeled-ATP, treating with calcineurin, washing, and then incorporating 32P in a second kinase reaction that labels predominantly those sites that are accessible to calcineurin.

[0209] For assays that use nonradioactive phosphorylated NFAT or 32P labeled NFAT in native form, mammalian cells or insect cells expressing high levels of recombinant protein after transformation with a baculovirus vector can be used to obtain sufficient NFAT in phosphorylated form. A method for preparation of fully phosphorylated native NFAT1 from mammalian cells is described in Shaw et al., Proc. Natl. Acad. Sci. USA 92:11205-11209 (1995). Fully phosphorylated NFAT1 can also be obtained by lysis of the cells in a detergent-containing buffer, provided that sufficient concentrations of phosphatase inhibitors, e.g., 60 mM sodium pyrophosphate, 10 mM EDTA, and 5 mM EGTA, are included in the lysis buffer. Since the inhibitors are subsequently washed away after NFAT is purified away from endogenous phosphatases, their inclusion at the lysis step does not compromise a subsequent enzymatic assay using calcineurin. Minor modifications of these procedures that may be necessary for isolation of phosphorylated NFAT from insect cells include, e.g., use of additional protease inhibitors, additional phosphatase inhibitors, or higher concentrations of the inhibitors. The NFAT expression construct introduced into these cells in a baculovirus vector preferably encodes a chimeric protein including an epitope tag or hexahistidine tag, or a fusion protein with glutathione-S-transferase, or some similar fusion protein providing for facile purification of the expressed protein. In some cases, phosphorylated NFAT or 32P-labeled NFAT can be obtained by coexpression of NFAT and a constitutively active kinase in bacteria, e.g., in E. coli.

[0210] In certain embodiments, determining dephosphorylation of NFAT can be accomplished by examining specific sites remaining phosphorylated in the NFAT protein after treatment with calcineurin. A compound is scored as positive if it increases the retention of covalently bound phosphate on a specific site or sites of NFAT. Preferably, the presence or absence of covalently bound phosphate is determined using antibodies, or a functionally equivalent reagent, e.g., genetically engineered antibodies, minibodies or aptamers, that discriminate between phosphorylated and unphosphorylated forms of a specific peptide in the context of the larger protein or protein fragment. NFAT peptides that can be used include, e.g., FQNIPAHYSPRT (SEQ ID NO:94), PAHYSPRTSPIM (SEQ ID NO:95), or SPRTSPIMSPRT (SEQ ID NO:96)(from the sequence FQNIPAHYSPRTSPIMSPRT (SEQ ID NO:97), residues 207 to 226 in murine NFATI) or PVPRPASRSSSP (SEQ ID NO:98), RPASRSSSPG (SEQ ID NO:99), or ASRSSSPGAKRR (SEQ ID NO:100)(from the sequence PVPRPASRSSSPGAKRR (SEQ ID NO:101), residues 239 to 255 in murine NFAT1). Antibodies to phosphorylated or dephosphorylated NFAT peptides can be raised, e.g., by immunization of rabbits. See e.g., Czernik et al, Methods Enzymol 201:264-283 (1991) for preparation and characterization of serum or monoclonal antibodies using short synthetic peptides (10-12 residues) corresponding to the sequence surrounding a phosphorylation site. The unphosphorylated peptides can be obtained by conventional methods of chemical synthesis, e.g., Merrifield solid phase synthesis. The phosphopeptides can be obtained, e.g., by in vitro phosphorylation of the synthetic peptides with kinase in instances where the synthetic peptide includes flanking residues that form a consensus site for the, kinase (Czernik et al, Methods Enzymol 201:264-283 (1991)), or, e.g., by chemical synthesis of peptides phosphorylated on serine or threonine residues (Perich J W, Methods Enzymol 201:225-233 (1991)). The antisera or monoclonal antibodies can be tested to determine whether they show the ability to discriminate between phosphorylated and unphosphorylated peptides, e.g., by dot immunoblotting or by ELISA (Czemik et al, Methods Enzymol 201:264-283 (1991)). To ensure that a specific antiserum or monoclonal antibody reagent discriminates between phosphopeptide and dephosphopeptide in the context of NFAT protein, and to select a high-affinity reagent with low background signal in the high-throughput screening assay, the candidate antiserum or monoclonal antibody can be further tested under the conditions to be used in the high-throughput screening assay.

[0211] Any antibody based assay can be used. Preferably, an automated assay that reflects the relative amount of phosphorylated or unphosphorylated peptide is used. For example, a very efficient method of monitoring dephosphorylation is to use fluorescence resonance energy transfer between two appropriately labeled antibodies to two distinct phosphopeptides, capable of simultaneous binding to the protein, which are added directly to the reaction after stopping the phosphatase incubation with, e.g., EGTA, another inhibitor of calcineurin activity, or by mild protein denaturation. Variants of this embodiment include, e.g., antibodies directed against the dephosphorylated forms of two distinct NFAT peptides, corresponding miniaturized antibodies (“minibodies”; Tramontano et al, J. Mol. Recognit. 7:9-24 (1994); Martin et al, EMBO J. 13:5303-5309 (1994); Martin et al, J. Mol Biol 255:86-97 (1996)), or peptide aptamers (Colas et al, Nature 380:548-550 (1996)) selected to recognize phosphorylated or dephosphorylated forms of NFAT peptides. In some variants of this embodiment, a single fluorescently labeled antibody, minibody, or peptide aptamer that binds to a phosphorylated or dephosphorylated form of an NFAT peptide is paired with fluorescently tagged NFAT in a fluorescence resonance energy transfer assay; or a fluorescently labeled antibody, minibody, or peptide aptamer directed to a phosphopeptide or dephosphopeptide is paired in a fluorescence resonance energy transfer assay with a second labeled antibody, minibody, or peptide aptamer that binds constitutively to NFAT or a peptide tag at a site unaffected by phosphorylation or dephosphorylation of the protein. In embodiments in which the antibodies, minibodies, or peptide aptamers are continuously present during the incubation with calcineurin, the reagents preferably are directed against the dephosphopeptide so that they will not interfere with access of calcineurin to the phosphopeptide.

[0212] In certain embodiments, the screening assay uses measurements of release of 32P from a reporter site introduced into recombinant NFAT, or measurements with antibodies to the phosphorylated or dephosphorylated forms of a reporter site introduced into recombinant NFAT. The inserted reporter site takes the form of a short peptide sequence, known to be an efficient substrate for a specific protein kinase, that is genetically engineered into NFAT. The inserted site that is used is able to be phosphorylated efficiently in vitro in its context within the NFAT protein, the phosphorylated site is dephosphorylated by calcineurin, and the efficiency of dephosphorylation is reduced by a 13-mer or 25-mer inhibitory peptide described herein, showing that the specific protein-protein recognition of NFAT by calcineurin is essential for dephosphorylation.

[0213] In certain embodiments, the interaction of NFAT with the enzyme active site of calcineurin, as distinct from the recognition site where the protein-protein interaction is disrupted by a 13-mer or 25-mer inhibitory peptide described herein, is assessed by examining the activity of calcineurin against a second substrate in the presence of NFAT. Because binding of NFAT to the recognition site brings substrate peptides within NFAT into proximity of the active site, and indeed into the active site as evidenced by their consequent dephosphorylation, NFAT exhibits competition with other substrates that are dephosphorylated by calcineurin. In the absence of binding to the recognition site, NFAT may still compete with other substrates, but only at significantly higher concentrations. Since the agents sought in this assay, like the 13-mer peptide, do not inhibit calcineurin activity against substrates other than NFAT, their presence in the assay will reduce competition by NFAT and cause an apparent stimulation of calcineurin activity against the assayed substrate.

[0214] Such an assay uses a standard calcineurin phosphatase assay. The concentration of NFAT required for competition depends on many factors, e.g., the substrate, assay time, temperature and assay buffer, which are determined for particular reaction conditions by simple testing of a range of NFAT concentrations in pilot experiments. The control reaction that shows the dependence of the competition on NFAT-calcineurin recognition is carried out with inclusion of a 13-mer or 25-mer inhibitory peptide described herein. In one embodiment, the measurement of calcineurin phosphatase activity is made by determining the release of 32P from a phosphopeptide substrate, e.g., 32P-RII peptide. In another embodiment, the enzymatic activity of calcineurin is determined by use of a biotinylated phosphopeptide substrate that can be captured, subsequent to the incubation with calcineurin, on a streptavidin-coated solid support and probed with an antibody specific for the dephosphopeptide. In certain embodiments, detection is by formation of a fluorescent product with, e.g., 4-nitrophenylphosphate (Molecular Probes, Eugene, Oreg.) or fluorescein diphosphate (Molecular Probes, Eugene, Oreg.) as substrate. Those skilled in the art are aware of many alternative ways to assess the enzymatic activity of calcineurin. An advantage of this embodiment is that such assays do not require phosphorylated NFAT.

[0215] In any of the dephosphorylation assays described herein, agents that inhibit NFAT dephosphorylation by preventing the specific interaction between calcineurin and NFAT are identified, as well as agents that act by a different mechanism, e.g., as general inhibitors of calcineurin. The latter general inhibitors can be eliminated, e.g., by a second screening assay which tests the agent's ability to inhibit dephosphorylation of other known substrates of calcineurin. The assay based on competition with a second substrate may identify general activators of calcineurin, which can likewise be eliminated, e.g., in a second screening assay that tests the agent's ability to augment dephosphorylation of the second substrate when the incubation is performed in the absence of NFAT. Further, to confirm that the mechanism of action is interference with the protein-protein interaction of NFAT and calcineurin, all compounds identified in the dephosphorylation assay can, e.g., be tested directly for their interference in the protein-protein interaction of NFAT and calcineurin using the assays described herein.

[0216] The invention also includes a method for high-throughput screening of candidate agents to identify an agent that inhibits conformational change in NFAT from dephosphorylation by calcineurin. Phosphorylated NFAT is provided. In certain embodiments, the phosphorylated NFAT is attached to a solid support. Calcineurin or a biologically active derivative thereof having enzymatic activity is provided. A candidate agent is provided. An oligonucleotide having an NFAT site is provided. The phosphorylated NFAT, calcineurin or biologically active derivative thereof, and the candidate agent are contacted with each other in reaction media under conditions that allow enzymatic activity of calcineurin. Specific binding of NFAT to the oligonucleotide having the NFAT site is determined. A reduction of binding is indicative of the usefulness of the agent in inhibiting conformational change in NFAT from dephosphorylation by calcineurin. Preferably, the reduction is assessed relative to the same reaction without addition of the candidate agent.

[0217] NFAT changes its conformation as a direct consequence of dephosphorylation by calcineurin in such a way as to dramatically increase its specific binding to DNA (Park et al, J. Biol Chem 270:20653-20659 (1995); Shaw et al, Proc Natl Acad Sci USA 92:11205-11209 (1995)). Specific binding of NFAT to DNA can be simply assessed in an assay that is suitable for high throughput screening. Thus, this alteration in DNA binding can be used to detect dephosphorylation of NFAT and to screen for compounds that are capable of inhibiting the dephosphorylation. NFAT in phosphorylated form, obtained as described above, is treated with calcineurin in the presence of a candidate agent to be tested. Preferably, control samples of phosphorylated NFAT are incubated (i) in the absence of calcineurin, (ii) in the presence of calcineurin with no added candidate agent, and (iii) in the presence of calcineurin and known inhibitors, e.g., the 13-mer peptide or 25-mer peptide as described herein, or CsA/cyclophilin complexes. At the end of the incubation, specific binding of NFAT to an oligonucleotide, e.g., a double-stranded oligonucleotide, e.g., DNA, incorporating an NFAT site, e.g., the distal NFAT site of the murine IL-2 promoter (Jain et al, Nature 356:801-804 (1992)) or the P1 site of the murine IL-4 promoter (Rooney et al, Immunity 2:473-483 (1995)), is measured. In one embodiment, this measurement is made by incubating the sample with biotinyl-DNA, incorporating the NFAT binding site, then further incubating with streptavidin-SPA beads and 125I-labeled antibody against NFAT. In this embodiment, scintillation counting of 125I label gives a measure of the NFAT-DNA complex that has formed. Compounds of interest in the assay are those that prevent or inhibit the increase in DNA binding that results from incubation of phosphorylated NFAT with calcineurin. In embodiments in which the compounds tested are not separated from NFAT before the DNA binding step, preferably, it is further shown that a compound of interest does not directly inhibit the ability of NFAT to bind to DNA, e.g., by examining DNA binding in the same assay when the test compound is added only after the incubation with calcineurin is completed, or by examining the effect of the compound on the binding of bacterially-expressed NFAT. As is known to one skilled in the art, there are many effectively equivalent methods for measuring the binding of NFAT to DNA including, e.g., recruitment of 3H-DNA to NFAT bound via anti-67.1 antiserum (Ho et al, J Biol Chem 269:28181-28186 (1994)) on protein A-SPA beads, competition by unlabeled NFAT with a fixed amount of 125I-NFAT for binding to biotinyl-DNA immobilized on streptavidin-SPA beads, inclusion in the DNA binding reaction of c-Fos and c-Jun proteins to increase the affinity of the interaction, and using another solid phase support.

[0218] In an alternative, the conformational change, and therefore dephosphorylation, may be detected directly by using a probe that recognizes specifically a region or determinant of NFAT that is exposed only after dephosphorylation. An example is the nuclear localization sequence (NLS) of NFAT, which is masked until dephosphorylation, but then becomes accessible for binding of other proteins, e.g., the importin proteins that direct dephosphorylated NFAT to the nucleus in cells. Exposure of the NLS, or of a tag peptide introduced into recombinant NFAT in place of the NLS, may be detected, e.g., in an immunoassay with an appropriate antibody. In another alternative, the conformational change in NFAT may be detected by fluorescence resonance energy transfer (FRET) using a recombinant NFAT protein labeled with appropriate fluorophores at two distinct sites, as has been illustrated for calmodulin (Miyawaki et al, Nature 388:883-887 (1997)), or by FRET between fluorophore-labeled minibodies directed to distinct sites on the surface of NFAT whose relative position changes as a result of the conformational change, or by alteration in the intrinsic fluorescence of NFAT upon dephosphorylation.

[0219] The invention also includes a method for high-throughput screening of candidate agents to identify an agent that inhibits calcineurin-dependent import of NFAT into the nucleus of a cell. Cells expressing NFAT are provided. A stimulant that activates NFAT through the calcium/calcineurin pathway is provided. A candidate agent is provided. The cells, stimulant and candidate agent are contacted with each other. The presence or absence of nuclear NFAT in the cells is determined. A reduction in nuclear NFAT is indicative of the agent inhibiting calcineurin-dependent import of NFAT into the nucleus of a cell. Preferably, the reduction is assessed relative to the same reaction without addition of the candidate agent.

[0220] This assay is based on the calcineurin-dependent difference in localization of NFAT in unstimulated and stimulated cells. Cells expressing NFAT, e.g., endogenous or recombinant NFAT, are incubated in the presence of a stimulant, e.g., calcium ionophore, a neurotransmitter, or a biologically active peptide, known to trigger activation of NFAT via the calcium/calcineurin pathway (for examples, see Table 1 in Rao et al, Annu. Rev. Immunol. 15:707-747 (1997)). Preferably, control samples of cells are incubated without addition of the stimulant, or in the presence of the stimulant and with known inhibitors of calcineurin-dependent NFAT activation, e.g., CsA and FK506. Determining the presence or absence of nuclear NFAT, and also, preferably cytoplasmic NFAT, can be accomplished by any method known to one skilled in the art. Preferably, localization to the nucleus in samples incubated in the presence of the candidate agent is compared with that of control samples incubated with the stimulant only. Agents are scored as positive if they interfere with the calcineurin-dependent import of NFAT to the cell nucleus.

[0221] Examples of determining the localization of NFAT include fixing the cells after the contacting step with histological fixative and examining by microscopy or other means capable of detecting the difference between cells having cytoplasmic NFAT and cells having nuclear NFAT. In some embodiments, NFAT is detected by immunocytochemical staining. In some embodiments, the localization of an NFAT fusion protein, e.g., NFAT-GFP, is detected by fluorescence microscopy. Localization of NFAT to the nucleus can be scored, e.g., by microscopy using visual inspection. In some embodiments, visual inspection is aided by automation, or the localization of NFAT is determined in an automated assay. In certain embodiments of an automated assay, cells are stained with, e.g., two distinct fluorophores, a first fluorophore that detects NFAT (e.g., fluorescently labeled NFAT or fluorescent antibodies that bind to NFAT) and a second fluorophore that labels cell nuclei. In these embodiments, the nuclear localization of NFAT is quantitated by assessing colocalization of the two labels, e.g., the average level of NFAT label is determined in pixels that show nuclear labeling above a designated threshold level that is easily determined by examining the positive and negative control samples. In yet other embodiments, line or raster scanning is used to excite fluorescence, or a method sensitive to the spatial frequency of the fluorescent signal is employed.

[0222] As with other assays based on detecting the calcineurin-NFAT interaction or its consequences in cells, assays based on the calcineurin-dependent import of NFAT into the cell nucleus have the advantage that the cell permeability of specific inhibitors identified in the assay is assured.

[0223] In some embodiments, these cell-based assays are used to screen, e.g., a retroviral expression library, or other peptide or protein expression libraries, for those recombinant proteins capable of interfering with the calcineurin-mediated activation of NFAT.

[0224] The cellular assay examining cytoplasmic or nuclear localization of NFAT is also useful as a diagnostic test to confirm normal physiological function in cells derived from an animal, e.g., human, to detect or classify pathological or abnormal function of immune system cells, or to identify stimuli or sources of activation of immune system cells. For example, an immune disorder can be detected or classified by documenting abnormal activation (constitutively nuclear NFAT protein) in a class of cells, or by documenting an abnormal failure to translocate NFAT in response to a stimulus. In another example, the source of an allergic response can be determined, e.g., by testing candidate allergens for their ability to induce nuclear translocation of NFAT in an indicator mast cell line stably expressing NFAT1(1-460)-GFP, where the indicator mast cells have first been exposed to IgE from an animal or a human, and where further exposure to an effective allergen will therefore cause activation of the cells through their Fc receptors and nuclear import of NFAT-GFP.

[0225] The invention includes a method for assessing the state of NFAT activation of immune system cells from an animal. Immune system cells isolated from an animal are provided. The presence or absence of nuclear NFAT in the cells is determined. The presence of nuclear NFAT in the cells is indicative of activation of NFAT in the cells. The cells can be isolated by any method known to those skilled in the art, e.g., by biopsy or aspiration. In certain embodiments, the cells are infiltrating cells at a site of inflammation or in a tumor. Preferably, the presence or absence of nuclear NFAT is determined by histological staining, e.g., immunocytochemical staining, of the cells.

[0226] The invention also includes a method for assessing the ability of immune system cells isolated from an animal to respond to an NFAT activating signal. Immune system cells from an animal are provided, the cells being unactivated for NFAT. A stimulant that activates NFAT is provided. The cells are contacted with the stimulant. The presence or absence of nuclear NFAT in the cells is determined. A reduction in nuclear NFAT is indicative of impairment of the ability of the cells to respond to an NFAT activating signal. Preferably, the reduction is assesssed relative to cells isolated from a normal animal. This assay can be used, e.g., to monitor the level of immune function in certain immunocompromised patients.

[0227] The invention also includes a method for identifying a stimulant that can activate NFAT in immune system cells isolated from an animal. Immune system cells isolated from an animal are provided. A candidate stimulant is provided. The cells are contacted with the candidate stimulant. The presence or absence of nuclear NFAT in the cells is determined. The presence of nuclear NFAT is indicative of the stimulant activating NFAT in the cells. In preferred embodiments, the stimulant is an allergen.

[0228] By allergen is meant an agent that elicits IgE-mediated reactions. This assay can be used, e.g., to monitor unrespon-siveness to a pathogen or tolerance to a specific antigen.

[0229] The invention also includes a method for identifying an allergen. An animal cell line expressing NFAT is provided. IgE from an animal, e.g., a human, is provided. A candidate allergen is provided. The cell line is contacted with the IgE. The cell line is contacted with the candidate allergen. Preferably, the cell line is contacted with the candidate allergen after the cell line is contacted with the IgE. The presence or absence of nuclear NFAT in cells of the cell line is determined. The presence of nuclear NFAT is indicative of the candidate allergen being an allergen.

[0230] Small Molecules for Inhibition of the Protein-Protein Interaction of Calcineurin and NFAT

[0231] As discussed above, the invention includes methods for high throughput screening of candidate agents, e.g., agents that are initially members of an organic chemical library, to identify agents that inhibit the protein-protein interaction between calcineurin and NFAT. The present invention also includes small organic molecules, e.g., nonpeptide molecules, that have been isolated using the methods described herein. It is contemplated that persons skilled in the art can readily modify the organic molecules specifically described herein to, e.g., obtain a molecule optimized for administration to an animal.

[0232] In general, organic molecules useful for the present invention have a molecular weight of less than 2500 Daltons (Da). The small molecules can be, e.g., from at least about 100 Da to about 2000 Da (e.g., between about 100 to about 2000 Da, about 100 to about 1750 Da, about 100 to about 1500 Da, about 100 to about 1250 Da, about 100 to about 1000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100 to about 250 Da).

[0233] Organic molecules useful for the present invention can exhibit the ability to interact with, e.g., bind to, calcineurin or a fragment thereof, with an affinity constant of at least about 2×104M−1, e.g., least about 105 M−1, at least about 106 M−1, at least about 107 M−1, or at least about 108 M−1, or stronger. In view of the common docking site on calcineurin for all NFAT-family proteins, inhibitors that bind to calcineurin can interfere with activation of all NFAT proteins, and can provide the beneficial effects of some currently used immunosuppressant drugs, e.g., CsA and FK506, potentially without undesirable side effects caused by such drugs.

[0234] Organic molecules useful for the present invention can exhibit the ability to interact with, e.g., bind to, NFAT or a fragment thereof, with an affinity constant of at least about 2×104 M−1, e.g., at least about 106 M−1 at least about 107 M−1, or at least about 108 M−1. Inhibitors of this class can be complementary to a surface of NFAT, rather than to the NFAT-docking site on calcineurin. The primary structure of NFAT proteins is only partially conserved in the domain that interacts with calcineurin. Hence, in some cases, such small molecules of the present invention can bind preferentially to one NFAT-family protein and/or to a subset of NFAT-family proteins, over others. Such a characteristic is desirable in treatment of certain disorders of the immune system, in preventing or treating myocardial disease without compromising immune function, and in treating conditions wherein pathological signalling by NFAT is predominantly caused by one type of NFAT protein or by a definable subset of NFAT proteins. Further, the conformation of NFAT is altered by dephosphorylation. Such small molecules can display a preference for binding to phosphorylated (inactive) NFAT or to dephosphorylated (activated) NFAT, making the small molecule capable of reducing calcineurin activity more effectively against one than the other, and preferentially modulating either the rate of activation of phosphorylated NFAT or the rate of inactivation of dephosphorylated NFAT. Such preferential binding could result in, for example, a selective attenuation of strong rapid transcriptional signalling via the calcineurin-NFAT pathway, with a lesser effect on weak sustained signalling, hence selectively reducing the expression of a subgroup of NFAT target genes or altering the time course of expression of NFAT target genes in a beneficial way.

[0235] Exemplary organic molecules of the present invention are illustrated below as formulas (I), (IV) and (III), and exemplary substitutions are given in accompanying Tables 2, 3, and 4. 6 TABLE 2 4 Compound R1 R2 R3 R4 R5 INCA1 H Ph Cl CHAc2 O INCA1A Me OEt Br H NMe INCA1B H Ph Br H O INCA1C Me OEt Br H O INCA1D H 4-MePh Cl Cl O INCA1E H 4-MePh Br H O

[0236] 7 TABLE 3 5 Cmpd R1 R2 R3 R4 R5 R6 R7 INCA2 Cl Cl — H H Double Bond INCA2A Cl Cl — H Me Double Bond INCA2B Cl H — H Me Double Bond INCA2C Br H — H Me Double Bond INCA2D Cl Cl — H Me Cl Cl INCA2E H H — H H Double Bond INCA2F Cl H — H Cl Double Bond INCA2G Br H — H Cl Double Bond INCA2H Cl Cl OMe H Me Double Bond INCA2I Cl Cl OMe H Cl Double Bond INCA2J H H — Me Me Double Bond INCA2K Cl Cl OBu H Me Double Bond INCA2L DDC H — H Me Double Bond INCA2M NMe2 H — H H Double Bond

[0237] 8 TABLE 4 6 Cmpd. R1 R2 R3 R4 R5 R6 R7 INCA6 O H H O H H H

[0238] Other representative inhibitory compounds containing scaffolds other than those of formulae (I), (II) and (III) are included in Appendix I, and are enumerated as Compounds INCA3, CAN4, INCA5, INCA7, INCA8, INCA9, INCA10, CAN33, CAN11, CAN30, CAN13, CAN22, CAN14, CAN15, CAN21, CAN16, CAN17, and CAN19.

[0239] As used herein, the term “halo” or “halogen” refers to any radical of fluorine, chlorine, bromine or iodine. The term “alkyl” refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C1-C10 indicates that the group may have from 1 to 10 (inclusive) carbon atoms in it. The term “lower alkyl” refers to a C1-C8 alkyl chain. The term “alkoxy” refers to an —O-alkyl radical. The term “alkylene” refers to a divalent alkyl (i.e., —R—). The term “alkylenedioxo” refers to a divalent species of the structure —O—R—O—, in which R represents an alkylene. The term “aminoalkyl” refers to an alkyl substituted with an amino. The term “mercapto” refers to an —SH radical. The term “thioalkoxy” refers to an —S-alkyl radical.

[0240] The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of aryl groups include phenyl, naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refers to alkyl substituted with an aryl. The term “arylalkoxy” refers to an alkoxy substituted with aryl.

[0241] The term “cycloalkyl” includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, wherein the cycloalkyl group additionally may be optionally substituted. Preferred cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0242] The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like. The termn “heteroarylalkyl” or the term “heteroaralkyl” refers to an alkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.

[0243] The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3 atoms of each ring may be substituted by a substituent. Examples of heterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

[0244] The term “oxo” refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

[0245] The term “substituents” refers to a group “substituted” on an alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl group at any atom of that group. Suitable substituents include, without limitation, halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups. In one aspect, the substituents are independently selected from the group consisting of C1-C6 alkyl, C3-Cg cycloalkyl, (C1-C6)alkyl(C3-C8)cycloalkyl, C2-C8alkenyl, C2-C8 alkynyl, cyano, amino, C1-C6alkylamino, di(C1-C6)alkylamino, benzylamino, dibenzylamino, nitro, carboxy, carbo(C1-C6)alkoxy, trifluoromethyl, halogen, C1-C6 alkoxy, C6-C10 aryl, (C6-C10)aryl(C1-C6)alkyl, (C6-C10)aryl(C1-C6)alkoxy, hydroxy, C1-C6 alkylthio, C1-C6 alkylsulfinyl, C1-C6 alkylsulfonyl, C6-C10 arylthio, C6-C10 arylsulfinyl, C6-C10 arylsulfonyl, C6-C10 aryl, (C1-C6)alkyl(C6-C10)aryl, and halo(C6-C10)aryl.

[0246] Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds. The term “stable,” as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).

[0247] As can be appreciated by the skilled artisan, further methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagentsfor Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

[0248] The compounds useful in the present invention can also be obtained as part of a library of organic compounds. Combinatorial techniques suitable for synthesizing the compounds described herein are known in the art as exemplified by Obrecht, D. and Villalgrodo, J. M., Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, Pergamon-Elsevier Science Limited (1998), and include those such as the “split and pool” or “parallel” synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czarnik, A. W., Curr. Opin. Chem. Bio., (1997) 1, 60.

[0249] In an alternate embodiment, the compounds described herein may be used as platforms or scaffolds that may be utilized in combinatorial chemistry techniques for preparation of derivatives of the organic compounds described herein and/or chemical libraries of compounds. Such derivatives and libraries of compounds have biological activity and are useful for identifying and designing compounds that inhibit the protein-protein interaction of calcineurin and NFAT.

[0250] Thus, one embodiment relates to a method of using the compounds described in the formulae herein for generating derivatives or chemical libraries comprising: 1) providing a body comprising a plurality of wells; 2) providing one or more compounds of the formulae described herein in each well; 3) providing an additional one or more chemicals in each well; 4) isolating the resulting one or more products from each well. An alternate embodiment relates to a method of using the compounds described in the formulae herein for generating derivatives or chemical libraries comprising: 1) providing one or more compounds of the formulae described herein attached to a solid support; 2) treating the one or more compounds of the formulae described herein attached to a solid support with one or more additional chemicals; 3) isolating the resulting one or more products from the solid support. In the methods described above, “tags” or identifier or labeling moieties may be attached to and/or detached from the compounds of the formulae herein or their derivatives, to facilitate tracking, identification or isolation of the desired products or their intermediates. Such moieties are known in the art. The chemicals used in the aforementioned methods may include, for example, solvents, reagents, catalysts, protecting group and deprotecting group reagents and the like. Examples of such chemicals are those that appear in the various synthetic and protecting group chemistry texts and treatises referenced herein.

[0251] The compounds of this invention may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included in the present invention. The compounds of this invention may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein (e.g., alkylation of a ring system may result in alkylation at multiple sites, the invention expressly includes all such reaction products). All such isomeric forms of such compounds are expressly included in the present invention. All crystal forms of the compounds described herein are expressly included in the present invention.

[0252] As used herein, the compounds of this invention, including the compounds of formulae described herein, are defined to include pharmaceutically acceptable derivatives or prodrugs thereof. A “pharmaceutically acceptable derivative or prodrug” means any pharmaceutically acceptable salt, ester, salt of an ester, or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention. Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Preferred prodrugs include derivatives where a group which enhances aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein, or to derivatives thereof.

[0253] The compounds of this invention may be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological compartment (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.

[0254] Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)4+ salts. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization.

[0255] The organic molecules described herein, or derivatives thereof, can be combined with a pharmaceutically acceptable carrier to create a pharmaceutical composition. The pharmaceutically acceptable carrier can be any solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic and absorption delaying agent, and any of the like that are physiologically compatible. The carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the organic molecule may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application.

[0256] The following non-limiting examples further illustrate the present invention.

EXAMPLES Example 1 The SPRIEITPS Sequence of NFAT1 is Involved in Nuclear Import of NFAT1

[0257] This example illustrates that mutations in the SPRIEITPS (SEQ ID NO:17) sequence in the conserved motif-2 (CM2) region (amino acid residues 110-118) of the NFAT1 conserved regulatory domain (amino acid residues 100-397) inhibit translocation of the mutant NFAT1 protein from the cytoplasm to the nucleus.

[0258] A triple CM2 mutant was generated which disrupts the sequence 110SPRIEITPS118 (SEQ ID NO:17) of NFAT1 by replacing each of the three amino acid residues Arg112 (R112), Glu114(E114), and Thr116(T116), with an alanine residue. These mutations were generated following the procedures described in Kunkel et al., Methods Enzymol. 154:367-382 (1987). The mutant proteins were expressed with an HA epitope tag in C1.7W2 murine T cells and in HeLa cells, and analyzed for nuclear translocation in response to ionomycin stimulation. The mutant proteins behaved identically in both cell types. Translocation of wild type and mutant NFAT1 to the nucleus was measured by immunocytochemistry as follows. HeLa cells expressing HA-tagged full length wild type NFAT1 or mutant NFAT1 were left unstimulated or activated with ionomycin (3 &mgr;M, 10 min). NFAT1 was detected with mouse anti-HA antibody (12CA5) and Cy3™ goat anti-mouse IgG, and visualized using a rhodamine filter set on a Zeiss Axioskop microscope at a magnification of 630×. Results indicated that the triple CM2 mutation impaired translocation of NFAT1 to the nucleus upon ionomycin stimulation. Wild type NFAT1, as well as the mutant ST21, having two mutations in this same region, in which the two serine residues flanking the SPRIEITPS (SEQ ID NO:17) motif were substituted by alanine residues, showed normal translocation.

[0259] The triple CM2 mutations also impaired nuclear translocation of a GFP fusion protein containing only the N-terminal domain of NFAT1 (amino acids 1-460), NFAT1(1-460)-GFP. 24 h after transfection with a plasmid encoding wild type or mutant NFAT-GFP, the HeLa cells or C1.7W2 murine T cells were left untreated or stimulated with ionomycin (2 &mgr;M, 10 min). Nuclear translocation of NFAT1 was visualized by fluorescence microscopy. This impairment of nuclear translocation indicated that the SPRIEITPS (SEQ ID NO:17) motif is involved in the nuclear import of NFAT1, and that the effect of the CM2 mutation does not require an intact DNA binding domain (amino acid residues 398-680 of murine NFAT1) or the C-terminal domain of NFAT1 (amino acid residues 681-923, 681-927 and 681-1064 in the three known isoforms of murine NFAT1).

[0260] The role in nuclear translocation of each of the amino acid residues, R112, E114 and T116, was also assessed individually. The single amino acid mutation T116 to A, in the SPRIEITPS (SEQ ID NO:17) sequence of NFAT1, inhibited translocation in response to ionomycin stimulation almost to the same extent as did the triple CM2 mutation. Single mutations of R112 and E114 also impaired translocation, but to a lesser extent than the triple CM2 mutation.

Example 2 The SPRIEITPS Sequence of NFAT1 is Required for Effective Dephosphorylation by Calcineurin

[0261] This example illustrates that mutations in the SPRIEITPS (SEQ ID NO:17) sequence of NFAT1 inhibit dephosphorylation of the mutant NFAT1 protein by calcineurin.

[0262] The inability of the CM2 mutant, containing the three mutations described in Example 1, to translocate to the nucleus correlated with its very limited dephosphorylation in stimulated cells. When transiently expressed either in HeLa cells or in C1.7W2 murine T cells, wild type NFAT1(1-460)-GFP fusion protein was dephosphorylated in response to ionomycin stimulation (3 &mgr;M, 10 min) as assessed by its shift in migration on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The more complete shift observed in C1.7W2 T cells under these conditions reflects the higher level of calcineurin activity in this cell line. In contrast, the triple CM2 mutant NFAT1 (1-460)-GFP showed no change in migration after ionomycin stimulation of HeLa cells, and only a slight shift after stimulation of C1.7W2 T cells, indicating that dephosphorylation was inhibited.

[0263] The difference in ionomycin sensitivity between HA-tagged wild type NFAT1 (1-460)-GFP and the corresponding triple CM2 mutant was tested in C1.7W2 T cells stimulated with a range of ionomycin concentrations (0.07 to 6 &mgr;M) for 10 min. Significant dephosphorylation of HA-tagged wild type NFAT1 was achieved in cells stimulated with 220 nM ionomycin, the dephosphorylation being complete with 660 nM ionomycin. In contrast, dephosphorylation of the CM2 mutant protein was incomplete even in cells stimulated with 6 &mgr;M ionomycin, a concentration 10 times higher than that required to induce complete dephosphorylation of wild type NFAT1. Dephosphorylation of both wild type NFAT1 and the CM2 mutant was inhibited by cyclosporin A (CsA)(500 nM), indicating that the dephosphorylation remained calcineurindependent. Immunocytochemical experiments confirmed that the CM2 mutant did not translocate to the nucleus even in C1.7W2 cells stimulated with 6 &mgr;M ionomycin.

[0264] The triple CM2 mutant also displayed significantly reduced sensitivity to treatment with exogenous calcineurin in vitro. Cytoplasmic extracts from HeLa cells expressing HA-tagged wild type NFAT1 or the triple CM2 mutant NFAT1 protein were incubated with calcineurin (200 nM-2.5 &mgr;M) and calmodulin for 30 min at 30° C. The samples were then resolved by SDS-PAGE and the phosphorylation state was analyzed by Western blotting (Shaw et al., Proc Natl Acad Sci USA 92:11205-11209 (1995); Luo et al., Proc Natl Acad Sci USA 93:8907-8912 (1996)) with anti-HA antibody. Dephosphorylation of wild type NFAT1 was apparent with 200 nM calcineurin, whereas the triple CM2 mutant was markedly less sensitive, with only partial dephosphorylation occurring in the presence of 2.5 &mgr;M calcineurin.

[0265] The role in NFAT1 dephosphorylation of each of the amino acid residues, R112, E114 and T116, was also assessed individually by Western blotting. The single amino acid substitution T116 to A, in the SPRIEITPS (SEQ ID NO:17) sequence of NFAT1, inhibited dephosphorylation in response to ionomycin stimulation almost to the same extent as did the triple CM2 mutation. Single mutations of R112 and E114 also impaired dephosphorylation, but to a lesser extent than the triple CM2 mutation.

Example 3 Peptides Spanning the SPRIRITPS Sequence of NFAT1 Interfere with Recognition and Dephosphorylation of NFAT1 by Calcineurin

[0266] This example illustrates that peptides spanning the SPRIEITPS (SEQ ID NO:17) sequence of NFAT1 interfere with recognition and dephosphorylation of NFAT1 by calcineurin.

[0267] The results from Examples 1 and 2 indicated that the CM2 mutations in the SPRIEITPS (SEQ ID NO:17) sequence impaired the ability of calcineurin to recognize NFAT1 as a substrate and to dephosphorylate it, either in vivo or in cell extracts. One possibility was that the SPRIEITPS (SEQ ID NO:17) sequence represented a region of NFAT-calcineurin contact and that mutation of this sequence impaired the targeting of calcineurin to NFAT.

[0268] To test whether the SPRIEITPS (SEQ ID NO:17) motif was directly involved in the interaction of NFAT with calcineurin, peptides spanning the CM2 motif of wild type and mutant NFAT1 were tested in an NFAT-calcineurin binding assay for their capacity to block calcineurin binding to NFAT1. Two wild type peptides were synthesized which spanned the SPRIEITPS (SEQ ID NO:17) motif, one containing 13 amino acid residues of murine NFAT1 (SEQ ID NO:22) and one containing 25 amino acid residues of NFAT1 (SEQ ID NO:29). In the case of the longer peptide, a tyrosine residue not present in the NFAT1 sequence was appended at the C terminus to facilitate chemical coupling to a carrier protein for production of antisera, and the peptide tested in this and subsequent examples (Examples 4 and 5 infra) was therefore a 26-mer. A corresponding 26-mer peptide incorporating the R112 to A, E114 to A, and T116 to A substitutions of the triple CM2 mutant NFAT1, and with the appended tyrosine residue, was synthesized for use as a control. It is evident from the results below, specifically from the identical effects of the 13-mer peptide and the 26-mer peptide on calcineurin binding and from the lack of effect of the 26-mer mutant peptide on calcineurin binding, that the C-terminal tyrosine residue has no role in inhibiting the protein-protein interaction.

[0269] 125I-labelled calcineurin (14 nM, −7×105 cpm) was incubated for 30 min at 4° C. with GST-tagged NFAT1 N-terminal domain (GST NFAT1(1-400)) that had been immobilized on glutathione-Sepharose™ beads (Pharmacia Biotech, Piscataway, N.J.), in the absence or presence of different concentrations (1 &mgr;M to 100 &mgr;M) of wild type 13-mer peptide, wild type 26-mer peptide, or CM2 mutant 26-mer peptide. At the end of the incubation, the glutathione Sepharose beads with bound NFAT1-calcineurin complexes were washed on 5 &mgr;m hydrophilic Durapore PVDF filters (Millipore Corporation, Bedford, Mass.) to remove unbound calcineurin, and NFAT1-bound calcineurin was quantitated using a gamma counter. Both peptides incorporating the wild type sequence SPRIEITPS (SEQ ID NO:17) inhibited the binding of calcineurin to NFAT1. The IC50 for inhibition by the wild type peptides was very similar, approximately 15 &mgr;M for both the 13-mer and 26-mer. In contrast, the mutated 26-mer peptide incorporating the sequence SPAIAIAPS (SEQ ID NO:82) did not inhibit the NFAT1-calcineurin interaction at concentrations up to 100 &mgr;M.

[0270] The ability of the SPRIEITPS peptides to inhibit dephosphorylation of NFAT1 by calcineurin in vitro was measured by Western blotting analysis. Cytosolic extracts from HeLa cells stably expressing HA-tagged N-terminal domain NFAT1(1-460)-GFP were incubated with calcineurin (200 nM) and calmodulin (600 nM) in the presence of different concentrations (16 &mgr;M to 2 mM) of wild type 13-mer, wild type 26-mer, and CM2 mutant peptides, during 30 min at 30° C. Controls were done with the calcineurin inhibitors sodium pyrophosphate (10 mM) and cyclosporinA/cyclophilin complexes (15 &mgr;M/5 &mgr;M). Samples were resolved by SDS-PAGE and dephosphorylation was assessed with anti-HA antibody 12CA5. Both the 13-mer and the 26-mer peptides with wild type sequence SPRIEITPS (SEQ ID NO:17) inhibited dephosphorylation of NFAT1 at concentrations in the range from 16 &mgr;M to 400 &mgr;M, whereas the 26-mer peptide with sequence SPAIAIAPS (SEQ ID NO:82) was only marginally inhibitory, and only at concentrations above 400 &mgr;M. Slightly higher concentrations of the peptides were required to inhibit the in vitro dephosphorylation than were required to inhibit NFAT-calcineurin binding, presumably due to some degradation of the peptides by enzymes present in the cell extracts.

Example 4 The SPRIEITPS Sequence Specifically Targets Calcineurin to NFAT Proteins

[0271] This example illustrates that peptides spanning the SPRIEITPS (SEQ ID NO:17) sequence do not interfere generally with calcineurin phosphatase activity.

[0272] The 13-mer and 26-mer SPRIEITPS peptides were tested to determine if they acted as general inhibitors of calcineurin phosphatase activity by assaying them on the dephosphorylation of a well characterized calcineurin substrate, the RII phosphopeptide (Blumenthal et al., J. Biol. Chem. 261:8140-8145 (1986)). 32P-RII phosphopeptide (100 &mgr;M; specific activity 1×109 cpm/&mgr;mole) was incubated with calcineurin (100 nM) and calmodulin (600 nM). In some samples, FK506/FKBP12 complexes (10 &mgr;M/10 &mgr;M), calcineurin autoinhibitory peptide (30 &mgr;M to 400 &mgr;M), or SPRIEITPS peptide, 13-mer or 26-mer at different concentrations (30 &mgr;M to 400 &mgr;M) were preincubated with calcineurin before addition of the calcineurin mixture to the RII phosphopeptide samples. The dephosphorylation reaction was allowed to proceed for 30 min at 30° C. after which the reaction was stopped by addition of excess 0.1% trichloroacetic acid, substrate peptide was removed by adsorption onto a cation exchange resin (AG50W; BioRad Laboratories, Hercules, Calif.), and released 32P label in the supernatant was measured in a liquid scintillation counter. Neither of the SPRIEITPS peptides inhibited dephosphorylation of the RII peptide by calcineurin in the same range of concentrations in which they inhibited NFAT1 dephosphorylation. In the same experiment, calcineurin activity was effectively inhibited by a peptide corresponding to its own autoinhibitory domain and by FK506/FKBP12 complexes.

[0273] Moreover, dephosphorylation of the RII regulatory subunit of cAMP-dependent protein kinase, a protein substrate of calcineurin (Blumenthal et al., Biol Chem 261:8140-8145 (1986)) from which the RII phosphopeptide is derived, was not inhibited by a SPRIEITPS peptide from NFAT1 as shown by an in vitro dephosphorylation assay with recombinant RII&agr; protein. A 6×Histagged RII&agr; protein (90 nM), 32P-labelled in vitro by the PKA catalytic subunit, was incubated with calcineurin (200 nM) and calmodulin (600 nM). In some samples, the calcineurin inhibitors sodium pyrophosphate (20 mM), CsA/cyclophilin complexes (15 &mgr;M/5 &mgr;M), FK506/FKBP12 complexes (4 &mgr;M/4 &mgr;M), or different concentrations of the 26-mer SPRIEITPS peptide (20 &mgr;M to 500 &mgr;M) were preincubated with calcineurin during 20 min on ice before adding 32P-labelled RII&agr; protein to the mixture. Dephosphorylation was allowed to proceed during 45 min at 30° C., samples were resolved by SDS-PAGE, and the gel was stained with Coomasie Brilliant Blue, dried, and autoradiographed. A phosphorimager was used to quantitate the level of 32P in each lane. Coomasie Brilliant Blue staining showed that equal amounts of the reaction were loaded in each lane. The dephosphorylation of RII&agr; was efficiently inhibited by the general phosphatase inhibitor sodium pyrophosphate and by the calcineurin inhibitors CsA/cyclophilin complexes and FK506/FKBP12 complexes. In contrast, the 26-mer SPRIEITPS peptide did not inhibit dephosphorylation when used at the concentrations (20 &mgr;M and 100 &mgr;M) required to inhibit NFAT1 dephosphorylation, and caused only slight inhibition (18% inhibition) at 500 &mgr;M.

[0274] Additionally, the 26-mer SPRIEITPS peptide did not inhibit the dephosphorylation of a different protein known to be a calcineurin substrate in vivo, the neuronal cytoskeleton protein Tau (Fleming and Johnson, J. Biochem 209:41-47 (1995); Yamamoto et al., J. Biochem 118:1224-1231 (1995)). Purified GST-Tau immobilized on glutathione-Sepharose beads was phosphorylated by MAP kinase, washed, and incubated with calcineurin (200 nM) and calmodulin (600 nM) during 90 min at 30° C. In some samples, the calcineurin inhibitors sodium pyrophosphate (10 mM) or CsA/cyclophilin complexes (15 &mgr;M/5 &mgr;M), or wild type 26-mer peptide at different concentrations (20 &mgr;M to 500 &mgr;M) were preincubated with calcineurin for 20 min on ice before the addition of Tau protein. Samples were resolved by SDS-PAGE and analyzed as above. Dephosphorylation of GST-Tau protein by calcineurin was not inhibited by the SPRIEITPS peptide at concentrations of 20 &mgr;M and 100 &mgr;M, and was minimally inhibited (10%) at 500 &mgr;M, a concentration at which the peptide fully inhibited dephosphorylation of NFAT1. The phosphatase inhibitor sodium pyrophosphate and the calcineurin inhibitor, CsA-cyclophilin complexes, efficiently inhibited dephosphorylation of the GST-Tau protein under these conditions.

[0275] In sum, a 13-residue or 26-residue peptide spanning the SPRIEITPS (SEQ ID NO:17) sequence of NFAT1 is a potent inhibitor of the interaction of NFAT1 with calcineurin, while not affecting either the phosphatase activity of the enzyme or its ability to dephosphorylate the other non-NFAT substrates tested.

Example 5 SPRIEITPS Peptides from NFAT1 Also Interfere with Activation of Other NFAT Family Members

[0276] This example illustrates that peptides spanning the SPRIEITPS (SEQ ID NO:17) sequence from NFAT1 interfere comparably with activation of NFAT2 and NFAT4 despite sequence differences among the NFAT proteins.

[0277] The SPRIEITPS (SEQ ID NO:17) sequence of NFAT1 is highly conserved in NFAT2, but only partially conserved in NFAT4 which has a CPSIQITSI (SEQ ID NO:20) sequence. NFAT1, NFAT2 and NFAT4 constitute the group of NFAT members expressed in immune cells. The inhibitory effect of the SPRIEITPS peptides, 13-mer and 26-mer, of NFAT1 on the binding of calcineurin to NFAT1, NFAT2 and NFAT4 was tested. Binding assays of 125I-calcineurin with GST-tagged N-terminal domains of NFAT1 (residues 1-400), NFAT2 (residues 1-418) and NFAT4 (residues 1-400), in the presence or absence of wild type SPRIEITPS peptides or the mutant control peptide, were done as described above. The results showed that the CM2 peptide inhibited the ability of 125I-calcineurin to bind to the N-terminal regions of NFAT2 and NFAT4 with a very similar concentration dependence. The IC50, for inhibition of NFAT2 and NFAT4 calcineurin binding by the 26-mer peptide was again approximately 15 &mgr;M, clearly indicative of a common calcineurin targeting mechanism involving the SPRIEITPS (SEQ ID NO:17) sequence in NFAT1 and the cognate SPRIEITSC (SEQ ID NO:18) and CPSIQITSI (SEQ ID NO:20) sequences in NFAT2 and NFAT4.

[0278] Similarly, the 13-mer SPRIEITPS peptide from NFAT1 inhibited dephosphorylation of the N-terminal domain of NFAT4 by calcineurin. The HA-tagged N-terminal domains of NFAT1 and NFAT4 were expressed as the fusion proteins HA-NFAT1(1-460)-GFP and HA-NFAT4(1-407)-GFP in HEK-293 cells. At 24 h after transfection, cytosolic extracts were prepared and equivalent aliquots were incubated without calcineurin or with increasing concentrations of calcineurin/calmodulin complexes (100 nM to 900 nM). Wild type 13-mer peptide of NFAT1 (100 &mgr;M or 400 &mgr;M final concentration in the dephosphorylation reaction) or mutant 26-mer peptide (400 &mgr;M final concentration) was added to the calcineurin/calmodulin preparations 20 min before mixing them with the NFAT-containing cell lysates. Dephosphorylation was allowed to proceed for 30 min at 30° C., and samples were analyzed by SDS-PAGE and Western blotting with anti-HA antibody. Lysates from HEK-293 cells expressing HA-NFAT1(1-460)-GFP or HA-NFAT4(1-407)-GFP incubated with calcineurin showed dephosphorylation of the NFAT proteins at all concentrations of calcineurin tested. The addition of wild type 13-mer SPRIEITPS peptide (100 &mgr;M or 400 &mgr;M) to the assay inhibited dephosphorylation, with more effective inhibition in the presence of 400 &mgr;M peptide, whereas the mutant peptide displayed little or no inhibition even with 400 &mgr;M peptide.

Example 6 An NFAT1 SPRIEITPS 19-mer Peptide Displays Immunosunpressive Properties In Vivo

[0279] This example illustrates that expression of a fusion protein including an NFAT1 SPRIEITPS 19-mer peptide inhibits NFAT1 activation in vivo as measured by dephosphorylation of NFAT1 in T lymphocytes, nuclear translocation of NFAT1 in T lymphocytes, and NFAT-mediated gene expression in T lymphocytes.

[0280] The ability of a GFP-SPRIEITPS-19 fusion protein to inhibit ionomycin-induced dephosphorylation of NFAT1 in T lymphocytes was tested as follows. Ionomycin-induced dephosphorylation of HA-tagged NFAT1(1-460)-GFP in T cells expressing the GFP-SPRIEITPS-19 fusion protein, or expressing related proteins used as controls, was examined by Western blotting analysis. The GFP-SPRIEITPS-19 expression vector was made by introducing a double-stranded oligonucleotide encoding the sequence KPAGASGPSPRIEITPSHEAYD (SEQ ID NO:102) in frame between the BsrGI and NotI sites located 3′ to the green fluorescent protein (GFP) coding sequence in the pEGFP-N1 expression vector (CLONTECH, Palo Alto, Calif.). The codons encoding residues AYD at the C-terminal end of the peptide sequence were included for convenience in subcloning, and a stop codon was introduced after the last codon for the peptide sequence. The GFP-SPAIAIAPS-19 construct was made by subcloning a double-stranded oligonucleotide encoding SGPSPAIAIAPSHEAYD (SEQ ID NO:103) between the BspEI and BsiWI sites in the GFP-SPRIEITPS-19 expression plasmid. Constructs expressing unmodified GFP, GFP-wild type peptide fusion protein, or GFP-mutant peptide fusion protein, were cotransfected together with expression vector encoding an HA-tagged N-terminal domain of NFAT1 (NFAT1(1-460)-GFP) into C1.7W2 murine T cells. 24 h post transfection, cells were left untreated or stimulated for 10 min with different concentrations of ionomycin. Whole cell lysates were analyzed for dephosphorylation of NFAT1 by Western blotting with anti-HA antibody and for expression of GFP proteins with anti-GFP antibody. The wild type GFP-SPRIEITPS-19 fusion protein efficiently inhibited ionomycin-induced dephosphorylation of NFAT1, whereas neither GFP alone nor the mutant GFP-SPAIAIAPS-19 protein had any inhibitory effect. Western blotting with anti-GFP antibody showed that the GFP, GFP-SPRIEITPS-19, and GFP-SPAIAIAPS-19 proteins were expressed at comparable levels. The inhibition of NFAT1 dephosphorylation by the wild type GFP-SPRIEITPS 19 protein was incomplete under strong stimulation conditions. This result is in agreement with in vitro assays where the concentration of SPRIEITPS peptide required to inhibit dephosphorylation of NFAT1 and NFAT4 was increasingly higher as the calcineurin concentration in the samples was increased.

[0281] The ability of GFP-SPRIEITPS-19 fusion protein to inhibit nuclear translocation of endogenous NFAT1 in T lymphocytes was tested as follows. C1.7W2 murine T cells expressing GFP, GFP-SPRIEITPS-19 (wild type peptide), or GFP-SPAIAIAPS-19 (mutant peptide) were stimulated with ionomycin (2 &mgr;M, 10 min) and processed for immunocytochemistry. NFAT1 was visualized with anti-T2B1, a rabbit anti-NFAT1 antiserum directed against the C-terminal peptide of NFAT1 isoform C (Wang et al., Ann. NY Acad. Sci. 766:182-194 (1995)) and Cy3TM labeled donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa.) using a rhodamine filter set. Simultaneous expression of the GFP constructs in individual cells was assessed by GFP fluorescence using a fluorescein filter set. GFP-SPRIEITPS-19 impaired ionomycin-induced nuclear translocation of NFAT1, whereas the control proteins, GFP and the mutant GFP-SPAIAIAPS-19, did not. Translocation was completely inhibited, however, only in those cells expressing higher levels of the GFP-SPRIEITPS-19 protein, indicating that, under these conditions of strong stimulation, effective inhibition required high intracellular concentration of the peptide.

[0282] The ability of GFP-SPRIEITPS-19 fusion protein to inhibit NFAT-driven gene transcription in Jurkat T cells was tested as follows. Jurkat human T cells (15×106 cells/transfection) were transfected with expression plasmids encoding GFP (9 &mgr;g plasmid DNA), wild type GFP-SPRIEITPS-19 fusion protein (variable amounts, supplemented with sufficient plasmid encoding GFP to bring the total plasmid DNA to 9 &mgr;g), or mutant GFP-SPAIAIAPS-19 fusion protein, together with an NFAT-driven luciferase reporter plasmid (2 &mgr;g). Aliquots of the transfected cells were stimulated 24 h after transfection with PMA (20 nM) and ionomycin (1 &mgr;M) for 6 hours and luciferase activity in cell lysates was measured. The results showed that GFP-SPRIEITPS-19 inhibited NFAT-mediated transcription in a concentration-dependent manner, up to greater than 60% inhibition. The mutant GFP-SPAIAIAPS-19 protein was only slightly inhibitory (−10%), and only at high concentrations. Western blotting analysis with anti-GFP antibody confirmed that the GFP proteins were expressed at equivalent levels, and that the amount of protein expressed was proportional to the amount of plasmid DNA transfected. The observation that the GFP-SPRIEITPS-19 fusion protein did not completely inhibit NFAT-driven transcription is consistent with the results described above, which showed that the inhibitory effect of the protein was balanced by the strength of the stimulation conditions, and consistent with the level of intracellular expression of the GFP-fusion protein.

[0283] In sum, these results showed that a peptide based on a calcineurin targeting motif of NFAT1 was able to inhibit NFAT1 activation and function in vivo.

Example 7 High-Throughput Screen for Inhibitors of Protein-Protein Interaction Between Calcineurin and NFAT (Using a Washing Step)

[0284] This example illustrates a high-throughput screen for inhibitors of protein-protein interaction between calcineurin and NFAT, which utilizes a washing step.

[0285] A fusion protein between glutathione S-transferase and NFAT1, GST-NFAT1(1-400) (Luo et al., Proc Natl Acad Sci USA 93:8907-8912 (1996)), is immobilized on glutathione-Sepharose beads (obtained from Pharmacia Biotech, Piscataway, N.J.) by incubation for 30 min at 4° C. in binding buffer (50 mM Tris phosphate pH 8.0, 150 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol, 1% Triton X-100, supplemented with 1 mM Na orthovanadate, 20 &mgr;M leupeptin, 10 &mgr;g/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride). For parallel incubations to monitor nonspecific binding (Luo et al., Proc Natl Acad Sci USA 93:8907-8912 (1996)), equivalent amounts of GST or GST-LSF, where LSF (Shirra et al., Mol Cell Biol 14:5076-5087 (1994)) is a DNA-binding protein unrelated to NFAT, are immobilized on glutathione-Sepharose beads. The beads are washed, resuspended in binding buffer, distributed to multiple wells, and the candidate agents are added. The agents may be supplied as a natural products library, e.g., microbial broths or extracts from diverse stains of bacteria, fungi, and actinomycetes (MDS Panlabs, Bothell, Wash.); a combinatorial chemical library, e.g., an Optiverse™ Screening Library (MDS Panlabs, Bothell, Wash.); an encoded combinatorial chemical library synthesized using ECLiPS™ technology (Pharmacopeia, Princeton, N.J.); or another organical chemical, combinatorial chemical, or natural products library assembled according to methods known to those skilled in the art and formatted for high-throughput screening. Total binding is measured in some samples in the absence of an added candidate agent. As a positive control for inhibition in the assay, binding is assessed in the presence of an effective concentration (200 &mgr;M) of a 13-mer or 26-mer peptide of the conserved regulatory domain of NFAT protein described herein which is capable of inhibiting protein-protein interaction between calcineurin and NFAT. The specificity of this positive control inhibition is monitored in incubations with similar concentrations of a mutant peptide described herein which is inactive in inhibiting such interaction. Each reaction is supplemented with 100 &mgr;M CaCl2, 800 nM calmodulin (Sigma, St. Louis, Mo.)(to confer calcium responsiveness on calcineurin) and 14 nM 125I-calcineurin (final concentrations). The incubation is carried out for 30 min at 4° C. Iodinated calcineurin is prepared by the following procedure. Calcineurin (100 &mgr;g) in 10 mM Tris phosphate pH 8.0, 120 mM NaCl, 0.1 mM EGTA, 5 mM MgCl2, is iodinated by reaction with 1 mCi carrier-free Na 125I (NEN Life Science Products, Boston, Mass.) in the presence of IODO-BEADS™ (Pierce, Rockford, Ill.) for 15 min at 4° C. The radiolabeled protein is separated from free Na125I on a spin column (BioRad Bio-spin 6; Bio-Rad Laboratories, Hercules, Calif.) and stored in aliquots at −80° C. until use.

[0286] After incubation, binding reactions are filtered through a 5 &mgr;m filter (hydrophilic Durapore PVDF filter; Millipore Corp., Bedford, Mass.) adapted to a multiwell format, to separate bound and unbound 125I-calcineurin. The filters are rapidly washed under continuous vacuum with 50 mM HEPES pH 7.0, 150 mM NaCl, 5 mM MgCl2, 200 &mgr;M CaCl2, 10% glycerol, 1% Triton X-100. The 125I-calcineurin retained by the immobilized GST fusion proteins is quantified by scintillation counting using a gamma counter. A candidate agent is scored as positive if it reduces the protein-protein interaction of calcineurin and NFAT as assessed by radiolabel bound in the assay.

[0287] Those skilled in the art will be aware of many alternative ways to carry out an equivalent assay. For example, the reactions can be suitably modified to use any of the proteins, protein fragments, peptides, or analogues described herein as materials for the assays. The roles of the proteins can be reversed, so that calcineurin is immobilized and NFAT is the radiolabeled compound. The reactions can be carried out with other solid supports or as a solution assay. Those skilled in the art will know that the optimal conditions of incubation and washing may change in such modified assays, and that small adjustments of the conditions may be necessary, including, e.g., changes in the concentrations of proteins, the temperature, salts, pH, or inclusion of additional inhibitors of peptidases and phosphatases in the incubation buffer. Likewise, use of other resins or solid supports may require the inclusion in the incubation of substances to block nonspecific binding to these materials.

[0288] This method of screening is capable of identifying organic molecule inhibitors, e.g., that bind to calcineurin and thereby inhibit the protein-protein interaction of calcineurin and NFAT. Skilled practitioners will also appreciate that this method, and other methods described herein (e.g., the method described in Example 8, below), can be used to identify inhibitors that bind to NFAT, instead of or in addition to calcineurin, and that such organic molecules can be used to inhibit the protein-protein interaction of calcineurin and NFAT.

Example 8 High-Throughput Screen for Inhibitors of Protein-Protein Interaction Between Calcineurin and NFAT (Without a Washing Step)

[0289] This example describes a high-throughput screen for inhibitors of protein-protein interaction between calcineurin and NFAT using a scintillation proximity assay.

[0290] Recombinant NFAT1 is immobilized on SPA beads (obtained from Amersham, Arlington Heights, Ill.)—e.g., by binding influenza haemagglutinin-tagged NFAT1 to a mouse monoclonal antibody directed against the haemagglutinin epitope, and thereby to SPA beads derivatized with an anti-mouse antibody (Amersham, Arlington Heights, Ill.); or by binding biotinylated NFAT1 to streptavidin SPA beads (Amersham)—and the beads are washed in the binding buffer described in Example 7 and distributed to replicate wells. Candidate agents are added as described in Example 7, and the reactions for total binding and for inhibition using the 13-mer or 26-mer peptide are constituted as described in Example 7. Each reaction is supplemented with 100 &mgr;M CaCl2, 800 nM calmodulin, and 14 nM 125I-calcineurin (final concentrations), and the incubation is carried out for 30 min at 4° C. Bound radioactivity is quantitated directly in the multiwell plate by scintillation counting. A candidate agent is scored as positive if it reduces the protein-protein interaction of calcineurin and NFAT as assessed by radiolabel bound in the assay.

Example 9 High-Throughput Screen for Inhibitors of Dephosphorylation of NFAT by Calcineurin

[0291] This example illustrates a high-throughput screen for inhibitors of dephosphorylation of NFAT by calcineurin.

[0292] Hexahistidine-tagged human NFAT1(1-415) is purified from bacterial lysates by incubation with Ni2+-NTA-agarose in 50 mM Tris pH 8.0, 150 mM NaCl, for 30 min at 4° C. For in vitro labeling with 32P, NFAT bound on the agarose beads is incubated in 20 mM HEPES pH 7.5, 20 mM MgCl2, 20 &mgr;M unlabelled ATP with the addition of 0.17 mCi/ml &ggr;-32P-ATP (NEN Life Science Products, Boston, Mass.) and 1700 units/ml of the MAP kinase ERK2 (New England Biolabs, Beverly, Mass.). After 20 min at 30° C., the beads are thoroughly washed to remove the kinase and unincorporated radiolabel, resuspended in phosphatase buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 2 mM MnCl2, 2 mM CaCl2, 15 mM 2-mercaptoethanol), and distributed to multiple wells. Candidate agents, or phosphatase buffer or diluent only, are added to individual reactions. The inhibitory 13-mer peptide or 26-mer peptide described herein is added to individual reactions in a range of final concentrations (100 &mgr;M to 2 mM) to serve as positive controls for inhibition. Each reaction is brought to a final volume of 30 &mgr;l with the addition of 150 nM calcineurin (Sigma Chemical Co., St. Louis, Mo.) and 500 nM calmodulin (Sigma), and incubated 20 min at 30° C. The supernatant is collected by filtration into a multiwell plate, and released 32P-phosphate is determined by scintillation counting.

[0293] Other formats for the assay involve different methods of separating free phosphate from phosphate covalently bound to protein, utilization of a variety of NFAT substrates, or utilization of NFAT purified from cell extracts. Likewise, radiolabel remaining bound to protein may be measured rather than measuring the radiolabel released. In some embodiments, a chromogenic assay for free phosphate (EnzChek Phosphate Assay Kit; Molecular Probes, Eugene, Oreg.) may be substituted for the radioactive assay, avoiding the use of radioactivity and the need for separation of free phosphate from protein after the incubation with calcineurin.

[0294] Other protein kinases are also suitable for preparation of 32P-labelled NFAT if they incorporate phosphate at sites that are targets for dephosphorylation by calcineurin, and if dephosphorylation of those sites by calcineurin is inhibited by the 13-mer or 26-mer inhibitory peptides described herein as is characteristic of the physiological dephosphorylation by calcineurin. In using other protein kinases, the particular conditions of the labeling reaction will depend on the optimal conditions for enzymatic activity of the kinase used. Likewise, for an optimal assay with different preparations of calcineurin, the concentration of calcineurin or of buffer components such as divalent ions, or reaction time or temperature, may require adjustments that can be determined by routine experimentation.

Example 10 Detection of NFAT Dephosphorylation by Calcineurin Using Antibodies to a Dephosphorylated NFAT Peptide

[0295] Phosphorylated NFAT1 or HA-tagged phosphorylated NFAT1 in dephosphorylation buffer (80 &mgr;l 100 mM HEPES pH 7.4, 100 mM NaCl, 20 mM potassium acetate, 2 mM magnesium acetate, 2 mM dithiothreitol, 0.1 mg/ml bovine serum albumin) is distributed, 0.3 ng/well, to the wells of a multiwell plate. To individual wells is added 20 &mgr;l of dephosphorylation buffer alone, of buffer containing a compound to be tested, or of buffer containing 13-mer or 26-mer inhibitory peptide (1-500 &mgr;M). Each reaction is brought to a final volume of 120 &mgr;l and a final concentration of 1 mM CaCl2, 150 nM calcineurin (Sigma Chemical Co., St. Louis, Mo.) and 500 nM calmodulin (Sigma Chemical Co., St. Louis, Mo.), and incubated 20 min at 30° C. The reaction is stopped by the addition of the calcineurin inhibitors EGTA and sodium pyrophosphate to concentrations of 5 mM and 30 mM, respectively. The contents of each well are transferred to a second multiwell plate coated for an ELISA with anti-dephosphopeptide antibody, and dephosphorylated NFAT is allowed to bind for 3 h at 20° C. The wells are washed three times with phosphate-buffered saline pH 7; incubated with alkaline phosphatase-labeled anti-67.1 antibody (Ho et al., J Biol Chem 269:28181-28186 (1994)) or antiHA tag antibody, as appropriate, 1 h at 20° C.; and again washed three times with phosphate-buffered saline pH 7. Reaction buffer containing the substrate p-nitrophenyl phosphate is added, the alkaline phosphatase reaction is allowed to proceed at 20° C. until color develops in the control samples that were dephosphorylated by calcineurin in the absence of an inhibitor, the reaction is stopped with 3 N NaOH, and absorbance is read at 405 nm. Compounds that inhibit the calcineurin-NFAT interaction are detected by a decreased absorbance, with a threshold decrease for example of 30%.

Example 11 Detection of the Calcineurin-Dependent Change in Intracellular Localization of NFAT1 Using an Antibody

[0296] Cells expressing NFAT1, e.g. PC12 cells, are plated in L15CO2 medium (Nardone et al., Proc Natl Acad Sci USA 91:4412-4416 (1994)) into replicate wells coated with poly-D-lysine in a 96-well plate. For optimal visualization the cells are allowed to attach to the substrate overnight. Individual wells are preincubated 20 min at 37° C. with medium alone, with medium containing a compound to be tested, or with medium containing known inhibitors of NFAT activation; and then further incubated 20 min at 37° C. with stimulus, e.g. ionomycin 20 &mgr;M for PC12 cells, in the continuing presence of test compound or inhibitor in those wells where a test compound or inhibitor is used. The assay is terminated by removal of medium and addition of fixative, 4% paraformaldehyde in 0.12 M phosphate buffer, and fixation is allowed to proceed 30 min at room temperature. The wells are washed 4 times with phosphate-buffered saline, and the fixed cells are permeabilized and preblocked for 30 min at room temperature with phosphate-buffered saline containing 5% fetal calf serum and 0.3% Triton X-100. The primary antibody incubation for immune staining is with anti-67.1 antiserum (Ho et al., J Biol Chem 269:28181-28186 (1994)), followed by washing and incubation with Cy3™-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa.). After washing to remove unbound second antibody, the samples are examined by fluorescence microscopy to determine the localization of immune staining for NFAT1, and by phase contrast microscopy to visualize cell nuclei and cytoplasm. With adequate stimulation, as verified by examination of those samples incubated with stimulus alone, not more than −1% of cells should have cytoplasmic NFAT1. A tested compound is scored positive if a larger fraction of cells displays predominantly cytoplasmic staining for NFAT1, or if significant numbers of cells display some cytoplasmic retention of NFAT1.

Example 12 Detection of the Calcineurin-Dependent Change in Intracellular Localization of NFAT1 Using an NFAT-GFP Fusion Protein

[0297] HeLa cell line NFAT16, a cell line stably expressing GFP-NFAT1(1-460) under control of the CMV promoter, is plated in a 96-well plate suitable for subsequent fluorescence microscopy, and incubated overnight in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, 10 mM HEPES, 2 mM L-glutamine, and 1 mg/ml neomycin to permit spreading of cells on the substrate for optimal visualization. A preincubation is initiated by withdrawal of the growth medium, and addition of the same medium containing (in individual wells) either a compound to be tested, a known inhibitor of NFAT activation and nuclear translocation (e.g., CsA), or no additive. After a 30 min preincubation, the incubation is supplemented with an additional 3 mM CaCl2 and with ionomycin to 3 &mgr;M final concentration, except that no ionomycin is added to the designated unstimulated control wells. The purpose of the additional Ca2+ is to ensure optimal activation of NFAT in this cell line. The incubation is continued at 37° C. for 10 min. The medium is aspirated, and the cells are fixed by treatment with 3% paraformaldehyde in 0.1 M sodium phosphate, pH 7.4, for 30 min at room temperature. Fixative is removed by washing three times, 5 min each, with phosphate-buffered saline. Localization of NFAT1(1-460)-GFP in the cells is examined by fluorescence microscopy using suitable excitation and emission filters for GFP, and nuclei and cytoplasm are visualized by phase contrast microscopy. Less than 5% of stimulated NFAT16 cells show predominantly cytoplasmic NFAT-GFP fluorescence under the stated conditions. A tested compound is scored positive if a larger fraction of cells, for example more than 10% of cells, displays predominantly cytoplasmic staining for NFAT1.

[0298] Many available cell lines, primary cells, or cells expressing recombinant NFAT display a calcineurin-dependent translocation of NFAT to the cell nucleus. Those skilled in the art will know that for adequate stimulation of different cell types, adjustments are made in the conditions of the assay, e.g., in the stimulus used, the concentration of stimulus, the time of incubation with stimulus, and the addition of CaCl2. In each case, appropriate assay conditions for the cells studied can be determined by routine experimentation.

Example 13 Selection of a Consensus Peptide Sequence that is Characteristic of Peptides that Bind Tightly to the SPRIEITPS-Binding Site of Calcineurin

[0299] This example illustrates the selection from combinatorial peptide libraries of peptide analogues of the SPRIEITPS peptide that display enhanced affinity for calcineurin.

[0300] The basic strategy was to select peptides from combinatorial peptide libraries based on their ability to bind to a GST fusion protein containing the calcineurin catalytic domain. The peptide libraries were biased toward binding at the NFAT recognition site on calcineurin by incorporating a partial consensus sequence. For example, as will be explained in more detail below, the first peptide library incorporated the calcineurin-binding PxIxIT sequence motif that is conserved in the NFAT proteins (FIG. 4A). The combinatorial peptide library method depends on the fact that the presence of certain amino acid residues, at an individual position within the peptide that contacts calcineurin or that influences the peptide conformation necessary for efficient interaction with calcineurin, will confer enhanced binding to the target site, whereas the presence of certain other amino acid residues will impair binding to the site.

[0301] In more detail, combinatorial peptide libraries were synthesized at the Tufts-New England Medical Center Peptide Synthesis Facility using N-&agr;-fluorenyl methoxycarbonyl-protected amino acids and standard BOP/HOBt coupling chemistry as described (M B Yaffe et al (1997) Cell 91, 961-971). GST-calcineurin is a glutathione S-transferase and calcineurin catalytic domain fusion protein, which was expressed from a plasmid derived by cloning a DNA fragment encoding residues 2-347 of human calcineurin A&agr; (GenBank accession number L14778) in-frame at the BamHI site of pGEX-6P vector (Amersham Pharmacia Biotech). The coding sequence of the abbreviated calcineurin cDNA was altered to encode a C-terminal peptide HPSWAPNFD, in place of HPYWLPNFM, in order to enhance stability of the truncated protein. The nucleotide and amino acid sequences of this modified calcineurin catalytic domain are shown in FIGS. 3A and 3B, respectively.

[0302] Library screening was performed using approximately 1 mg of GST-calcineurin fusion protein immobilized on 100 &mgr;l of glutathione-agarose resin in buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2, 300 &mgr;M CaCl2, 100 &mgr;M sodium vanadate, 100 &mgr;M EGTA, 1 mM dithiothreitol, and 1% Triton X-100. After allowing the mixture of peptides to bind to the immobilized protein, the resin was washed twice with buffer and twice with PBS, bound peptides were eluted with 30% acetic acid, and the mixture of eluted peptides was sequenced. An aliquot of the peptide library that had not been selected on the calcineurin affinity resin was also sequenced to provide baseline data on the recovery of individual amino acids at each randomized position. A numerical index of selection (“preference value”) for each amino acid residue at a given randomized position was obtained (M B Yaffe et al (1997) Cell 91, 961-971; Yaffe M B and Cantley L C (2000) Methods Enzymol 328, 157-170) by comparing the relative abundance (mole percent) of that residue at the corresponding sequencing cycle of the selected peptide mixture with the relative abundance at the same sequencing cycle of the unselected library. A preference value greater than 1.0 indicates selection on the calcineurin column, and a preference value of around 2.0 or greater indicates moderately strong to strong selection (M B Yaffe et al (1997) Cell 91, 961-971; Yaffe M B and Cantley L C (2000) Methods Enzymol 328, 157-170).

[0303] FIGS. 4A-4D depict a summary of the evolution of an optimized peptide inhibitor capable of disrupting the NFAT-calcineurin interaction. A first combinatorial peptide library, with the sequence MAxxxPxIxITxxHKK, was targeted to the docking site on calcineurin by incorporation of the sequence PxIxIT (see FIG. 4A) and was randomized at seven positions where the sequence is not fully conserved within the NFAT family (FIG. 4B). In FIG. 4B, residues in the nondegenerate positions of the peptide library are shown in the single-letter code, and the essential targeting residues are boxed. Within the library, in aggregate, the randomized positions denoted “X” contained roughly equimolar amounts of all naturally-occurring amino acid residues except cysteine. This first library was selected for binding to GST-calcineurin, and the data were analyzed as previously described. Amino acid residues selected at the degenerate positions of the first library are shown in FIG. 4B, with residues that were strongly selected presented in bold face type and underlined, and with preference values indicated in parentheses. Thus, in the first library, the peptide pool eluted from the GST-calcineurin column showed moderate selection for glycine, serine, and lysine at position 3; no preferred residues at position 4; selection for histidine or aliphatic residues at position 5; and moderate selection for the polar residues threonine, lysine, glutamine, and glutamate at position 7. At position 9 there was relatively weak selection for aliphatic residues, notably valine. At positions 12 and 13 there was selection for glycine and proline respectively, suggesting that the NFAT binding site in calcineurin imposes a turn at the C-terminal end of the PxIxIT motif.

[0304] To refine the peptide selection further, a second degenerate peptide library was synthesized, in this case targeted to the same binding site on calcineurin by incorporation of an alternative set of fixed residues based on the initial round of library selection, but now randomized at positions that had been fixed previously (FIG. 4C). In FIG. 4C, the new targeting residues are boxed, and the randomized positions are again indicated by “X”. “Z” denotes a mixture of the nonnatural amino acid residues p-fluorophenylalanine, p-chlorophenylalanine, 2-naphthylalanine, tetrahydroisoquinoline-3-carboxylic acid, and cyclohexylalanine. As is evident in this figure, positions 3 and 5 were fixed as glycine and histidine, respectively, while positions 12 and 13 were fixed as glycine and proline to favor the putative turn. Position 7 contained 50% threonine and 50% a mixture of all other residues, to probe whether threonine and polar residues were indeed preferred at this position. Position 8 contained the 19 natural amino acid residues (omitting cysteine) and 5 additional nonnatural amino acid residues with large aromatic or cyclic groups, to determine whether binding affinity could be improved by substitution of a large hydrophobic side chain for the isoleucine side chain that is naturally conserved at this position. Position 10 was fixed as isoleucine, positions 9 and 11 were randomized, and the two C-terminal lysine residues of the first library were replaced by glutamate residues to eliminate any bias that might have been present in the first library because of their positive charge.

[0305] Selection of the second library over the calcineurin affinity matrix yielded strongly preferred residues at most of the randomized positions (FIG. 4C). A striking feature in this round of selection was that bulky or &bgr;-branched hydrophobic residues (valine, isoleucine, leucine) were highly preferred at positions 7 and 9 for binding to the target site. Proline was preferred at position 4, echoing its occurrence at this position in NFAT1, although not in NFAT2-NFAT4. Isoleucine was stringently preferred at position 8, with lesser selection for other hydrophobic residues and no selection for the nonnatural amino acid residues, consistent with the invariance of isoleucine at this position of the PxIxIT sequence in all four NFAT proteins. Finally, there was strong selection for the conserved threonine of the PxIxIT sequence at position 11, with a weaker preference for serine. The findings can be summarized in the sequence of a consensus peptide, MAGPHPVIVITGPHEE (FIG. 4D).

[0306] These results identified a consensus peptide sequence, containing the PxIxIT motif, that is characteristic of peptides that bind tightly to the SPRIEITPS-binding site of calcineurin.

Example 14 VIVIT Peptide Strongly Inhibits Dephosphorylation of NFAT, but does not Inhibit Calcineurin Enzyme Activity

[0307] This example illustrates that a synthetic peptide with the consensus sequence determined in Example 13, MAGPHPVIVITGPHEE, is an effective inhibitor of NFAT recognition and dephosphorylation, and like SPRIEITPS does not interfere with calcineurin enzymatic activity generally.

[0308] Based on the results described in Example 13, a predicted optimal peptide was synthesized, with the amino acid sequence MAGPHPVIVITGPHEE, and its effect on the interaction of calcineurin with NFAT was examined in biochemical assays. This peptide is referred to hereinafter as “VIVIT 16-mer peptide”, and this peptide and other peptides based on the consensus or alternative consensus residues determined in Example 13 are generically referred to as “VIVIT” peptides.

[0309] FIGS. 5A-5C illustrate that the optimised VIVIT peptide is a potent inhibitor of the interaction between NFAT and calcineurin. In FIG. 5A, the protein-protein interaction between calcineurin and NFAT was assayed by incubating 200 nM calcineurin that had been activated with 600 nM calmodulin and 2 mM CaCl2 with 3 &mgr;g GST or GST-NFAT1(1-415) as described (F J Garcia-Cozar et al (1998) J Biol Chem 273, 23877-23883). NFAT1-bound calcineurin was collected by incubation with glutathione-Sepharose resin and detected by Western blotting with a polyclonal antibody to the calcineurin A chain (C Loh et al (1996) J Biol Chem 271, 10884-10891). The peptides SPRIEIT-13 or SPAIAIA-25 (J Aramburu et al (1998) Mol Cell 1, 627-637) or the VIVIT 16-mer peptide were included in the assay where indicated, in the amounts indicated above each lane (in &mgr;M).

[0310] The assay demonstrated specific calcineurin binding to GST-NFAT1, but not to GST (FIG. 5A, compare lane 2 with lane 1, respectively). Both the SPRIEITPS peptide (lanes 3-6), which recapitulates the docking sequence in NFAT1, and the VIVIT 16-mer peptide (lanes 7-10) inhibited the binding of activated calcineurin to GST-NFAT1. The mutant peptide SPAIAIA did not inhibit calcineurin binding to GST-NFAT1 when tested at 100 &mgr;M (compare lane 11 with lane 2).

[0311] The VIVIT 16-mer peptide also inhibited the calcineurin-mediated dephosphorylation of NFAT (FIG. 5B). Lysates of HeLa cells stably expressing HA-NFAT1(1-460)-GFP (hereinafter “HA-NFAT1”) were incubated with 200 nM calcineurin plus 600 nM calmodulin and 2 mM CaCl2 for 30 min at 30 C. in buffer as described (J Aramburu et al (1998) Mol Cell 1, 627-637). The phosphorylation status of NFAT was evaluated by SDS-polyacrylamide gel electrophoresis and Western blotting with anti-HA antibody 12CA5. For these experiments, reaction conditions were chosen that produced only limited dephosphorylation of NFAT (FIG. 5B, lane 2) in order to maximize the sensitivity of the assay in detecting inhibition. The SPRIEIT-13 (lanes 3-6), VIVIT 16-mer (lanes 7-9), and SPAIAIA-25 (lane 10) peptides were again included in the assay where indicated, in the amounts indicated above each lane (in &mgr;M). To demonstrate the effect of completely inhibiting calcineurin phosphatase activity, the general phosphatase inhibitor sodium pyrophosphate (NaPPI) was used at 10 mM (lane 1). Both the VIVIT peptide and the SPRIEITPS peptide were effective at inhibiting NFAT dephosphorylation.

[0312] Inhibition of NFAT dephosphorylation by VIVIT peptide was not due to occlusion of the catalytic site of calcineurin. This point had previously been established for the SPRIEITPS peptide (see Example 4), and was tested here by measuring calcineurin phosphatase activity with 32P-labelled phospho-RII peptide as substrate (J Aramburu et al (1998) Mol Cell 1, 627-637). SPRIEIT-13, SPAIAIA-25, and VIVIT 16-mer peptides were included in the incubation as indicated. The results are plotted in FIG. 5C as radiolabel released from 32P-phospho-RII peptide (counts per minute×10−3). Where peptide was present, its concentration (&mgr;M) is indicated in FIG. 5C; CsA/cyclophilin A complexes (CsA/Cyp) were present at 10 &mgr;M. Even at 100 &mgr;M, a concentration that completely blocks calcineurin-NFAT1 binding and NFAT1 dephosphorylation (FIGS. 5A and 5B), the VIVIT peptide did not inhibit calcineurin phosphatase activity towards the RII phosphopeptide (FIG. 5C). In contrast, CsA/cyclophilin A complexes inhibited calcineurin phosphatase activity by approximately 95% in this assay, consistent with the established mode of action of CsA/cyclophilin A complexes, which is interference with calcineurin activity against all its peptide and protein substrates. These results showed that VIVIT peptide is a selective inhibitor, and blocks the dephosphorylation of NFAT but not of RII phosphopeptide.

Example 15 VIVIT Peptide Selectively Inhibits NFAT-Dependent Expression of Reporter Genes

[0313] This example illustrates that expression of a fusion protein including the VIVIT 16-mer peptide selectively inhibits NFAT-mediated reporter gene expression, but not NF-&kgr;B mediated reporter gene expression, in T lymphocytes.

[0314] The general strategy in this Example was to examine transcriptional signalling in cells having effective concentrations of VIVIT peptide in the cell cytoplasm. Since the VIVIT peptide itself is not cell permeant, it was delivered into cells by expression of a GFP-VIVIT fusion protein. Luciferase reporter plasmids were cotransfected into the same cells, and the effects of VIVIT peptide on NFAT-dependent and NF-&kgr;B-dependent transcription were examined after appropriate stimulation of the cells.

[0315] In more detail, Jurkat cells were transfected (J Aramburu et al (1998) Mol Cell 1, 627-637; C Luo et al (1996) Mol Cell Biol 16, 3955-3966) with 0.25 &mgr;g per 106 cells luciferase reporter plasmid; with 0.25 &mgr;g per 106 cells expression plasmid for NFAT1, NFAT2, or NFAT4; with 40 ng per 106 cells human growth hormone (hGH) expression plasmid as an internal reference for transfection efficiency; and with 0.5 &mgr;g per 10 cells, or other indicated amount, GFP, GFP-SPRIEIT, or GFP-VIVIT expression plasmids. The reporter plasmids contained three tandem copies of the distal NFAT-AP 1 site of the murine IL-2 promoter (3×NFAT-Luc), two copies of a consensus NF-&kgr;B site (2×NF-&kgr;B-Luc), the human IL-2 promoter, or the human TNF&agr; promoter (C Luo et al (1996) Mol Cell Biol 16, 3955-3966; F Mercurio et al (1997) Science 278, 860-866). The GFP expression vector was pEGFP-N1 (Clontech). The GFP-SPRIEIT expression vector was previously described (J Aramburu et al (1998) Mol Cell 1, 627-637), and the GFP-VIVIT expression vector was constructed by subcloning an oligonucleotide encoding the sequence MAGPHPVIVITGPHEE in-frame with green fluorescent protein (GFP) at the N-terminus of GFP. Twenty-four hours after transfection, cells were left unstimulated, stimulated for 6 hours with 20 nM phorbol 12-myristate 13-acetate (PMA) and 1 &mgr;M ionomycin, or stimulated for 6 hours with 0.2 &mgr;g/ml immobilized anti-CD3 (HIT3a, Pharmingen) and 0.5 &mgr;g/ml soluble anti-CD28 (CD28.2, Pharmingen). CsA was added 30 min before the stimuli. Luciferase activity in cell lysates was measured using a luciferase assay system (Promega) and was normalized to the concentration of hGH in the cell culture medium determined with a radioimmunometric assay kit (Hybritech).

[0316] FIGS. 6A-6D are bar graphs that generally illustrate that VIVIT peptide is a selective and potent inhibitor of NFAT activity, and that it does not substantially affect calcineurin activity or other calcineurin-dependent processes. FIG. 6A illustrates that VIVIT peptide inhibits NFAT-dependent gene expression. In the left panel panel of FIG. 6A are graphed data from Jurkat cells that had been cotransfected with a 3×NFAT-Luc reporter plasmid and with expression plasmids encoding murine NFAT1, GFP, GFP-SPRIEIT, or GFP-VIVIT as indicated. In the right panel are data from Jurkat cells that had been cotransfected with a 3×NFAT-Luc reporter plasmid and with expression plasmids encoding murine NFAT1, human NFAT2, or human NFAT4 as indicated. Twenty-four hours after transfection, cells were left unstimulated (Unstimulated; open bars) or were stimulated for 6 hours with PMA and ionomycin (PMA+iono; solid bars), cell lysates were prepared, and luciferase activity attributable to endogenous NFAT (Endog.) or to overexpressed NFAT proteins was measured.

[0317] Little luciferase was detected in lysates from unstimulated cells. As shown in the left panel of FIG. 6A, stimulation with PMA and ionomycin induced luciferase through activation of endogenous NFAT, and the induced expression was partially blocked by SPRIEIT and almost completely blocked by VIVIT. Even when NFAT1, NFAT2, or NFAT4 was overexpressed in the cells (right panel of FIG. 6A), VIVIT efficiently inhibited luciferase expression. Since luciferase induction is largely attributable to an individual NFAT protein in the latter cases, this test provides clear confirmation that the cellular action of VIVIT is on the calcineurin-NFAT pathway.

[0318] FIG. 6B illustrates that delivery of increasing concentrations of the GFP-VIVIT expression plasmid inhibited activation of an NFAT reporter but not activation of an NF-KB reporter. Jurkat cells were cotransfected with 3×NFAT-Luc (left panel) or with 2×NF-&kgr;B-Luc (right panel) reporter plasmid, and with the indicated amounts (&mgr;g plasmid per 106 cells) of GFP and GFP-VIVIT expression plasmids, the total amount of plasmid DNA added being kept constant. Twenty-four hours after transfection, cells were left untreated (open bars) or were stimulated for 6 hours with PMA and ionomycin (solid bars).

[0319] These data show that basal levels of luciferase were unaffected by VIVIT peptide, but that stimulated expression of the NFAT reporter gene was blocked by VIVIT. The maximal effect of GFP-VIVIT on NFAT-dependent reporter activity was as great as that of CsA, inhibiting reporter activity to the level observed in unstimulated cells. On the other hand, stimulated expression of NF-&kgr;B reporter was unaffected by VIVIT.

[0320] The finding that activation of an NF-&kgr;B reporter is not affected by VIVIT is of special interest, because NF-&kgr;B activation in T cells is calcineurin-dependent and is sensitive to FK506 and CsA (B Frantz et al (1994) EMBO J 13, 861-870; K Kalli et al (1998) Mol Cell Biol 18, 3140-3148). The calcineurin dependence of reporter activity was verified in Jurkat T cells transfected with 3×NFAT-Luc and 2×NF-&kgr;B-Luc reporter plasmids (FIG. 6C, left and right panels, respectively). Cells were untreated, or were stimulated with PMA and ionomycin (P+I), in the absence or presence of CsA at the concentrations indicated. CsA in fact produced a concentration-dependent inhibition of NF-&kgr;B reporter gene expression, with IC50 less than 5 nM and maximal inhibition at 100 nM. This set of experiments indicates that calcineurin-NFKB signalling is fully functional in cells expressing VIVIT, and that VIVIT has a more selective action than CsA, discriminating between transcriptional signalling pathways downstream of calcineurin.

[0321] FIG. 6D illustrates that VIVIT peptide inhibited NFAT-dependent activation of the IL-2 and TNF&agr; promoters (FIG. 6D). Jurkat cells were cotransfected with GFP or GFP-VIVIT expression plasmid and with a luciferase reporter plasmid driven either by the human IL-2 promoter (left panel) or by the human TNF&agr; promoter (right panel). Cells were unstimulated (open bars), stimulated with PMA and ionomycin (solid bars), or stimulated with anti-CD3 and anti-CD28 (hatched bars). GFP-VIVIT, but not GFP, efficiently inhibited gene expression driven by the IL-2 and TNF&agr; promoters in T cells stimulated with PMA and ionomycin or with anti-CD3 plus anti-CD28. These results showed that VIVIT peptide inhibits calcineurin-NFAT transcriptional signalling in T cells, and moreover that VIVIT peptide is a more selective inhibitor than CsA, since it does not inhibit calcineurin-NF-&kgr;B transcriptional signalling in T cells.

Example 16 VIVIT Peptide Inhibits Expression of NFAT-Dependent, but not NFAT-Independent, Cyclosporin-Sensitive Genes

[0322] This example illustrates that expression of a fusion protein including the VIVIT 16-mer peptide selectively inhibits NFAT-mediated expression of endogenous cytokine genes in T lymphocytes.

[0323] Studying the effect of VIVIT peptide on endogenous gene expression presents the challenge that the cell population examined must uniformly express VIVIT. Thus, to determine whether the VIVIT peptide inhibits expression of endogenous NFAT-dependent genes, Jurkat T cells were cotransfected with GFP or GFP-VIVIT expression plasmids, together with an expression plasmid encoding a selectable cell surface marker (murine CD4) that allowed isolation of a homogeneous population of transfected cells. The purified cells were then stimulated, and the induced expression of multiple cytokine mRNAs was analyzed by multiprobe RNase protection assay.

[0324] In a more detail, human Jurkat T cells were cotransfected with 0.75 &mgr;g per 106 cells murine CD4 expression plasmid (mCD4/mL3T4, kind gift of Dr Dan R Littman, New York University) and 0.75 &mgr;g per 106 cells GFP or GFP-VIVIT expression plasmid. Cells expressing mCD4 were selected using magnetic beads (Dynabeads L3T4, Dynal Biotech) as described (A Kiani et al (1997) Immunity 7, 849-860). Magnetic bead selection of mCD4-expressing cells yielded a population that was greater than 90% GFP-positive, whereas mCD4-nonexpressing cells were less than 5% GFP-positive, indicating that the selection isolated the desired population of cells expressing GFP or GFP-VIVIT. RNA was prepared from the selected cells, and multiprobe RNase protection assays were performed using RiboQuant multiprobe kits (Pharmingen, San Diego, Calif.) as described (A Kiani et al (1997) Immunity 7, 849-860). The results are shown in FIGS. 7A to 7C.

[0325] FIGS. 7A-7C are pictures of autoradiograms of RNA gels generally illustrating that VIVIT peptide blocks NFAT-dependent expression of endogenous cytokine genes. FIGS. 7A-7C show protected segments of complementary RNA probes, which indicate the presence and level of specific cytokine mRNAs in the corresponding samples. In FIG. 7A, lanes 1-4 represent Jurkat T cells that had not been transfected with expression plasmids. For example, FIG. 7A shows that IL-2 and IL-13 mRNAs were undetectable in resting cells (lane 1) and were induced in cells stimulated with PMA and ionomycin (lane 2). The mRNAs were undetectable in resting cells treated with CsA (lane 3), and their induction was prevented in the presence of CsA (lane 4). Cells transfected with mCD4 expression vector only, and selected using magnetic beads, did not behave differently than the untransfected cells with regard to expression of these cytokines (FIG. 7A, compare lanes 9 and 10 with lanes 1 and 2). Thus, the presence of the cell surface protein mCD4 and its interaction with anti-mCD4 on the magnetic beads did not in itself have a stimulatory or inhibitory influence on cytokine gene expression. Similarly, cells expressing unmodified GFP protein (together with mCD4) did not behave differently than cells without GFP (FIG. 7A, compare lanes 5 and 6 with lanes 9 and 10), or than Jurkat cells expressing no exogenous protein (FIG. 7A, compare lanes 5 and 6 with lanes 1 and 2). In contrast, cells expressing GFP-VIVIT displayed much-attenuated induction of IL-2 and IL-13 mRNAs (FIG. 7A, compare lanes 7 and 8 with lanes 5 and 6). Levels of mRNA from the housekeeping genes L32 and GAPDH, included as a control for sample loading, showed little variation from lane to lane.

[0326] FIG. 7B illustrates a similar analysis for the mRNAs encoding IL-3 (which was also probed in the experiment of FIG. 7A), TNF&agr;, GM-CSF, and MIP-1&agr;. The untransfected cell controls are not shown for this experiment. The cytokine mRNAs probed were not detected in unstimulated cells expressing mCD4 and selected with magnetic beads, but were induced by treatment with PMA and ionomycin (FIG. 7B, lanes 5 and 6). Expression of unmodified GFP had no effect on induction of the mRNAs (lanes 1 and 2), but expression of GFP-VIVIT was strongly inhibitory (lanes 3 and 4).

[0327] In FIG. 7C, the first set of control cells had been transfected with the empty expression vectors (FIG. 7C, lanes 1-4). The mRNAs examined, TNF&bgr;, LT-&bgr;, and TNF&agr; mRNAs, were induced by treatment with PMA and ionomycin (lanes 1 and 2), and their induction was largely or completely blocked by CsA (lanes 3 and 4). Basal and induced mRNA levels were unaffected by GFP (lanes 5 and 6). TNF&agr; mRNA induction was blocked by GFP-VIVIT, as it was in the experiment of FIG. 7B, but TNF&bgr; and lymphotoxin-&bgr; (LT-&bgr;) mRNAs were insensitive to GFP-VIVIT (lanes 7 and 8).

[0328] Thus the VIVIT peptide inhibited inducible expression of IL-2, IL-13, IL-3, TNF&agr;, GM-CSF, and MIP-1&agr; mRNAs in Jurkat T cells, consistent with published work indicating the presence of functional NFAT sites in the promoter/enhancer regions of these genes (see A Rao et al (1997) Annu Rev Immunol 15, 707-747; G R Crabtree (1999) Cell 96, 611-614). In contrast, the expression of TNF&bgr; and lymphotoxin-&bgr;, although sensitive to cyclosporin A, was unaffected by VIVIT peptide. This latter result, demonstrating signalling that is inhibited by CsA and not inhibited by VIVIT, is most easily understood as reflecting calcineurin transcriptional signalling pathway(s) that are not routed through NFAT.

[0329] These results show that VIVIT peptide inhibits transcriptional signalling through the calcineurin-NFAT pathway, but not through other calcineurin-dependent pathways, in T cells.

Example 17 High-Throughput Screening Assay for Inhibitors of the Protein-Peptide Interaction Between Calcineurin and VIVIT Peptide

[0330] This example illustrates the validation of a high-throughput screening assay for compounds that inhibit binding of the optimized VIVIT peptide to calcineurin.

[0331] The principle of the assay is to assess binding to calcineurin of a VIVIT peptide, covalently labelled with the fluorophore Oregon Green™ 488, by measuring the polarization of Oregon Green fluorescence. The fluorescence lifetime of Oregon Green is ˜4 ns, and this label is therefore well suited (see, for example, C R Cantor and P R Schimmel (1980) Biophysical Chemistry, Part II: Techniques for the study of biological structure and function, pp. 454-465, W. H. Freeman and Company, San Francisco, 1980) to discriminate between rotational movements of the free VIVIT peptide and the peptide bound to a recombinant calcineurin with MW ˜40 kDa.

[0332] The reagents utilized for the assay were a recombinant human calcineurin catalytic domain and an Oregon Green-labelled VIVIT peptide. Recombinant calcineurin was expressed in E. coli strain DH5&agr; as a glutathione S-transferase-calcineurin fusion protein, using an expression construct prepared by subcloning the cDNA encoding a modified human calcineurin(2-347) (see FIGS. 3A-3B) into pGEX-6P. The calcineurin cDNA coding sequence was altered to produce three amino substitutions (Y341 S, L343A, and M347D) near the carboxy terminus of the encoded protein in order to enhance the stability of the truncated protein. The GST-calcineurin fusion protein was purified from bacterial lysates by batch affinity chromatography on glutathione-Sepharose resin, eluted, and dialyzed to remove low molecular weight contaminants. Calcineurin(2-347) was cleaved from the fusion protein using PreScission™ protease (Amersham Pharmacia Biotech) under conditions specified by the supplier, and repurified on glutathione-Sepharose resin to remove GST and PreScission protease. The resulting human calcineurin catalytic domain had the sequence, not including an initial four residues encoded by the pGEX linker, shown in FIG. 3B. Unlabelled VIVIT 14mer peptide (GPHPVIVITGPHEE-amide) was synthesized at Tufts-New England Medical Center Peptide Synthesis Facility, purified by reversed phase HPLC, and the identity of the purified peptide verified by mass spectrometry. VIVIT peptide (2 mg) was combined with 1.5 mg Oregon Green™ 488 carboxylic acid, succinimidyl ester 5-isomer (Molecular Probes) and 51 &mgr;l N,N-diisopropylethylamine (Aldrich) in 190 &mgr;l anhydrous N,N-dimethylformamide (Aldrich), and incubated at room temperature for 18 h in the dark with occasional mixing. The labelled peptide, denoted OG-VIVIT, was purified by C18 reversed phase HPLC and its identity verified by mass spectrometry.

[0333] Saturable binding of OG-VIVIT to calcineurin was demonstrated by addition of increasing concentrations of calcineurin to an assay solution consisting of 30 nM OG-VIVIT in phosphate-buffered saline (PBS) containing 0.1% bovine gamma globulin (BGG) (Sigma). Triplicate samples for each concentration of calcineurin were transferred, 10 &mgr;l per well, to the wells of a black 384-well plate (LJL) and fluorescence polarization determined employing an Analyst plate reader (Molecular Devices) using the fluorescein filter set. Results are expressed in millipolarization units (mP), where polarization (P) is given, in terms of measured fluorescence intensities polarized parallel (Iparallel) and perpendicular (Iperpendicular) to the exciting light, by the standard definition:

P=(Iparallel−perpendicular)/(parallel+Iperpendicular).

[0334] The typical polarization in this assay was 35-45 mP for free peptide, and ˜200 mP for peptide bound to calcineurin. The data of FIG. 8A indicate a saturable protein-peptide interaction with Kd ˜0.5 &mgr;M.

[0335] The ability of an unlabelled competitor to displace OG-VIVIT from calcineurin was demonstrated by addition of increasing concentrations of unlabelled VIVIT peptide in an assay mixture consisting of 30 nM OG-VIVIT and 0.5 &mgr;M or 1 &mgr;M calcineurin(2-347) in PBS/0.1% BGG. Samples were analyzed as described in the preceding paragraph, and the resulting polarization data were plotted against the concentration of unlabelled VIVIT peptide, as shown in FIG. 8B. Because a robust signal for uncompeted binding in this assay requires the use of calcineurin at a concentration that is near or above its Kd for binding fluorescent VIVIT, the IC50 measured in the assay is shifted slightly to higher concentrations than the true Ki. After correcting for this effect, the data were consistent with a Ki for unlabelled peptide binding of 0.5 &mgr;M. The agreement between the Kd value measured for calcineurin in the direct binding assay and the Ki value measured for unlabelled peptide in the competition assay is expected for protein-ligand binding at a single, saturable site.

[0336] The binding assay was reproducible and showed little variation among replicate wells. Assay quality was assessed as signal-to-noise ratio (S:N) and Z′ parameter using the expressions

S:N=(&mgr;b−&mgr;f)/(&sgr;b2+&sgr;f2)0.5

[0337] and

Z′=1−3 (&sgr;b+&sgr;f)/(&mgr;b−&mgr;f),

[0338] respectively (J H Zhang et al (1999) J Biomol Screen 4, 67-73), where &mgr;b and &mgr;f are the mean polarization measured for bound and free peptide, and &sgr;b and &sgr;f are the corresponding standard deviations. Under the stated conditions, the assay had excellent characteristics, as expressed in a signal-to-noise ratio of 18.11 and a Z′ parameter of 0.78.

[0339] These results demonstrate that binding of a fluorescent VIVIT peptide to calcineurin can be detected by measurement of the change in its fluorescence polarization, and that competition by an unlabelled compound included in the assay can be readily detected in a multiwell format suitable for high-throughput screening.

Example 18 Identification of Inhibitors of the Calcineurin-VIVIT Peptide Interaction by High-Throughput Screening of a Diverse Chemical Library

[0340] This example illustrates the implementation of the high-throughput screening assay described in the preceding example to identify low-molecular-weight organic molecules that inhibit the calcineurin-VIVIT peptide interaction.

[0341] The fluorescence polarization assay for inhibitors of calcineurin-VIVIT interaction was used to screen a diverse chemical library of 16320 compounds (DiverSet E library from ChemBridge). The assay mixture, consisting of 30 nM OG-VIVIT and 0.5 &mgr;M calcineurin(2-347) in PBS/0.1% BGG, was distributed in 10 &mgr;l aliquots to the wells of black 384-well assay plates (LJL). In the case of free peptide control samples, calcineurin was omitted. The library stocks were dissolved at a nominal concentration of 10 mM in DMSO, and formatted for convenient robotic transfer on 384-well plates at the Institute of Chemistry and Cell Biology, Harvard Medical School. To initiate the competition assay, 40 nl volumes were pin-transferred from each well of a library stock plate to the corresponding wells of an assay plate, utilizing a robotic device. After a minimum 10 min incubation at room temperature to allow the binding to reach equilibrium, fluorescence polarization data were collected employing an Analyst plate reader (Molecular Devices).

[0342] As a first step in data analysis, library compounds that themselves exhibited substantial fluorescence in the assay were excluded from consideration, since the polarization value obtained from these samples cannot accurately reflect the signal from OG-VIVIT. Library compounds that contributed substantially to the fluorescence signal were identified by plotting a histogram of total sample fluorescence, which is proportional to (Iparallel+2Iperpendicular), where Iparallel and Iperpendicular are the fluorescence intensities parallel and perpendicular to the exciting light, and excluding those compounds whose total sample fluorescence exceeded the 99th percentile of a Gaussian fit to the main peak of OG-VIVIT fluorescence in the histogram of all samples. A total of 13445 of the library compounds met this criterion and were further analyzed.

[0343] The signature of active compounds, i.e., those that displace VIVIT peptide from calcineurin, is a decrease in polarization value, and hence the presence of the corresponding data points in the leftmost tail of the distribution of polarization values from the assay. Those library compounds that had not been excluded by the criterion of total sample fluorescence were ranked on the basis of their raw polarization scores in the assay; on the basis of a percentile score for each sample relative to a Gaussian fitted to a histogram of all samples on the same plate; and on the basis of the projected IC50 of the tested compound computed from its nominal concentration of 40 &mgr;M, the known concentrations of calcineurin and fluorescent VIVIT peptide, and the known Kd of the calcineurin-peptide interaction. For example, 66 compounds (0.4% of the compounds screened) had a projected IC50 below 100 &mgr;M, which is a hit rate comparable to that in other high-throughput screens. A set of 11 library compounds that ranked as best candidate inhibitors in the three ranking methods was selected for more detailed study.

[0344] Stocks of candidate inhibitor compounds and their structural analogues were purchased from ChemBridge or Asinex. The structure and purity of individual compounds were verified by 1H NMR, mass spectrometry, or thin-layer chromatography. The selected compounds were individually tested at a series of concentrations in the fluorescence polarization assay in which the compounds were incubated with 1 &mgr;M calcineurin and 60 nM OG-VIVIT. Each of the 11 compounds produced a concentration-dependent displacement of OG-VIVIT from calcineurin, confirming the results of the high-throughput screen. Illustrative competition data are shown in FIGS. 9A and 9B. The concentrations of test compounds denoted INCA1 and INCA2 in FIG. 9A, and INCA6 in FIG. 9B, are plotted on the horizontal axis, and the polarization signal (mP) from OG-VIVIT is plotted on the vertical axis. Compound NEG2 in FIG. 9B is one of several negative control compounds chosen from the chemical library on the basis of their failure to inhibit calcineurin-peptide interaction in the high-throughput screen. Displacement of OG-VIVIT from calcineurin was complete at high concentrations in the case of INCA1, INCA2, and INCA6, as shown in FIGS. 9A and 9B. With some other competitors the displacement of peptide was incomplete, reaching a plateau at 50-90% displacement. The latter compounds may be intrinsically less effective in displacing the peptide ligand, or may be insoluble in aqueous buffer at the concentrations that would be necessary for full displacement. Because calcineurin concentration in these experiments was not negligible relative to the Kd for OG-VIVIT binding and the probable Ki for some of the inhibitory compounds, the competition experiment was repeated for several inhibitory compounds and their structural analogues with varied calcineurin concentrations, and the data fitted to a three-state equilibrium binding model (in which binding of VIVIT and inhibitor to calcineurin is mutually exclusive) or a four-state model (in which a ternary calcineurin-VIVIT-inhibitor complex is permitted) in order to estimate the Ki for binding of each compound to calcineurin. The precise Ki values estimated will depend on the model used, but the estimates vary within a relatively small range. For example, the Ki values estimated for INCA1 fell between 0.5 &mgr;M and 2 &mgr;M. Compounds whose inhibitory activity was confirmed in the secondary assay were termed “INCA” (inhibitor of NFAT-calcineurin association) coupled with a unique numerical designation for each compound. INCA1, INCA2, and INCA6 can be represented generally as follows:

[0345] INCA 1: 7

[0346] wherein R1 is H, R2 is Ph, R3 is C1, R4 is CHAc2, and R5 is O;

[0347] INCA 2: 8

[0348] wherein R1 is C1, R2 is C1, R3 is 0, R4 is H, and R5 is H; and

[0349] INCA 6: 9

[0350] wherein R1 is O, R2 is 4, R3 is H, R4 is 0, and R5 is H.

[0351] In addition to INCA1, INCA2, and INCA6, several other compounds were isolated using the screen described above, the structures of which are presented in Appendix I. The estimated IC50 and Ki values for INCA1, INCA2, and INCA6, as well the other isolated compounds, are provided in Table 5, below. 9 TABLE 5 Description of Calcineurin-NFAT inhibitors Compound Estimated IC50 (&mgr;M) Ki (&mgr;M) INCA1 1 0.5-2 INCA2 3 0.1-0.5 3 5 ˜20 4 10 Not determined 5 17 ˜20 INCA6 17 0.8-5 7 22 ˜5 8 25 ˜20 9 33 ˜100 10 38 ˜20 11 39 Not determined 12 43 ˜30 13 46 Not determined 14 46 Not determined 15 48 Not determined 16 48 Not determined 17 49 Not determined 18 51 Not determined 19 51 ˜200 21 57 Not determined 22 58 Not determined 30 72 Not determined 33 74 Not determined

[0352] These results demonstrate the isolation of a few compounds in a diverse chemical library that were able to inhibit the calcineurin-VIVIT peptide interaction when present at low micromolar concentrations, which can be further tested in established assays of calcineurin-NFAT interaction and NFAT activation.

Example 19 The inhibitors INCA1, INCA2, and INCA6 Bind to Calcineurin

[0353] This example illustrates that binding of several of the INCA compounds to calcineurin detected in the absence of VIVIT peptide utilizing NMR spectroscopy.

[0354] In the fluorescence polarization assay, binding of the inhibitory compounds to calcineurin is inferred from their ability to displace fluorescent VIVIT peptide from the protein. To demonstrate independently that these compounds bind to calcineurin, NMR titration spectroscopy (J W Peng et al (2001) in Methods in Enzymology, volume 338, pp 202-230, T L James, V Dotsch, and U Schmitz, eds., Academic Press, San Diego, 2001) was employed.

[0355] 1H NMR binding experiments were performed at 25° C. in buffered D2O, with calcineurin concentration in the sample 0-20 &mgr;M and tested compound concentration 0-20 &mgr;M. T2-filtered 1H NMR spectra were obtained on a Bruker Avance 600 instrument with CryoPhobe using low-power presaturation of residual water. Preacquisition delays included up to five spin echos to suppress fast-relaxing broad protein resonances and were times to yield in-phase signals for J-coupled spins. Chemical shifts were referenced to internal standard 5 &mgr;M 2,2-dimethylsilapentane-5-sulfonic acid (DSS).

[0356] Portions of the T2-filtered 1H NMR spectra of 10 &mgr;M INCA1, INCA2, and INCA6 alone are displayed in the upper trace of each panel in FIGS. 10A, 10B, and 10C, respectively. The INCA1 resonance labelled “a” in FIG. 10A corresponds to the R1 proton and resonances labelled “b,” “c,” and “d,” correspond to protons at the ortho, para, amd meta positions of R2, with the R1 and R2 substituents designated as in Example 18. The INCA2 resonances from the naphthoquinonimine core (a and c) and from the benzenesulfonamide substituent (b, d, and e) are shown in FIG. 10B. The INCA6 resonances from the benzene rings are shown in FIG. 1C. A corresponding portion of the NMR spectrum taken in the presence of 20 &mgr;M calcineurin is displayed in the lower trace of each panel. Addition of calcineurin caused the resonances from aromatic protons of INCA1 and the signal from the six methyl protons, which is at a chemical shift of 2.26 ppm and therefore is not shown in this segment of the spectrum, to disappear. Likewise, addition of calcineurin caused the resonances arising from the four aromatic protons of the naphthoquinone of INCA2 and the resonances from aromatic protons of INCA6 to disappear. The methyl resonance of DSS, which was present in all experiments, was not affected in any way by the presence of calcineurin, and was used to scale spectra for comparison. Loss of the signals on addition of calcineurin can be attributed to additional relaxation processes that become available on binding to the protein, and indicates direct interaction of INCA compounds with calcineurin.

[0357] In NMR titration spectroscopy, saturable binding is confirmed by the concentration dependence of the effect on resonances. In another series of experiments with calcineurin and INCA compounds, the peak height dimunition was titratable by increasing calcineurin concentration at a single concentration of INCA compound, leading to a diminution in the signal that was linearly proportional to the calcineurin concentration up to a stoichiometric addition of calcineurin. A linear relation, rather than a conventional sigmoidal binding curve, is expected in this case, given that the estimated Kd values of the interaction are at least an order of magnitude below the 10-20 &mgr;M INCA compound concentration utilized in these NMR experiments.

[0358] These results showed that the inhibitors INCA1, INCA2, and INCA6, which displace VIVIT peptide from calcineurin, have a direct molecular interaction with calcineurin in the absence of VIVIT peptide.

Example 20 INCA2 Selectively Inhibits NFAT Dephosphorylation In Vitro

[0359] This example illustrates that INCA2 selectively inhibits the dephosphorylation of NFAT by recombinant calcineurin in vitro.

[0360] A method for studying dephosphorylation of NFAT in vitro uses NFAT that has been phosphorylated in mammalian cells, since recombinant NFAT isolated from bacteria is not phosphorylated, and it has not been demonstrated that NFAT can be stoichiometrically phosphorylated on the correct sites by protein kinases in vitro. NFAT1 is present in its phosphorylated form in cell lysates from unstimulated T cells, and can be detected by SDS-polyacrylamide gel electrophoresis and Western blotting using an antiserum directed to an NFAT1 peptide (anti-67.1; A Ho et al (1994) J Biol Chem 269, 28181-28186). Dephosphorylation produces a change in mobility that is visible as a shift in the immunostained NFAT band.

[0361] Cell lysates were made from C1.7W2 T cells washed in PBS/2 mM EGTA, 10 mM iodoacetamide, collected by centrifugation, and resuspended at 80×106 cells/ml in chilled lysis buffer consisting of 10 mM KAc, 2 mM MgAc2, 2 mM EGTA, 100 mM HEPES/NaHEPES pH 7.4, 0.2% NP-40, 10 mM iodoacetamide, 2 mM PMSF, 20 &mgr;g/ml aprotinin, and 50 &mgr;M leupeptin. After incubation for 10 minutes on wet ice, the nuclei and cell debris were pelleted in a refrigerated microcentrifuge at 4° C. by centrifugation for 5 minutes at 13,500 rpm, and the resulting supernatant recovered with a prechilled pipette tip, rapidly frozen using a dry ice/ethanol bath, and stored at −80° C.

[0362] For the in vitro dephosphorylation incubation, 500 nM recombinant human calcineurin (A and B chains; A Mondragon et al (1997) Biochemistry 36, 4934-4942) and 600 nM calmodulin (Sigma) was preincubated for 15 min on ice with varied concentrations of INCA2, to allow binding of inhibitor to equilibrate, in a reaction buffer consisting of 150 mM NaCl, 100 mM HEPES pH 7.4, 2 mM MgAc2, 1 mM CaCl2, 1 mg/ml BGG, 10 mM dithiothreitol, 2 mM PMSF, 20 &mgr;g/ml aprotinin, and 50 &mgr;M leupeptin. Control reactions contained calcineurin alone, or calcineurin with DMSO at a concentration comparable to that resulting from dilution of the INCA2 stock. The dephosphorylation reaction was initiated by addition of 0.6 &mgr;l lysate per 20 &mgr;l reaction. After 30 minutes at 30° C., the reaction was stopped by adding 5× Laemmli buffer containing sodium pyrophosphate and EDTA at final concentrations 20 mM and 3 mM respectively. Samples were analyzed by SDS-polyacrylamide gel electrophoresis according to Laemmli (U K Laemmli (1970) Nature 227, 680-685) using a 6% separating gel, transferred to nitrocellulose membrane, and probed with an antiserum to NFAT1.

[0363] The results are presented in FIG. 1A. FIGS. 11A-11B illustrate generally that INCA2 blocks dephosphorylation of NFAT by calcineurin and that the indicated INCA compounds (in FIG. 11B) do not block dephoshorylation of RII phosphopeptide. In FIG. 11A, the unincubated lysate sample (lane 1) represents the position of phosphorylated NFAT. There was no dephosphorylation when incubation was carried out in the presence of the phosphatase inhibitor sodium pyrophosphate (lanes 3 and 8). Calcineurin in the absence of inhibitors produced a clear dephosphorylation, evidenced by a shift in the NFAT band (lanes 4 and 7). INCA2 at micromolar concentrations (lanes 5 and 6) blocked dephosphorylation of NFAT as effectively as VIVIT peptide (lane 2). There was a slight apparent dephosphorylation of NFAT in both the VIVIT and INCA2 samples in the experiment shown, compared with lanes representing unincubated lysate or lysate incubated in the presence of the general phosphatase inhibitor sodium pyrophosphate, reflecting the use of a relatively high concentration of recombinant calcineurin, which appears to dephosphorylate sites in NFAT outside the N-terminal regulatory region. The similar slight dephosphorylation in the sample incubated with VIVIT peptide shows, however, that dephosphorylation of these sites does not depend on recognition of the SPRIEITPS docking site on NFAT.

[0364] To determine whether INCA inhibitors caused a general inhibition of calcineurin enzyme activity, in vitro assays were performed measuring the release of free inorganic phosphate from the standard calcineurin substrate RII phosphopeptide. The phosphatase activity of calmodulin-activated calcineurin was assayed photometrically using phosphorylated RII peptide as substrate and Malachite green for detection (BIOMOL Research Laboratories). Assays were carried out according to instructions provided by the supplier of the kit, in 96-well plates for 15 min at 25° C., with 6.8 nM calcineurin, 250 nM calmodulin, and 150 &mgr;M phosphorylated RII peptide, without further additions or in the presence of 100 &mgr;M INCA compound, 100 &mgr;M VIVIT peptide, or 1 &mgr;M cyclosporin A/cyclophilin A complex. DMSO concentration was kept constant in all samples. Release of free phosphate, calibrated to known standards, was 12-18 pmol/min under the conditions described and was not affected by the presence of dimethyl sulfoxide (DMSO), VIVIT peptide, INCA2, or any of the other 10 INCA compounds described in Example 18. The data are plotted as a bar graph in FIG. 11B, which also includes data for two of the negative control compounds from the chemical library (NEG-1 and NEG-2). Under the same conditions cyclosporin A/cyclophilin A (CsA/CypA) complex completely blocked calcineurin enzyme activity, demonstrating that the assay would report a true calcineurin inhibitor.

[0365] These results showed that INCA2, like the SPRIEITPS and VIVIT peptides, inhibits dephosphorylation of NFAT by calcineurin, but does not directly inhibit the phosphatase activity of the enzyme.

Example 21 INCA6 Inhibits Calcineurin-Dependent NFAT Activation in T Cells

[0366] This example illustrates that INCA6 inhibits the activation of NFAT elicited in T cells by treatment with the calcium ionophore ionomycin.

[0367] Dephosphorylation and nuclear import of NFAT are the earliest experimental indicators of the physiological activation of NFAT in stimulated cells (KT-Y Shaw et al (1995) Proc Natl Acad Sci USA 92, 11205-11209). These indicators were assessed by SDS-polyacrylamide gel electrophoresis and by immunocytochemistry in control and INCA6-treated T cells. It is confirmed here that these early steps in activation are blocked by the calcineurin inhibitors cyclosporin A and FK506.

[0368] For the dephosphorylation assay, C1.7W2 T cells were pretreated in Dulbecco's modified Eagle medium, supplemented with 10% fetal calf serum, 10 mM HEPES, and 2 mM glutamine, at 37 C. with INCA6 (5-40 &mgr;M) for 10 min, then stimulated for an additional 15 min with 1 &mgr;M ionomycin in the continued presence of inhibitor. Control incubations omitted INCA6, ionomycin, or both. DMSO was present at equal concentrations in all incubations. Cells were collected by centrifugation and lysed in Laemmli sample buffer. Samples were analyzed by SDS-polyacrylamide gel electrophoresis according to Laemmli (U K Laemmli (1970) Nature 227, 680-685) using a 6% separating gel, transferred to nitrocellulose membrane, and probed with an antiserum to NFAT1.

[0369] The results are presented in FIG. 12A. Dephosphorylation of NFAT is evident in the shift of the stained NFAT band the sample from cells that were stimulated with ionomycin in the absence of inhibitor (FIG. 12A, lane 2). Dependence of the dephosphorylation on calcineurin is demonstrated by blockade of the shift when cells were pretreated with CsA/FK506 (1 &mgr;M/100 nM) (not shown). Cells pretreated with INCA6 show a concentration-dependent blockade that is partial with 10 &mgr;M INCA6 (lane 5), nearly complete with 20 &mgr;M INCA6 (lane 4), and total with 40 &mgr;M INCA6 (lane 3). The inhibitor does not produce a general impairment of intracellular signalling, since activation of the PMA-MAP kinase signalling pathway was not blocked by INCA6 under these conditions. Activation of the latter pathway was monitored by examining phosphorylation of p44/p42 MAP kinase. For this assay the cells were pretreated and incubated as for the NFAT dephosphorylation assay, except that stimulation was for 15 min with 20 nM PMA. Cells were collected by centrifugation, lysed in Laemmli sample buffer, and analyzed by Western blotting with an anti-phospho-p44/p42 MAP kinase antibody (Cell Signaling Technology). The results of the MAP kinase phosphorylation assay are presented in FIG. 12B.

[0370] For an immune staining assay, C1.7W2 T cells growing on coverslips in Dulbecco's modified Eagle medium, supplemented with 10% fetal calf serum, 10 mM HEPES, and 2 mM glutamine, at 37 C. were pretreated with medium alone, with INCA6 (20 &mgr;M or 40 &mgr;M), or with CsA/FK506 (1 &mgr;M/100 nM) for 10 min, then stimulated for an additional 15 min with 1 &mgr;M ionomycin, in medium alone or in the continued presence of inhibitor as appropriate. Medium was removed, and the cells were fixed, permeabilized, and stained using the anti-67.1 antiserum against NFAT1 (A Ho et al (1994) J Biol Chem 269, 28181-28186) and a fluorescent second antibody. The results are presented in FIG. 13. The fluorescence microscopy micrographs presented in FIG. 13 demonstrate that NFAT immune staining was localized to the cytoplasm in unstimulated cells, localized to the nucleus in cells stimulated with ionomycin in the absence of inhibitor, but localized to the cytoplasm in cells stimulated with ionomycin in the presence of either concentration of INCA6 or in the presence of cyclosporin A/FK506 (CsA/FK506).

[0371] These results showed that exposure of C1.7W2 T cells to INCA6 prevented the early changes that are hallmarks of calcineurin-dependent activation of NFAT in stimulated cells.

Example 22 INCA6 Inhibits Induction of Cytokine mRNAs that are Targets of NFAT in T Cells

[0372] This example illustrates that, under stimulation conditions that mimic physiological stimulation of T cells, INCA6 is as effective as CsA/FK506 in inhibiting the early induction of several cytokine mRNAs that are the targets of calcineurin-NFAT signalling.

[0373] The effects of INCA6 on transcription were examined in the case of several cytokine mRNAs that are known targets of calcineurin-NFAT signalling. C1.7W2 T cells were preincubated for 10 min. in medium alone, in medium containing INCA6, or in medium containing CsA and FK506, then stimulated under conditions that normally initiate signalling through the calcium-calmodulin-calcineurin-NFAT pathway. Cells in control samples that had not been pretreated with inhibitor were left unstimulated, or were stimulated for 45 min with 20 nM PMA alone, or with 20 nM PMA and 1 &mgr;M ionomycin. Cells in parallel samples that had been pretreated for 10 min with inhibitor were then further incubated for 45 min without stimulation or with PMA and ionomycin, in the continued presence of inhibitor. At the end of the incubation, cells were harvested and total cellular RNA was extracted using Ultraspec RNA-binding resin (Biotecx Laboratories). Levels of cytokine mRNAs were analyzed by RNase protection assays performed with RiboQuant multiprobe kits (Pharmingen) following the instructions of the manufacturer. Briefly, for each sample 10 &mgr;g of target RNA was hybridized overnight to a 32P-labelled RNA probe that had been synthesized in vitro from a template set representing multiple murine cytokine mRNAs, and then the unhybridized probe and other single-stranded RNAs were digested with RNases. The protected RNAs were purified, and probes for the individual cytokine mRNAs were resolved on a denaturing 6% polyacrylamide gel. Transcript levels were quantified by autoradiography and by use of a phosphorimager (Molecular Dynamics). Probes for the individual cytokine transcripts were identified by the length of the respective protected fragments. The results are presented in FIGS. 14A to 14C, which are pictures of RNA gel autoradiograms. Data were corrected for minor variations in RNA loading by normalizing the data for each sample to the intensity of the signal for the housekeeping transcripts L32 and GAPDH (see lower panels of FIGS. 14A, 14B, and 14C).

[0374] FIGS. 14A-14C are pictures of autoradiograms of RNA gels illustrating that INCA6 blocks the NFAT-dependent expression of endogenous cytokine genes. FIGS. 14A-14C show protected segments of complementary RNA probes, which indicate the presence and level of specific cytokine mRNAs in the corresponding samples. As illustrated in FIG. 14A, GM-CSF mRNA was detectable in unstimulated cells, and its level was unchanged in cells stimulated with PMA alone. The level of GM-CSF mRNA was sharply increased by treatment with PMA and ionomycin. The basal level of GM-CSF mRNA was not affected by treatment with INCA6 or with CsA/FK506 (1 &mgr;M/100 nM), but the induction was partially prevented by 20 &mgr;M INCA6, and fully blocked by 40 &mgr;M INCA6 or by CsA/FK506. M-CSF mRNA showed only a modest induction over its basal level on treatment for 45 min with PMA and ionomycin, and so was not further analyzed. The mRNAs for the housekeeping genes L32 and GAPDH are present at similar levels in all lanes, indicating little variation in mRNA loading on the gel.

[0375] FIG. 14B shows that there was a slight elevation of TNF&agr; mRNA level on stimulation with PMA alone, and a more pronounced induction on stimulation with PMA and ionomycin. The induction of TNF&agr; mRNA was inhibited by 20 &mgr;M or 40 &mgr;M INCA6 and by CsA/FK506. IFN&ggr; mRNA was induced only in cells stimulated with the combination of PMA and ionomycin. Here again, induction was inhibited by INCA6 and by CsA/FK506. The level of TNF&bgr; mRNA was unaltered by any condition tested.

[0376] FIG. 14C documents that lymphotactin (Ltn), MIP1&bgr;, and MIP-1&agr; mRNAs were induced in response to stimulation with PMA and ionomycin, that induction was partially blocked by 20 &mgr;M INCA6, and that induction was fully blocked by 40 &mgr;M INCA6 or by CsA/FK506. RANTES mRNA and interferon &ggr;-inducible protein of 10 kDa (IP-10) mRNA served as controls that were unaffected by the stimulation conditions assayed and by the inhibitors.

[0377] Recapitulating, treatment of C1.7W2 T cells with PMA and ionomycin caused rapid induction of the mRNAs for GM-CSF, IFN&ggr;, TNF&agr;, lymphotactin, MIP1&agr; and MIP1&bgr;. Consistent with previous work, the increase in levels of these mRNAs was blocked by inhibiting calcineurin with CsA/FK506. Cytokine mRNA induction was likewise inhibited by 20 &mgr;M or 40 &mgr;M INCA6, with 40 &mgr;M INCA6 reducing mRNA levels to those of unstimulated cells. Levels of TNF&bgr;, RANTES, and IP-10 mRNAs, which are not downstream targets of NFAT, were not increased at early times by treatment with PMA and ionomycin and were unaffected by INCA6.

[0378] These results showed that exposure of C1.7W2 T cells to 1NCA6 prevented the induced transcription of cytokine genes that are downstream targets of NFAT.

Example 23 Certain Structural Analogues of INCA1, INCA2 and INCA6 are Inhibitors of the Calcineurin-VIVIT Peptide Interaction

[0379] This example illustrates that the three inhibitors INCA1, INCA2, and INCA6 are representative of three families of organic compounds that interfere with the calcineurin-VIVIT interaction.

[0380] The ability of commercially available structural analogues of INCA1, INCA2, and INCA6 to inhibit calcineurin-VIVIT interaction was evaluated using the fluorescence polarization assay of Example 18. Each compound was incubated, at a range of concentrations, with calcineurin and OG-VIVIT, and the ability of the tested compound to displace bound peptide from calcineurin was determined by measuring the polarization of OG-VIVIT fluorescence. FIGS. 15A-15C illustrate three general chemical structures of inhibitors of the protein-protein interaction between calcineurin and NFAT, typical chemical modifications that can be made thereto, and the inhibitory activity exhibited by such modified compounds. The general structures of INCA1 (FIG. 15A), INCA2 (FIG. 15B), and INCA6 (FIG. 15C) are presented, and the modifications to each R group for each analog are indicated. In FIG. 15B, the dashed line in the ring system represents a single bond for compound 2D and a double bond for other compounds; the dashed line between the nitrogen atom and the ring bearing substituents R1-R3 represents a single bond for compounds 2H, 2I, and 2K, and a double bond for other compounds. In FIG. 15C, the dashed lines represent single bonds for compounds 6A, 6B, and 6D, and double bonds for other compounds. Ac=acetyl; DDC=4,4-dimethyl-2,6-dioxocyclohexyl; Et=ethyl; Me=methyl; and Ph=phenyl. Ki values were estimated as described in Example 18 for those compounds that showed inhibitory activity and are indicated in the last column of FIGS. 15A-15C.

[0381] In many cases the inhibitory potency was only marginally affected by conservative changes in ring substituents. However, certain changes caused moderate to dramatic losses of potency. For example, reduction of the vicinal keto groups of INCA1 to hydroxyl groups, or their replacement by halogen substituents, resulted in inactive compounds. Likewise, introduction of bulky substituents at R1 in INCA2, or reduction of INCA6 to the hydroquinone or methoxyquinone, caused a pronounced decrease in or a loss of inhibitory activity. These distinctive structure-activity profiles are the signature of a specific ligand-protein interaction.

[0382] These results demonstrate that structural modifications to INCA compounds result in additional organic compounds that could be useful in inhibiting calcineurin-NFAT signalling.

Example 24 Gene Targets of NFAT and Identification of Same

[0383] Transcriptional targets of the NFAT proteins vary depending on the cellular context, including signals impinging on the cell, the structure of cellular chromatin, and the presence in the cell of other constitutively active or induced transcription factors. In a given cellular context, NFAT induces expression of a specific panel of genes related to cell differentiation or cell function. NFAT works in concert with other transcription factors, and it is generally observed that blockade of NFAT activation or ablation of its binding site(s) in DNA can reduce or eliminate target gene expression.

[0384] Familiar target genes of NFAT are cytokine and activation genes in the immune system. Cell surface receptors that activate immune cells, e.g. the T cell receptor, the macrophage Fc&ggr; receptor, or the mast cell Fc&egr; receptor, signal through NFAT to induce expression of cytokine genes, e.g. the IL2, TNF&agr;, IL4, IL5, IL13, and GM-CSF genes, depending on cellular context, and a variety of other activation genes, some of which are listed herein. Immunosuppressants CsA and FK506 has been used to block induction of this array of genes by inhibiting signalling in the calcineurin-NFAT pathway.

[0385] Under other conditions, NFAT has a central role in initiating immune tolerance or anergy in T cells and B cells. Due to the cellular context and the presence or absence of specific environmental inputs, NFAT signalling is addressed in this case to a different set of target genes, including diacylglycerol kinase-&agr;, Itch, Cbl-b, and Jumonji.

[0386] Yet another set of targets is controlled by NFAT in the heart, when physiological stresses lead to initiation of pathological cardiac hypertrophy. Genes known to be targets of NFAT in this process are the atrial natriuretic factor (ANF), B-type natriuretic peptide (BNP), and adenylosuccinate synthetase-1 genes. Given the critical role that NFAT proteins have been shown to play in initiation and development of cardiac hypertrophy, it is unlikely that its direct targets are limited to these few indicator genes. A skilled practitioner will be able to determine, through methods known in the art, which of the many other genes induced during myocardial hypertrophy are direct NFAT targets.

[0387] NFAT proteins also participate in osteoclast differentiation and bone resorption. The osteoclast markers TRAP and calcitonin receptor are NFAT target genes and, again, routine experimentation will show which other genes expressed by osteoclasts are direct NFAT targets.

[0388] NFAT targets also include binding sites that support reactivation of latent viruses and viral growth, for example the reactivation of latent Kaposi's sarcoma-associated herpesvirus (KSHV; also termed human herpesvirus-8) and the replication of HIV.

[0389] Among other NFAT targets are genes that have functions in physiological and pathological processes in a variety of tissues, for example the COX-2 and iNOS genes. Biological processes where regulation of these genes by NFAT is implicated include inflammation, angiogenesis, and tumor invasion.

[0390] Whether any specific gene or set of genes is an NFAT target can be determined using methods known to those of ordinary skill in the art. Several such methods are described below.

[0391] In one method, a constitutively active NFAT protein is expressed in cells and transcription of the gene is assayed by RT-PCR to determine whether or not it is influenced by NFAT expression (see, e.g., Macian et al., Cell 109: 719-731 (2002)).

[0392] In a another method, cells are transfected or infected to express high levels of GFP-VIVIT, then stimulated to induce an increase in intracellular calcium levels, with or without activation of other intracellular signalling pathways. Under these conditions GFP-VIVIT can block the induced transcription of genes that are direct or indirect targets of NFAT (see, e.g., Aramburu et al., Science 285: 2129-2133 (1999)).

[0393] In another method, gene expression is evaluated by RT-PCR or DNA array analysis in cells lacking one or more members of the NFAT family, and compared to expression in wildtype cells expressing all NFAT family members (see, e.g., Macian et al., Cell 109: 719-731 (2002)).

[0394] In another method, genes that are potentially direct targets of NFAT are identified by using bioinformatic techniques to find noncoding (putative regulatory) sequences that are highly conserved across species (see, e.g., Loots et al., Genome Res. 12: 832-839 (2002). These regions may then be examined for the presence of NFAT:AP-1, NFAT-dimer, or high affinity NFAT binding sites according to criteria known in the art (see, e.g., Kel et al., J Mol Biol. 288: 353-376 (1999) and Rao et al., Annual Review of Immunology 15: 707-747 (1997)) or derived from known NFAT-binding sequences. Candidate NFAT target sites can then be analyzed experimentally.

[0395] In another method, regions within a gene that contain regulatory sequences can be identified experimentally by analyzing the gene locus for the presence of inducible DNase I hypersensitive sites (see, e.g., Cockerill, Methods Mol Biol. 130: 29-46 (2000), Carey et al., Transcriptional regulation in eukaryotes: Concepts, strategies and techniques. Chapter 10, In vivo analysis of an endogenous control region. Cold Spring Harbour Laboraotry Press (2000), Agarwal et al., Immunity 9: 765-775 (1998), and Agarwal et al., Immunity 12:643-652 (2000)), and candidate NFAT sites in these regions can be analyzed experimentally.

[0396] In another method, the proximal promoter of the gene, or a putative regulatory region found by either of the above two techniques, is incorporated into a reporter plasmid, the plasmid is transfected into cells with or without co-expressed NFAT, the cells are stimulated appropriately to activate NFAT, and reporter activity is assessed. If the regulatory region contains functional NFAT binding sites, reporter activity will be induced under the same stimulation conditions that activate NFAT; reporter activity will be influenced by expression of exogenous NFAT or constitutively-active NFAT; and mutation of NFAT-binding sites in the regulatory region will abrogate induction of reporter activity (see, e.g., Carey et al., Transcriptional regulation in eukaryotes: Concepts, strategies and techniques, Chapter 5, Functional assays for promoter analysis. Cold Spring Harbour Laboraotry Press (2000))

[0397] Another method involves determining whether the promoter or other regulatory regions of the gene are represented in a chromatin immunoprecipitation assay, in which NFAT proteins and other DNA-binding proteins are crosslinked to the genomic regions to which they are bound in living cells, following which immunoprecipitation is performed using specific antibodies to NFAT, and the presence in the immunoprecipitate of the genomic sequences of interest is determined by PCR (see, e.g., Avni et al., Nature Immunol 3: 643-651(2002).

[0398] Those skilled in the art will be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims.

Claims

1. A pharmaceutical composition comprising a therapeutically effective amount of an organic molecule capable of inhibiting protein-protein interaction between calcineurin and NFAT, and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, wherein the agent inhibits dephosphorylation of NFAT by calcineurin.

3. The pharmaceutical composition of claim 1, wherein the molecular weight of the organic molecule is less than 2500 Da.

4. The pharmaceutical composition of claim 1, wherein the molecular weight of the organic molecule is between about 100 and 2000 Da.

5. The pharmaceutical composition of claim 1, wherein the molecular weight of the organic molecule is between about 200 and 1500 Da.

6. The pharmaceutical composition of claim 1, wherein the molecular weight of the organic molecule is between about 300 and 1000 Da.

7. The pharmaceutical composition of claim 1, wherein the organic molecule binds calcineurin with an affinity constant of at least about 2×104 M−1.

8. The pharmaceutical composition of claim 1, wherein the organic molecule binds calcineurin with an affinity constant of at least about 106 M−1.

9. The pharmaceutical composition of claim 1, wherein the organic molecule binds calcineurin with an affinity constant of at least about 107 M−1.

10. The pharmaceutical composition of claim 1, wherein the organic molecule binds calcineurin with an affinity constant of at least about 108 M−1.

11. The pharmaceutical composition of claim 1, wherein the organic molecule is a compound selected from the group consisting of:

formula (I):

10

wherein:

R1 is hydrogen, C1-C20 alkyl optionally substituted with 1-20 R6, C3-C8 cycloalkyl optionally substituted with 1-3 R6, aryl optionally substituted with 1-4 R6, heterocyclyl optionally substituted with 1-3 R6; heteroaryl optionally substituted with 1-4 R6; C2-C8 alkenyl, or C2-C8 alkynyl, cyano, nitro, carboxy, carbo(C1-C6)alkoxy, trihalomethyl, halogen, C1-C6 alkoxy, hydroxy, aryloxy, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, or ureido;

R2 is C1-C20 alkyl optionally substituted with 1-20 R6, C3-C8 cycloalkyl optionally substituted with 1-3 R6, aryl optionally substituted with 1-4 R6, heterocyclyl optionally substituted with 1-3 R6, heteroaryl optionally substituted with 1-4 R6, C1-C6 alkoxy, or hydroxy;

R3 is hydrogen or halogen;

R4 is hydrogen, C1-C20 alkyl optionally substituted with 1-20 R6, C3-C8 cycloalkyl optionally substituted with 1-3 R6, aryl optionally substituted with 1-4 R6, heterocyclyl optionally substituted with 1-3 R6, heteroaryl optionally substituted with 1-4 R6, or halogen;

R is NR7, O or S;

R6 is halogen, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido; and

R7 is C1-C6 alkyl;

formula (II):

11

wherein:

R1 and R2 are each independently hydrogen, halogen, amino, C1-C6alkylamino, di(C1-C6)alkylamino, arylamino, diarylamino, or 4,4-dimethyl-2,6-dioxocyclohexyl;

R3 is NR11 or O;

R4, R5 and R8 are each independently hydrogen, C1-C6 alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;

R6 is hydrogen, halogen, or when taken together with R7 forms a double bond between the carbon atoms to which they are attached;

R7 is hydrogen, halogen, or when taken together with R6 forms a double bond between the carbon atoms to which they are attached;

R9 is OR13, or when taken together with R10 forms a double bond between the carbon and nitrogen atoms to which they are attached;

R10 is hydrogen, or when taken together with R9 forms a double bond between the carbon and nitrogen atoms to which they are attached;

R11 is SO2R12; and

R12 is aryl optionally substituted with alkyl;

R13 is alkyl or aryl; and

formula (III):

12

wherein,

R1 and R4 are each independently O or NR8;

R2 and R3 are each independently hydrogen, halogen, or R2 and R3 together combine to form aryl optionally substituted with 1-4 R9;

R5 is hydrogen, halogen, carboxy, acylamino, alkoxycarbonyl, carboxy, alkylcarbonyl, acyloxy, or cyano;

R6, R7 and R9 are each independently hydrogen, C1-C6 alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;

R8 is SO2R10; and

R10 is aryl optionally substituted with alkyl.

12. A method for inhibiting protein-protein interaction between calcineurin and NFAT, comprising:

providing calcineurin and NFAT;

providing the pharmaceutical composition of claim 1; and

contacting the calcineurin, NFAT, and pharmaceutical composition together, such that the protein-protein interaction between calcineurin and NFAT is inhibited.

13. A method of inhibiting an immune response in an animal, comprising administering to the animal the pharmaceutical composition of claim 1.

14. A method for treating a disease or condition involving hyperactivity or inappropriate activity of the immune system, comprising:

identifying an animal suffering from a disease or condition involving hyperactivity or inappropriate activity of the immune system; and

administering to the animal a therapeutically effective amount of the pharmaceutical composition of claim 1, to thereby treat the disease or condition involving hyperactivity or inappropriate activity of the immune system.

15. The method of claim 14, wherein the organic molecule is a compound selected from the group consisting of:

formula (I):

13

wherein:

R1 is hydrogen, C1-C20 alkyl optionally substituted with 1-20 R6, C3-C8 cycloalkyl optionally substituted with 1-3 R6, aryl optionally substituted with 1-4 R6, heterocyclyl optionally substituted with 1-3 R6; heteroaryl optionally substituted with 1-4 R6; C2-C8 alkenyl, or C2-C8 alkynyl, cyano, nitro, carboxy, carbo(C1-C6)alkoxy, trihalomethyl, halogen, C1-C6 alkoxy, hydroxy, aryloxy, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, or ureido;

R2 is C1-C20 alkyl optionally substituted with 1-20 R6, C3-C8 cycloalkyl optionally substituted with 1-3 R6, aryl optionally substituted with 1-4 R6, heterocyclyl optionally substituted with 1-3 R6, heteroaryl optionally substituted with 1-4 R6, C1-C6 alkoxy, hydroxy;

R3 is hydrogen or halogen;

R4 is hydrogen, C1-C20 alkyl optionally substituted with 1-20 R6, C3-C8 cycloalkyl optionally substituted with 1-3 R6, aryl optionally substituted with 1-4 R6, heterocyclyl optionally substituted with 1-3 R6, heteroaryl optionally substituted with 1-4 R6, or halogen;

R5 is NR7, O or S;

R6 is halogen, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido; and

R7 is C1-C6 alkyl;

formula (II):

14

wherein:

R1 and R2 are each independently hydrogen, halogen, amino, C1-C6alkylamino, di(C1-C6)alkylamino, arylamino, diarylamino, or 4,4-dimethyl-2,6-dioxocyclohexyl;

R3 is NR11 or O;

R4, R5 and R8 are each independently hydrogen, C1-C6 alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;

R6 is hydrogen, halogen, or when taken together with R7 forms a double bond between the carbon atoms to which they are attached;

R7 is hydrogen, halogen, or when taken together with R6 forms a double bond between the carbon atoms to which they are attached;

R9 is OR13, or when taken together with R10 forms a double bond between the carbon and nitrogen atoms to which they are attached;

R10 is hydrogen, or when taken together with R9 forms a double bond between the carbon and nitrogen atoms to which they are attached;

R11 is SO2R12; and

R12 is aryl optionally substituted with alkyl;

R13 is alkyl or aryl; and

formula (III):

15

wherein,

R1 and R4 are each independently O or NR8;

R2 and R3 are each independently hydrogen, halogen, or R2 and R3 together combine to form aryl optionally substituted with 1-4 R9;

R5 is hydrogen, halogen, carboxy, acylamino, alkoxycarbonyl, carboxy, alkylcarbonyl, acyloxy, or cyano;

R6, R7 and R9 are each independently hydrogen, C1-C6 alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;

R8 is SO2R10; and

R10 is aryl optionally substituted with alkyl.

16. The method of claim 14 wherein the disease or condition involving hyperactivity or inappropriate activity of the immune system is selected from the group consisting of: an acute immune disease, a chronic immune disease, and an autoimmune disease.

17. A method for treating a disease involving excessive or inappropriate activation of NFAT, or a molecular target thereof, comprising:

identifying an animal suffering from a disease involving excessive or inappropriate activation of NFAT or a molecular target thereof, and

administering to the animal a therapeutically effective amount of the pharmaceutical composition of claim 1, to thereby treat the disease involving excessive or inappropriate activation of NFAT or molecular target thereof.

18. A process of making an agent that inhibits protein-protein interaction between calcineurin and NFAT, the process comprising:

carrying out a method to identify an agent that inhibits protein-protein interaction between calcineurin and NFAT, wherein the method comprises:

providing a first compound selected from the group consisting of calcineurin or a biologically active derivative thereof, and NFAT or a biologically active derivative thereof;

providing a second compound selected from the group consisting of calcineurin or a biologically active derivative thereof, and NFAT or a biologically active derivative thereof, wherein the second compound is different from the first compound, and wherein the second compound is labeled;

providing a candidate agent;

contacting the first compound, the second compound, and the candidate agent with each other; and

determining the amount of label bound to the first compound, wherein a reduction in interaction between the first compound and the second compound as assessed by label bound is indicative of usefulness of the candidate agent in inhibiting protein-protein interaction between calcineurin and NFAT; and

manufacturing the agent, to thereby make an agent that inhibits protein-protein interaction between calcineurin and NFAT.

19. The process of claim 18, wherein the first compound is calcineurin and the second compound is a biologically active derivative of NFAT.

20. The process of claim 18, wherein the biologically active derivative of NFAT comprises the amino acid sequence GPHPVIVITGPHEE.

21. A method of manufacturing an agent that inhibits protein-protein interaction between calcineurin and NFAT, the method comprising:

providing an organic compound capable of inhibiting protein-protein interaction between calcineurin and NFAT;

providing at least one pharmaceutically acceptable carrier; and

combining the organic compound with the pharmaceutically acceptable carrier, to thereby manufacture an agent that inhibits protein-protein interaction between calcineurin and NFAT.

22. The method of claim 21, further comprising the step of manufacturing the agent into a form suitable for administration to an animal via a route selected from a group consisting of: oral, parenteral, topical, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal.

23. A method for inhibiting protein-protein interaction between calcineurin and NFAT, comprising:

providing calcineurin and NFAT;

providing an organic molecule capable of inhibiting protein-protein interaction between calcineurin and NFAT, wherein the organic molecule is a compound selected from the group consisting of:

formula (I):

16

wherein:

R1 is hydrogen, C1-C20 alkyl optionally substituted with 1-20 R6, C3-C8 cycloalkyl optionally substituted with 1-3 R6, aryl optionally substituted with 1-4 R6, heterocyclyl optionally substituted with 1-3 R6; heteroaryl optionally substituted with 1-4 R6; C2-C8 alkenyl, or C2-C8 alkynyl, cyano, nitro, carboxy, carbo(C1-C6)alkoxy, trihalomethyl, halogen, C1-C6 alkoxy, hydroxy, aryloxy, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, or ureido;

R2 is C1-C20 alkyl optionally substituted with 1-20 R6, C3-C8 cycloalkyl optionally substituted with 1-3 R6, aryl optionally substituted with 1-4 R6, heterocyclyl optionally substituted with 1-3 R6, heteroaryl optionally substituted with 1-4 R6, C1-C6 alkoxy, hydroxy;

R3 is hydrogen or halogen;

R4 is hydrogen, C1-C20 alkyl optionally substituted with 1-20 R6, C3-C8 cycloalkyl optionally substituted with 1-3 R6, aryl optionally substituted with 1-4 R6, heterocyclyl optionally substituted with 1-3 R6, heteroaryl optionally substituted with 1-4 R6, or halogen;

R5 is NR7, O or S;

R6 is halogen, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido; and

R7 is C1-C6 alkyl;

formula (II):

17

wherein:

R1 and R2 are each independently hydrogen, halogen, amino, C1-C6alkylamino, di(C1-C6)alkylamino, arylamino, diarylamino, or 4,4-dimethyl-2,6-dioxocyclohexyl;

R3 is NR11 or O;

R4, R5 and R8 are each independently hydrogen, C1-C6 alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;

R6 is hydrogen, halogen, or when taken together with R7 forms a double bond between the carbon atoms to which they are attached;

R7 is hydrogen, halogen, or when taken together with R6 forms a double bond between the carbon atoms to which they are attached;

R9 is OR13, or when taken together with R10 forms a double bond between the carbon and nitrogen atoms to which they are attached;

R10 is hydrogen, or when taken together with R9 forms a double bond between the carbon and nitrogen atoms to which they are attached;

R11 is SO2R12; and

R12 is aryl optionally substituted with alkyl;

R13 is alkyl or aryl; and

formula (III):

18

wherein,

R1 and R4 are each independently O or NR8;

R2 and R3 are each independently hydrogen, halogen, or R2 and R3 together combine to form aryl optionally substituted with 1-4 R9;

R5 is hydrogen, halogen, carboxy, acylamino, alkoxycarbonyl, carboxy, alkylcarbonyl, acyloxy, or cyano;

R6, R7 and R9 are each independently hydrogen, C1-C6 alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;

R8 is SO2R10; and

R10 is aryl optionally substituted with alkyl; and

contacting the calcineurin, NFAT, and organic molecule together such that protein-protein interaction between the calcineurin and the NFAT is inhibited.