ANTI-ANGIOGENIC PEPTIDES AND USES THEREOF
Provided are peptide sequences derived from prostate serum antigen (PSA). The peptides are provided in cyclic and linear form. Methods for using the peptides for inhibition of angiogenesis, such as angiogenesis in a tumor, are provided.
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This application claims priority to U.S. provisional patent application No. 61/513,019, filed Jul. 29, 2011, the disclosure of which is incorporated herein by reference.
FIELD OF THE INVENTIONThe present invention relates generally to cancer therapy and more specifically to novel peptides for use as anti-angiogenic/anti-cancer agents.
BACKGROUND OF THE INVENTIONProstate cancer is a major contributor to cancer mortality in American males causing projected death of approximately 27,360 men in 2009 (Jemal et al., Cancer J. Clin., 59: 225-249, 2009). Therapeutic modalities such as radical prostatectomy and radiotherapy are sometimes curative for localized disease, yet no treatments for metastatic prostate cancer that significantly increases patient survival are available.
Angiogenesis is critical in tumor progression and metastasis in most if not all solid tumors, since a functional vascular supply is required for the continued growth of solid tumors and the spread of cancer cells. Small non-growing tumors may remain dormant for years and the angiogenic switch to aggressive metastatic phenotype involves a change in the local equilibrium between factors inducing blood vessel formation and those inhibiting the process.
There is an ongoing and unmet need to develop compositions and methods for inhibiting angiogenesis as a therapeutic modality for treating solid tumors, and in particular, for prophylaxis and/or therapy of prostate tumors. The present invention meets this need.
SUMMARY OF THE INVENTIONThe present invention provides peptides and methods of using the peptides for inhibiting angiogenesis. Thus, the invention provides compositions and methods for use in therapeutic interventions for treating diseases that include but are not limited to cancer.
The peptides of the invention include but are not limited to peptides comprising or consisting of amino acid sequences disclosed herein, fragments of the amino acid sequences, and modifications of peptides comprising amino acid sequences derived from prostate specific antigen (PSA). Each peptide and peptide that can be modified as described herein and which is encompassed within the scope of the invention has the capability to inhibit human endothelial cell migration, such as endothelial cell migration in a gelatinous protein mixture, such as that sold under the name Matrigel. Thus, in one embodiment, a peptide encompassed within the scope of the invention is a peptide that can inhibit cell network formation in a gelatinous protein mixture.
In particular embodiments, the amino acids of the peptides provided by the invention comprise or consist of the following sequences:
Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp Ser Ser His (SEQ ID NO:1). This peptide is also referred to herein as Peptide #1 (linear) and as “PSA-P1L.” It is not provided as a cyclic peptide. A modified version of this peptide is provided and is referred to herein as PSA-P1C (constrained, or cyclized). It has the sequence Cys Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp Ser Ser His Cys (SEQ ID NO:7). In this peptide, a cysteine was engineered at the N-terminus and at the C-terminus of Peptide #1. These facilitate formation of a disulfide bond between the N- and C-terminal cysteine residues. The result is a cyclic (“constrained” or cyclized) peptide wherein the N- and C-termini are connected to one another via a disulfide bond.
In another embodiment, Peptide #2 is provided. It has the sequence Gly Trp Gly Ser Ile Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln (SEQ ID NO:2).
In another embodiment, Peptide #3 is provided. It has the sequence Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys (SEQ ID NO:3).
In another embodiment, Peptide #3 is modified to replace the Cys that is endogenous to PSA with Ala, to provide Peptide 3*. Peptide 3* has the sequence Asn Asp Val Ala Ala Gln Val His Pro Gln Lys Val Thr Lys (SEQ ID NO:8).
In another embodiment, Peptide #4 is provided. It has the sequence Gly Arg Trp Thr Gly Gly Lys Ser Thr Cys Ser (SEQ ID NO:4).
In another embodiment, Peptide #5 is provided. It has the sequence Ser Glu Pro Cys Ala Leu Pro Glu Arg Pro Ser (SEQ ID NO:5).
In another embodiment, Peptide #6 is provided. It has the sequence Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Cys Ala (SEQ ID NO:6). Peptide #6 is also referred to herein as “PSA-P3C”. The two Cys in PSA-P3C can cyclize (constrain) the peptide via a disulfide bond. In one embodiment, Peptide #6 is modified so that the two Cys residues in it are replaced by Ala to provide a peptide which has the sequence Asn Asp Val Ala Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Ala Ala (SEQ ID NO:9). This peptide is referred to herein as “PSA-P3L” and is not provided in cyclic form.
The method comprises administering a composition comprising one or more peptides comprising or consisting of an amino acid sequence as described herein to an individual in an amount effective to inhibit angiogenesis, and/or in an amount effective to inhibit the growth and/or metastasis of a tumor. Other diseases that involve undesirable or irregular angiogenesis are also contemplated for treatment. Compositions comprising the peptides are also provided. Such compositions include but are not necessarily limited to pharmaceutical preparations.
The present invention provides isolated peptides derived from PSA that are expected to be capable of inhibiting angiogenesis in vivo, compositions comprising the peptides, and methods of using the peptides to inhibit angiogenesis.
In more detail, PSA (also referred to in the art as KLK3) is widely recognized by the lay population as the serum biomarker utilized for screening for the presence, aggressiveness and/or recurrence of prostate cancer. However, recent observations have called into question the utility of PSA as a screening or diagnostic tool, and the biology of PSA production and function in the human prostate is not consistent with a causal role for PSA in the progression of prostate cancer. PSA is at 106-fold greater levels in seminal fluid, and 103-fold greater levels in the prostate tissue, than the level of PSA present in the serum of post-pubertal males. The PSA in seminal fluid and prostate tissue is largely enzymatically active and is not complexed with chaperone/carrier proteins, whereas, PSA in serum is largely enzymatically inactive and sequestered through binding to chaperone/carrier proteins. Furthermore, prostate production of PSA falls with age, prostate cancer progression and androgen deprivation therapy, whereas, the serum levels of PSA measured in the screening test rise even though prostate production of PSA is falling.
PSA was cloned and characterized as a product of the human prostate gland, however, PSA is produced by many organs in the human, in both males and females. In females, PSA is produced in benign breast, endometrial and ovarian tissues, and malignancies of these tissues, as well as in non-hormonally regulated tissues and their cancers, such as the parotid gland. Across all of these malignancies, including prostate cancer, tissue levels of PSA in the cancer tissue are inversely correlated with prognosis.
PSA is well known to undergo proteolytic processing from a full length protein that initially contains a signal peptide sequence (residues 1-17 of the full length protein), a sequence corresponding to an activation peptide (residues 18-24), and the processed PSA protein, which ordinarily consists of 237 amino acids spanning residues 25-261 of the full length protein. The 237 amino acid protein (25-261) were used in the studies that include PSA as described herein. The full length (i.e., unprocessed) protein consists of the amino acid sequence:
The principal biological function of PSA is currently believed to be in liquification of seminal fluid. PSA has also been shown to have significant transcriptional regulatory activity, modulating expression of multiple potent anti-angiogenic genes and processes. The present invention provides evidence that the transcriptional regulatory, anti-tumorigenic, and anti-angiogenic activity of human PSA are independent of the enzymatic activity of PSA, in that these biological endpoints are observed at equal molar concentrations of enzymatically inactive PSA. We demonstrate using established techniques for evaluating anti-angiogenic agents that the anti-angiogenic activity is exhibited by small peptides that we have derived from human PSA. Thus, the present invention provides a plurality of peptides derived from PSA, each of which is expected to be useful for inhibition of angiogenesis, either alone, or in combination with each other. Each peptide and derivative(s) thereof that are encompassed within the scope of the present invention has the capability to inhibit human endothelial cell migration in a gelatinous protein mixture. Thus, the invention provides a method of inhibiting human endothelial cell migration comprising contacting human endothelial cells with a composition comprising a peptide described herein, wherein the migration is inhibited subsequent to the contacting of the human endothelial cells with the composition. Also provided is a method of inhibiting angiogenesis and/or tumor growth and/or metastasis. The method comprises administering a composition comprising one or more of the peptides of the invention to an individual in an amount effective to inhibit angiogenesis, and/or inhibit tumor growth and/or inhibit metastasis. Also provided pharmaceutical preparations comprising the peptides, which can be used in the methods of the invention.
Without intending to be bound by any particular theory, it is considered that PSA is an endogenous anti-neoplastic factor in many human tissues and their malignancies, and that the peptides described herein will provide anti-angiogenic activity targeted to the immature/angiogenic tumor vascular network, thus inhibiting angiogenesis, and/or tumor progression and/or metastasis. The peptides described herein are believed to represent ideal therapeutic modalities in that they are a derived from a constitutively expressed protein that should not elicit an immune response, and systemic administration is expected to provide direct delivery to the therapeutic target, namely, the endothelial cells of the cancer.
Data presented herein show that a subset of synthetic peptides derived from human PSA inhibit angiogenesis in the Matrigel endothelial cell tube formation assay. It is noteworthy that tube formation in Matrigel by cultured endothelial cells, usually HUVEC, is an established in vitro surrogate for angiogenesis that is proposed to require both migration and differentiation by the endothelial cells. The “tube formation” assay has been utilized extensively to evaluate in vitro the anti-angiogenic capacity of peptides, proteins and pharmacologic agents (Print, C., et al., Soluble factors from human endometrium promote angiogenesis and regulate the endothelial cell transcriptome. Hum Reprod, 2004. 19(10): p. 2356-66; Delves, G. H., et al., In vitro inhibition of angiogenesis by prostasomes. 2005. Prostate Cancer Prostatic Dis. 8(2): p. 174-8). Thus, results presented herein are strongly indicative that the peptides of the invention will be suitable for inhibition of angiogenesis, and thus can be expected to be useful for prophylaxis and/or therapy of human cancers, including but not limited to prostate cancer, breast cancer, ovarian cancer, cervical cancer and parotid cancers. In certain embodiments, the invention provides a method for inhibiting the migration and/or differentiation of human endothelial cells comprising contacting the cells with a composition comprising one or more peptides of the invention.
The data in Table #1 shows that PSA levels in prostate gland as expressed in prostate cancer tissue are lower than in benign tissue in the same gland. At the time of surgery, prostate tissue, that is removed, is checked by pathology to determine what portion of tissue is benign and what portion is malignant. Thus, Table 1 establishes that tissue PSA is down regulated in cancerous tissue. The significance of the data presented in this Table, without intending to be bound by any particular theory, is that since it is generally accepted that serum PSA levels increase in prostate cancer, it is not a logical hypothesis that PSA is an endogenous antiangiogenic factor. However, we have determined that malignant tissues actually make less PSA than normal tissue (as described in the Table) which actually reduces expression of the endogenous antiangiogenic agent.
The following peptide sequences illustrate embodiments of peptides encompassed by the present disclosure. It is notable that Peptides #2, #4 and #5 did not demonstrate reproducible anti-angiogenic activity. Thus, not all fragments of PSA exhibit anti-angiogenic properties.
Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp Ser Ser His (SEQ ID NO:1). This peptide is also referred to in the description of the invention and the Examples as Peptide #1 and as “PSA-P1L.” This peptide is not cyclized. We modified this peptide to provide the peptide referred to herein as “PSA-P1C.” It has the sequence Cys Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp Ser Ser His Cys (SEQ ID NO:7). To derive PSA-P1C, we engineered a cysteine at the N-terminus and at the C-terminus of Peptide #1, which enabled formation of a disulfide between the N- and C-terminal residues. The result is a cyclic peptide wherein the N- and C-termini are connected to one another via a disulfide bond. In all cases where PSA-P1L is tested in the data presented herein it was tested in the cyclic form.
Peptide #2 is provided and has the sequence Gly Trp Gly Ser Ile Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln (SEQ ID NO:2).
In another embodiment, Peptide #3 is provided and has the sequence Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys (SEQ ID NO:3).
We modified Peptide #3 to replace the Cys that is endogenous to PSA with Ala, resulting in Peptide 3*. Peptide 3* has the sequence Asn Asp Val Ala Ala Gln Val His Pro Gln Lys Val Thr Lys (SEQ ID NO:8).
Peptide #4 has the sequence Gly Arg Trp Thr Gly Gly Lys Ser Thr Cys Ser (SEQ ID NO:4). Peptide #5 has the sequence Ser Glu Pro Cys Ala Leu Pro Glu Arg Pro Ser (SEQ ID NO:5).
In another embodiment, Peptide #6 is provided. It has the sequence Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Cys Ala (SEQ ID NO:6). Peptide #6 is also referred to herein as “PSA-P3C.” The two Cys in PSA-P3C are used to cyclize the peptide via a disulfide bond. PSA-P3C was tested in the experiments described herein in cyclic form. We modified Peptide #6 to test a similar peptide in linear form. To do this, the two Cys residues in Peptide #6 were replaced by Ala to provide a peptide which has the sequence Asn Asp Val Ala Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Ala Ala (SEQ ID NO:9). This peptide is referred to herein as “PSA-P3L”.
It is expected that the amino acids sequences of the peptides presented herein and described as suitable for use in the invention can be modified and still retain desirable properties, such as anti-angiogenic properties. Such modifications can be determined in a variety of ways, such as by almandine scanning mutagenesis. Illustrative, non-limiting examples of peptide derivatives based upon Peptide #1 that can be produced in this manner, and are therefore included within the scope of the invention, are illustrated by the following sequences:
The invention includes peptides of various lengths, and with various amino acid substitutions, based on the amino acid sequences of the peptides provided by the invention. For example, Peptide #1 (or any other peptide described herein) can be modified by conservative amino acid substitutions that are based generally on relative similarity of R— group substituents. Non-limiting examples of such substitutions contemplated in the present invention include, but are not limited to: gly or ser for ala; lys for arg; gln or his for asn; glu for asp; ser for cys; asn for gln; asp for glu; ala for gly; asn or gln for his; leu or val for ile; ile or val for leu; arg for lys; leu or tyr for met; thr for ser; tyr for trp; phe for tyr; and ile or leu for val. Thus, peptides that comprise any single conservative amino acid substitution, or any combination of conservative amino acid substitutions, are included in the invention, so long as they retain their anti-angiogenic properties. In certain embodiments, peptides of the invention which comprise conservative amino acid substitutions retain their capability to inhibit tubule formation by HUVECs.
Non-conservative substitutions that enhance desirable characteristics of the peptides, such as their anti-angiogenic activity, circulation time, bioavailability, stability, binding to cell surface receptors, regulation of gene transcription, targeting imaging or therapeutic modalities to the tumor vasculature, etc., are also included in the invention.
In addition to amino acid substitutions, peptides of the invention may include fragments of any of the peptides described herein. Any peptide encompassed within the scope of the invention can comprise or consist of from 4-20 amino acids, inclusive, and including all integers there between. For example, Peptide #1 fragments encompassed within the scope of the invention include but are not limited to:
Each peptide described herein can also be truncated by any number of amino acids at its C-terminus, and by any number of amino acids at both the N- and C-termini, so long as the remaining sequence retains its anti-angiogenic activity. Each of the foregoing fragments can also be subjected to the aforementioned amino acid substitutions, and thus, fragments having such substitutions are included within the scope of the invention. Any peptide provided by the invention can comprise one, two, three, four, or five conservative amino acid substitutions. Any of the peptides provided by the invention can be at least four amino acids in length.
To the extent that any of the peptides/fragment sequences per se have been previously described, then the present invention contemplates their use in methods for inhibition of angiogenesis, and/or for the prophylaxis and/or treatment of tumors and/or metastasis, and in pharmaceutical prepartions. It is also contemplated that the peptides of the present invention may include additional amino acids, and may include modified amino acids that can improve any desirable property of the peptides. Thus, the peptides could be covalently or non-covalently associated with any desirable moiety that would be expected to improve their functional capabilities in accordance with the method of the invention.
The peptides of the invention can be prepared by any technique known to those skilled in the art or by techniques hereafter developed. For example, the peptides can be prepared using the solid-phase synthetic technique (Merrifield, J. Am. Chem. Soc., 15:2149-2154 (1963); M. Bodanszky et al., (1976) Peptide Synthesis, John Wiley & Sons, 2d Ed.; Kent and Clark-Lewis in Synthetic Peptides in Biology and Medicine, p. 295-358, eds. Alitalo, K., et al. Science Publishers, (Amsterdam, 1985). The synthesis of peptides by solution methods may also be used, as described in The Proteins, Vol. II, 3d Ed., p. 105-237, Neurath, H., et al., Eds., Academic Press, New York, N.Y. (1976). The synthesized peptides may be substantially purified by preparative high performance liquid chromatography or other comparable techniques available in the art. The composition of the synthetic peptides can be confirmed by an technique for amino acid composition analysis.
In one embodiment, the peptides are provided as cyclic peptides.
For use in angiogenesis inhibition, prophylaxis and/or therapy of tumors and/or metastasis, the peptides can be administered in a conventional dosage form prepared by combining the peptides with a standard pharmaceutically acceptable carrier according to known techniques. Some examples of pharmaceutically acceptable carriers can be found in: Remington: The Science and Practice of Pharmacy (2005) 21st Edition, Philadelphia, Pa. Lippincott Williams & Wilkins.
In one embodiment, the invention comprises at least one of the peptides provided by the invention in a pharmaceutical preparation, such as a composition comprising at least one pharmaceutically acceptable carrier, which can be provided in a pharmaceutically acceptable bufferIn certain embodiments, compositions of the invention can include distinct peptides, and can include mixtures of linear (non-cyclic) and cyclic peptides.
Various methods known to those skilled in the art may be used to introduce the compositions of the invention to an individual. These methods include but are not limited to intracranial, intrathecal, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, intranasal and retrograde routes.
It will be recognized by those of skill in the art that the form and character of the particular dosing regime employed in the method of the invention will be dictated by the route of administration and other well-known variables, such as rate of clearance, the size of the individual and the stage of the particular disease being treated. Based on such criteria, one skilled in the art can determine an amount of any of the particular peptides described herein that will be effective to inhibit angiogenesis and/or tumor growth for any particular individual. It is generally considered that the amount of peptide administered will range from microgram to milligram amounts.
The method of the invention can be performed in conjunction with conventional anti-cancer therapies. Such therapies can include but are not limited to chemotherapies, such as androgen deprivation therapy, surgical interventions, and radiation therapy. The compositions of the invention could be administered prior to, concurrently, or subsequent to such anti-cancer therapies.
The following Examples are meant to illustrate, but not limit the invention.
Example 1This Example demonstrates PSA-induced inhibition of gene expression in human prostate cancer cells.
The human prostate cancer cell lines LNCaP, DU-145 and PC-3 have low, moderate and high metastatic potential, and we reported that expression of pro-angiogenic factors correlated with the metastatic potential (Aalinkeel R, et al., 2004. Cancer Res 64:5311-5321). Constitutive expression of multiple pro-angiogenic growth factors, including: VEGF, IL-8 and TGF-β, as well as MMP-9 and tissue inhibitors of metalloproteinase 1 and 4, was significantly greater in the highly metastatic PC-3M cell line compared to LNCaP cells, with a low intrinsic metastatic potential (Aalinkeel R, et al., 2004. Cancer Res 64:5311-5321).
PSA purified to homogeneity from human seminal plasma is enzymatically active, and free of contaminating hk2, and complexes with carrier proteins/chaperones (Bindukumar, B., et al., J Chromatogr B Analyt Technol Biomed Life Sci, 2004. 813(1-2): p. 113-20). Gene expression array analysis of PC-3M cells versus LNCaP cells treated with 10 μM PSA, revealed that 136 genes were up-regulated and 137 genes were down-regulated. QPCR analysis demonstrated that urokinase-type plasminogen activator (uPA), VEGF, cysteine-rich angiogenic inducer-61, EphA2, TGF-β2, were significantly down-regulated in the highly metastatic PC-3M cells, whereas, IFN-γ and several interferon related genes, including 2,5 oligo-adenylated synthetase-2, were up-regulated (
This Example provides an illustration of proteomic profiling of the effect of PSA on human prostate cancer cells. In this regard, a proteomic approach was employed to identify proteins whose expression was modulated in PC-3M cells treated with PSA. 2D-DIGE coupled with HPLC-tandem mass spectrometry (MS/MS) identified a total of 41 proteins significantly changed (p<0.05) in abundance in PC-3M cells in response to PSA treatment. Proteins from 26 gel spots were identified [
This Example demonstrates enzymatic activity of isolated PSA. In particular, enzymatic activity of PSA isolated from human seminal plasma (f-PSA) and from human prostate cancer tissue specimens (Tissue-PSA: T-PSA) was evaluated routinely using a substrate (Mu-His-Ser-Ser-Lys-Leu-Gln-AFC) (Calbiochem) that is highly specific for PSA. In connection with the human prostate cancer tissue specimens, it should be noted that the majority of PSA in serum is complexed with serine protease inhibitors and is enzymatically inactive, while essentially all of the PSA in seminal fluid is in a free form (f-PSA) and is enzymatically active. PSA in the tissue microenvironment (T-PSA) is largely free and enzymatically active. T-PSA levels are lower in advanced prostate cancer than benign prostate, and T-PSA levels correlate with prognosis in prostate cancer, as well as in breast cancer; the higher the T-PSA level, the better the prognosis.
Hydrolysis of the fluorogenic substrate was quantitated using a LS-45 Luminescence Spectrometer from Perkin Elmer) using the FL-WinlabR program. Human prostate cancer tissue specimens were obtained from the Pathology Resource Network at Roswell Park Cancer Institute under an IRB approved protocol. Both f-PSA and T-PSA demonstrated enzymatic activity in the assay, however, T-PSA routinely demonstrated roughly 50% of the activity of f-PSA from seminal plasma per microgram of protein, probably reflecting loss of activity during extraction from tissue. Enzymatic activity against the fluorescent substrate of both f-PSA and T-PSA were inhibited completely by pre-incubation with zinc chloride (5 μM).
Example 4This Example demonstrates that enzymatic activity of PSA is not required for its effects on modulating angiogenesis related gene expression.
Enzymatically active PSA, and PSA inactivated by incubation with Zinc2+ [activity inhibited >75% by 5.0 μM of Zinc, inset
This Example demonstrates the effect of enzymatically active f-PSA and T-PSA on endothelial tube formation in the Matrigel Assay.
This Example demonstrates that illustrative PSA-mimetic peptides provided by the invention have inhibitory effect upon endothelial tube formation by HUVEC in the Matrigel Assay. The 3-D model of human PSA, the amino acid sequence of the initial five candidate peptides, and their color-coded location on the exterior of the PSA protein molecule in aqueous solution, are presented in
The stability of a peptide against proteolytic degradation is an important factor for the use of peptides in vivo. Peptides are frequently modified to prevent enzymatic degradation. Several approaches have been used to achieve peptide stability including use of D-amino acids, peptidomimetics or cyclization. For this Example we prepared a cyclic variant of peptide #3 and compared the biological activities of the linear and cyclic peptide. Peptide #3 (181Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys) (SEQ ID NO:3) had one cysteine residue (#184) in its structure and it was replaced with alanine during synthesis. It is demonstrated that linear peptide #3 at 10 μM concentration significantly inhibits HUVEC tube formation in Matrigel (
Linear and cyclic peptides were evaluated for their ability of inhibit HUVEC tube formation in an antiangiogenic assay in Matrigel. The results are shown in
The following Example provides a description of our characterization of two putative binding sites for PSA on receptor(s) of Human Umbilical Vein Endothelial cells. Illustrative mass spectroscopy results are shown in
Biotinylation of f-PSA
The procedure for biotin labeling of proteins is known in the art. In brief, 10 mM of Sulfo-NHS-LC-Biotin (Pierce, Rockford, Ill.) was dissolved in PBS, pH 7.4 and added in 20-fold molar excess to 5 mg of f-PSA and incubated at room temperature for 30 min. Excess reactive biotin was quenched by the adding 1M Tris, pH 7.2 at 1/10th the volume of the biotin labeling reaction, and incubated at room temperature for 10 mM Biotinylated PSA was separated from excess biotin by centrifugation at 2000 rpm using a concentrator with a 10,000 MW cut off membrane (Millipore Corp., Billerica, Mass.). The labeled PSA was aliquoted in 0.5 mL volumes and frozen at −80° C. Biotinylated PSA was detected by ELISA using anti-PSA antibodies and by Western Blot using HRP-conjugated Streptavidin (Pierce, Rockford, Ill., USA).
Biotinylated PSA and Isolation of PSA-Target Complexes
The details of the protocol for isolation of receptor targets using biotinylated PSA and crosslinking approach are as follows: Approximately 30×106 HUVECs were grown on petri dishes (10 cm, BD Falcon, Franklin Lakes, N.J.) to 90% confluency. Media was removed and the cells were washed once with PBS. Cells were incubated with serum free media containing 10 μM biotin labeled PSA and 1:100 (v/v) protease inhibitor cocktail (Sigma, St. Louis, Mo.) for 15 minutes at 4° C. The binding solution was removed and 25 mM of dithiobis[succinimidyl-propionate] (DSP) crosslinker, dissolved in DMSO, was added at final concentration of 10 mM to each dish and incubated for 30 min at room temperature. The crosslinking reaction was stopped by adding 1M Tris, pH 7.2 for a final concentration of 20 mM for 15 minutes at room temperature. Cells were removed from the dishes by using a cell scraper (Costar, Corning, N.Y.) and washed with PBS. Cells collected were centrifuged at 1000 rpm and the supernatant discarded. The cell pellet was lysed using mammalian protein extraction reagent (M-PER) (Pierce, Rockford, Ill.) containing 1:100 (v/v) protease inhibitor cocktail for 15 minutes at room temperature and then centrifuged at 14,000 rpm for 10 minutes. The supernatant was collected to a new tube for further processing. To isolate the PSA-target complex, the cell debris pellet was mixed with non-reducing loading buffer or NP-40 (Sigma, St. Louis, Mo.) at a 1:1 (v/v) ratio and incubated in a 60° C. water bath to re-solubilize membrane proteins, which may contain the biotin labeled PSA crosslinked to the target molecule(s). The supernatant was collected and run on SDS/PAGE, western blot analysis, and probed with streptavidin-HRP for the PSA-target complex. Based on the presence of a band higher than 33 kDa, the re-solubilized sample was run on SDS/PAGE, stained with Deep Purple Total Protein Stain (GE Healthcare Life Sciences, Piscataway, N.J.), the band of interest was cut out, and sent for mass spectroscopy analysis.
Trypsin Digestion
The solubilized cellular fraction was run under non-reducing conditions on an SDS-PAGE gel, stained with Deep Purple Total Protein Stain (GE Healthcare, Piscataway, N.J.) and scanned using a Typhoon 9410 Imager (GE Healthcare, Piscataway, N.J.). Protein bands of interest were excised manually and placed in 0.6 mL microtube (Axygen, pre-washed with 18MΩ·cm water (Milli-Q) and methanol). In-gel trypsin digestion was performed according to standard operating procedures routinely used in the Roswell Park Cancer Institute
Proteomics Facility. In brief, gel pieces were de-stained with 200 μL 50% acetonitrile/100 mM ammonium bicarbonate solution for 30 minutes with constant mixing (MixMate, Eppendorf, Hauppauge, N.Y.). After removal of the de-stain solution, gel pieces were dehydrated in 100 μL acetonitrile for 15 minutes at room temperature (RT) and dried in a Speedvac concentrator (Eppendorf, Hauppauge, N.Y.). Dried gel pieces were reduced with 10 mM DTT (Sigma, St. Louis, Mo.)/100 mM sodium bicarbonate solution at RT for 45 minutes. After removal of this solution, samples were alkylated with 200 μL 50 mM iodoacetamide (Sigma, St. Louis, Mo.)/100 mM sodium bicarbonate at RT for 30 minutes under dark conditions. After removal of alkylation solution, samples were washed with 200 μL of 100 mM sodium bicarbonate and incubated with 200 μL of 50% acetonitrile/100 mM sodium bicarbonate at RT for 10 minutes. Samples were dehydrated with 100 μL acetonitrile and dried in a Speedvac concentrator and digested with trypsin (Promega, Madison, Wis., 10 ng/μL in 10% acetonitrile/40 mM sodium bicarbonate, 30-50 μL) at 37° C. for 16 hours. The digests were extracted twice with 100 μL 50% acetonitrile/0.1% TFA at RT for 60 minutes with constant mixing. The extracts were pooled and dried in a Speedvac concentrator and each sample was then reconstituted with 8 μL of 2% formic acid (FA).
LC-MS/MS Analysis
Trypsin digested samples (5 μL each) were analyzed by LC nanoelectrospray-tandem mass spectrometry (LC-ESI-MS/MS) using a nanoACQUITY UPLC (Waters Corp, Milford, Mass.) coupled through a nebulization-assisted nanospray ionization source to a Q-ToF Premier mass spectrometer (Waters/Micromass, Milford, Mass.) The LC consisted of a trap column (Symmetry C18, 5μ, 180μ×20 mm, Waters, Milford, Mass.), followed by separation on an analytical column (Atlantis C18, 3μ, 100μ×10 cm, Walters, Milford, Mass.). Samples were loaded, trapped, and washed at a flow rate of 3 μL/min with 98% solvent A (water containing 0.1% FA)/2% solvent B (acetonitrile containing 0.1% FA) for 5 minutes. Peptides were eluted with a gradient of 98% A/2% B to 40% A/60% B for 40 minutes at 0.4 μL/min, 10% A/90% B for 7 minutes at 1.5 μL/minute, and then 10% A/90% B at 1.5 μL/minute for 8 minutes. Throughout the gradient, the mass spectrometer was programmed (Data Dependent Acquisition experiment, DDA) to monitor ions with m/z in the range of 300-1500, and ions with +2 to +4 charges only were selected for MS/MS experiments using the preset DDA collision energy parameters.
Database Search and Peptide and Protein Identification
MS/MS spectra were processed and transformed to the PKL file formation using Proteinlynx Global Server v2.3 (Waters/Micromass, Milford, Mass.) and the default parameters of MaxEnt3 (Waters/Micromass, Milford, Mass.). The PKL files were used to search the Homo sapiens subset of the Swiss-Prot database (containing 20,352 sequences) using a locally installed version of MASCOT (Matrix Science, v 2.2.2). The search parameters were as follows: trypsin as the proteolytic enzyme with 2 possible missed cleavages, carboxyamidomethylation of cysteine as a fixed modification, NHS-LC-Biotin of lysine, and 3-(carbamidomethylthio)propanoyl of lysine as a variable modification, the allowable mass error was 100 ppm for peptides and 100 mDa for fragment ions, peptide charge was set to 2+ and 3+, the instrument was set to ESI-QUAD-TOF. The mascot default significance threshold of p<0.05 for assignments was used in the searches and a minimum of two unique peptides were used as a criteria for a match. This example suggests there is/are proteins on the cell membrane that are specific binding sites for intact PSA and that these receptors modulate the biological effect of PSA.
Example 9The following materials and methods were used in obtaining some of the data presented herein.
Fmoc-Based Solid-Phase Peptide Synthesis: Peptides were prepared by manual solid-phase peptide synthesis. Fmoc groups for Na protection were cleaved by 8 min treatment with 20% piperidine in DMF followed by the second treatment with the same reagent for 10 min. After the Fmoc cleavage, the rink-resin was washed with DMF (×6). The next residue was then incorporated with the DIPC/HOBt coupling protocol [Fmoc-amino acid (3 equiv), DIPC (3 equiv), and HOBt (3 equiv)]. After gentle agitation (1 hr) and washing with DMF (×6), part of the peptide-resin was subjected to the Kaiser test. On completion of the assembly, the peptide-resin was successively washed with DMF (×3), DCM (×4) and then dried in vacuo.
Cleavage and Deprotection. The cleavage cocktail (Trifluoroacetic acid/H2O=95/5, v/v; 3 ml/100 mg of resin) is added to the washed resin. Complete deprotection was achieved in the cocktail in 4 hrs at 30° C. Following the cleavage reaction, TFA was removed by evaporation.
Disulfide Bond Formation. After cleaved from resin, the crude linear peptide was obtained, which was characterized by RP-HPLC and Maldi-TOF MS. Disulfide formation was realized by oxidation in air (pH˜8), which was monitored by RP-HPLC, MS and free sulfhydryl detection (DTNB method).
Purification by Preparative RP-HPLC and Purity Assessment by Analytical HPLC. A crude peptide sample was purified by preparative RP-HPLC (HP1100, Agilent) using a column of Daiso C18 (10 μm, 100 Å, 50×250 mm). A solvent system consisting of solvent A (0.05% TFA, 2% CH3CN in water) and solvent B (90% CH3CN/H2O) at a flow rate of 25 mL/min was used for elution, and the absorbance was detected at 220 nm. The solvent was removed by lyophilization to afford a fluffy powder as a final purified peptide. The chemical structure was characterized by MALDI-TOF-MS, and the purity of the purified material was assessed by analytical HPLC (C18-4.6×250 mm, flow rate of 1 mL/min), and the absorbance was detected at 220 nm
Example 10Using peptides made and tested as described above, the data presented in
The foregoing description of specific embodiments is for the purpose of illustration and is not to be construed as restrictive. From the teachings of the present invention, those skilled in the art will recognize that various modifications and changes may be made without departing from the spirit of the invention.
Claims
1. A peptide having a sequence selected from Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp Ser Ser His (SEQ ID NO:1), Cys Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp Ser Ser His Cys (SEQ ID NO:7), Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys (SEQ ID NO:3), Val Ala Ala Gln Val His Pro Gln Lys Val Thr Lys (SEQ ID NO:8), Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Cys Ala (SEQ ID NO:6), and Asn Asp Val Ala Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Ala Ala (SEQ ID NO:9).
2. The peptide of claim 1, wherein the peptide is cyclic.
3. The peptide of claim 2, wherein the peptide has the sequence of SEQ ID NO:6, and wherein the two Cys in the sequence of SEQ ID NO:6 are connected to one another via a disulfide bond, or wherein the peptide has the sequence of SEQ ID NO:7, and wherein the two Cys in the sequence of SEQ ID NO:7 are connected to one another via a disulfide bond.
4. The peptide of claim 1, wherein the peptide comprises one, two, three, or four conservative amino acid substitutions, and wherein the peptide is capable of inhibiting tubule formation by human umbilical vein endothelial cells.
5. The peptide of claim 1, wherein the peptide is present in a composition comprising a pharmaceutically acceptable carrier.
6. The peptide of claim 5, wherein the peptide is cyclic.
7. The composition of claim 6, wherein the isolated peptide has the sequence of SEQ ID NO:6, and wherein the two Cys in the sequence of SEQ ID NO:6 are connected to one another via a disulfide bond, or wherein the isolated peptide has the sequence of SEQ ID NO:7, and wherein the two Cys in the sequence of SEQ ID NO:7 are connected to one another via a disulfide bond.
8. A method of inhibiting angiogenesis in an individual comprising administering a composition comprising a peptide of claim 1 to an individual in an amount effective to inhibit the angiogenesis in the individual.
9. The method of claim 8, wherein the inhibition of angiogenesis comprises inhibition of angiogenesis in a tumor in the individual.
10. The method of claim 8, wherein the peptide is cyclic.
11. The method of claim 10, wherein the peptide has the sequence of SEQ ID NO:6, and wherein the two Cys in the sequence of SEQ ID NO:6 are connected to one another via a disulfide bond, or wherein the isolated peptide has the sequence of SEQ ID NO:7, and wherein the two Cys in the sequence of SEQ ID NO:7 are connected to one another via a disulfide bond.
12. The method of claim 8, wherein the composition comprises more than one peptide of claim 1.
13. A method of inhibiting human endothelial cell migration comprising contacting human endothelial cells with a composition comprising a peptide of claim 1, wherein the migration is inhibited subsequent to the contacting of the human endothelial cells with the composition.
14. The method of claim 13, wherein the peptide is cyclic.
15. The method of claim 14, wherein the peptide has the sequence of SEQ ID NO:6, and wherein the two Cys in the sequence of SEQ ID NO:6 are connected to one another via a disulfide bond, or wherein the isolated peptide has the sequence of SEQ ID NO:7, and wherein the two Cys in the sequence of SEQ ID NO:7 are connected to one another via a disulfide bond.
Type: Application
Filed: Jul 23, 2012
Publication Date: Oct 16, 2014
Applicant: HEALTH RESEARCH, INC. (Buffalo, NY)
Inventors: Kailash C. Chadha (Williamsville, NY), Gary J. Smith (Buffalo, NY)
Application Number: 14/235,563
International Classification: C07K 7/08 (20060101); A61K 38/10 (20060101); A61K 38/08 (20060101); C07K 7/06 (20060101);
