Patents Assigned to Discoverx Corporation
-
Patent number: 8467970Abstract: A method of screening biologically active agent based on the analysis of complex biological responses in culture. Methods for selecting cells and culture conditions for such screens are provided, as well as the identification of an optimized set of discrete parameters to be measured, and the use of biomap analysis for rapid identification and characterization of drug candidates, genetic sequences acting pathways, and the like. A feature of the invention is simultaneous screening of a large number of cellular pathways, and the rapid identification of compounds that cause cellular responses.Type: GrantFiled: November 17, 2003Date of Patent: June 18, 2013Assignee: DiscoveRx CorporationInventors: Ellen L. Berg, Eugene C. Butcher, Jennifer Melrose
-
Publication number: 20120329075Abstract: Methods and materials are disclosed for use in an enzyme fragment complementation assay using complementary fragments of ?-galactosidase to study the trafficking of proteins in a cell. Compounds that bind to a target peptide have been found to affect protein folding and therefore trafficking. ?-Galactosidase fragments, an enzyme donor (ED) and an enzyme acceptor (EA), are fused to a target peptide and to an intracellular compartment protein, wherein the compartment is involved in intracellular trafficking. Contacting the cell with a compound that binds to the target peptide results in enhanced movement of the protein through the cellular trafficking pathway comprised of the endoplasmic reticulum, Golgi apparatus, the plasma membrane, endosomes, etc. Using this approach, compounds that bind to a target peptide and alter its ability to traffic through the normal cellular pathway can be readily detected.Type: ApplicationFiled: June 22, 2012Publication date: December 27, 2012Applicant: DISCOVERX CORPORATIONInventors: Thomas S. Wehrman, Daniel Bassoni, William Raab
-
Patent number: 8211655Abstract: A method for determining ligand activation of receptors using cells expressing genetic constructs of a fusion protein of at least a binding domain of an auxiliary protein and a fragment of ?-galactosidase, a fusion protein of an endosome-associated protein and a complementary fragment of ?-galactosidase, and a wild-type receptor. The receptors are characterized by binding to the auxiliary protein-binding domain upon activation by an agonist and then endocytosing associated with an endosome to which the endosome-associated protein binds. Cells are incubated with a candidate ligand followed by lysis with a lysing medium comprising a substrate for the ?-galactosidase. The enzyme product is then detected as a measure of the activation of the receptor.Type: GrantFiled: February 11, 2010Date of Patent: July 3, 2012Assignee: Discoverx CorporationInventors: Thomas S. Wehrman, William Raab, Chin Yee Loh
-
Publication number: 20120045769Abstract: Provided herein are assays useful, for example, for determining the activity of a protein involved in a cellular process. In some embodiments, the activity of the protein is assessed using a nucleic acid tag, and in particular, by detecting the presence of a nucleic acid tag. Such assays can be used, for example, to study the effects of test compounds as modulators, e.g., inhibitors, agonists and antagonists, of protein activity.Type: ApplicationFiled: April 23, 2010Publication date: February 23, 2012Applicant: DISCOVERX CORPORATIONInventors: Daniel Kelly Treiber, Warren G. Lewis, Lisa M. Wodicka
-
Publication number: 20120040372Abstract: Methods for detecting phosphorylation of receptor tyrosine kinases (“RTKs”) upon activation and the modulation of activation by a candidate compound are provided. The method employs cells comprising two fusion products: (1) an RTK fused to a small fragment of ?-galactosidase and (2) a phosphotyrosine binding peptide fused to the large fragment of ?-galactosidase, where the 2 fragments weakly complex to form an active enzyme, and optionally a construct for a cytosolic RTK phosphorylating kinase, when the RTK does not autophosphoryate. To detect phosphorylation a ?-galactosidase substrate is added to the cells, whereby product formation indicates the occurrence of phosphorylation.Type: ApplicationFiled: October 25, 2011Publication date: February 16, 2012Applicant: DISCOVERX CORPORATIONInventors: Wei Feng, William Raab, Philip Achacoso, Thomas Wehrman, Keith R. Olson
-
Patent number: 8101373Abstract: Truncated fragments of the small fragment of ?-galactosidase are provided that have low affinity for the large fragment of ?-galactosidase and provide for robust signals when two fusion proteins are complexed due to the binding of the proteins to which the ?-galactosidase fragments are fused. The truncated fragments do not interfere with the complexing of the two proteins and allow for the two proteins to function and be responsive to candidate compounds that affect complex formation.Type: GrantFiled: October 10, 2008Date of Patent: January 24, 2012Assignee: Discoverx CorporationInventors: Thomas Scott Wehrman, Keith R. Olson
-
Patent number: 8067155Abstract: Methods for detecting phosphorylation of receptor tyrosine kinases (“RTKs”) upon activation are provided. The method employs cells comprising two fusion products: (1) an RTK fused to a small fragment of ?-galactosidase and (2) a phosphotyrosine binding peptide fused to the large fragment of ?-galactosidase, where the 2 fragments weakly complex to form an active enzyme, and optionally a construct for a cytosolic RTK phosphorylating kinase, when the RTK does not autophosphoryate. To detect phosphorylation a ?-galactosidase substrate is added to the cells, whereby product formation indicates the occurrence of phosphorylation.Type: GrantFiled: August 6, 2009Date of Patent: November 29, 2011Assignee: DiscoveRx CorporationInventors: Wei Feng, William Raab, Philip Achacoso, Thomas Wehrman, Keith R. Olson
-
Publication number: 20100203555Abstract: A method for determining ligand activation of receptors using cells expressing genetic constructs of a fusion protein of at least a binding domain of an auxiliary protein and a fragment of ?-galactosidase, a fusion protein of an endosome-associated protein and a complementary fragment of ?-galactosidase, and a wild-type receptor. The receptors are characterized by binding to the auxiliary protein-binding domain upon activation by an agonist and then endocytosing associated with an endosome to which the endosome-associated protein binds. Cells are incubated with a candidate ligand followed by lysis with a lysing medium comprising a substrate for the ?-galactosidase. The enzyme product is then detected as a measure of the activation of the receptor.Type: ApplicationFiled: February 11, 2010Publication date: August 12, 2010Applicant: DISCOVERX CORPORATIONInventors: Thomas S. Wehrman, William Raab, Chin Yee Loh
-
Publication number: 20100151496Abstract: Methods and genetic constructs are provided for detecting the binding of nuclear hormone receptors to a coactivator/corepressor. The methods employ enzyme fragment complementation using fragments of ?-galactosidase as the detection system. Cells are transformed to express the large fragment of ?-galactosidase fused to a member of the complex with NHR for initiation of transcription and have it localized in the nucleus and to express the small fragment of ?-galactosidase fused to the nuclear hormone receptor for binding to the member upon stimulation with a ligand.Type: ApplicationFiled: June 23, 2009Publication date: June 17, 2010Applicant: DiscoveRx CorporationInventors: Thomas S. Wehrman, Chin Yee Loh, Mahesh Mathrubutham, Keith R. Olson
-
Publication number: 20100120063Abstract: Sensitive assays for candidate compounds affecting GPCR activity are provided using a cell containing fusion proteins comprising a first fusion protein comprising (a) a target GPCR fused to a small fragment of ?-galactosidase through a linker comprising a phosphorylation site or (b) a GPCR or a protein of interest, where the GPCR and protein of interest form a complex and one of them is fused to the small fragment of ?-galactosidase; and a second fusion protein comprising arrestin fused to a large fragment of ?-galactosidase. In (a), the affinity of the small and large fragments is optimized based on the background to signal ratio and the absolute signal observed. The assay is performed using a ?-galactosidase substrate that provides a detectable optical signal.Type: ApplicationFiled: October 10, 2009Publication date: May 13, 2010Applicant: DISCOVERX CORPORATIONInventors: Daniel Bassoni, Keith R. Olson, Thomas S. Wehrman
-
Publication number: 20100041052Abstract: Methods for detecting phosphorylation of receptor tyrosine kinases (“RTKs”) upon activation are provided. The method employs cells comprising two fusion products: (1) an RTK fused to a small fragment of ?-galactosidase and (2) a phosphotyrosine binding peptide fused to the large fragment of ?-galactosidase, where the 2 fragments weakly complex to form an active enzyme, and optionally a construct for a cytosolic RTK phosphorylating kinase, when the RTK does not autophosphoryate. To detect phosphorylation a ?-galactosidase substrate is added to the cells, whereby product formation indicates the occurrence of phosphorylation.Type: ApplicationFiled: August 6, 2009Publication date: February 18, 2010Applicant: DISCOVERX CORPORATIONInventors: Wei Feng, William Raab, Philip Achacoso, Thomas Wehrman, Keith R. Olson
-
Patent number: 7608415Abstract: Improved methods of determining the intracellular state of a protein as well as modifications of the protein are provided by introducing a surrogate fusion protein comprising a member of an enzyme fragment complementation complex and a target protein. After exposing cells transformed with the surrogate fusion protein to a change in environment, e.g. a candidate drug, the cells are lysed, the lysate separated into fractions or bands, conveniently by gel electrophoresis and transferring the proteins by Western blot to a membrane. The bands on the membrane are developed using the other member of the enzyme fragment complementation complex and a substrate providing a detectable signal. The method is found to provide high sensitivity and the ability to observe modifications of the target protein.Type: GrantFiled: June 29, 2005Date of Patent: October 27, 2009Assignee: Discoverx CorporationInventors: Joseph Horecka, Peter Fung, Richard M. Eglen
-
Publication number: 20090098588Abstract: Truncated fragments of the small fragment of ?-galactosidase are provided that have low affinity for the large fragment of ?-galactosidase and provide for robust signals when two fusion proteins are complexed due to the binding of the proteins to which the ?-galactosidase fragments are fused. The truncated fragments do not interfere with the complexing of the two proteins and allow for the two proteins to function and be responsive to candidate compounds that affect complex formation.Type: ApplicationFiled: October 10, 2008Publication date: April 16, 2009Applicant: DiscoveRx CorporationInventors: Thomas Scott Wehrman, Keith R. Olson
-
Patent number: 7479377Abstract: A system is provided an enzyme donor (“ED”) fused a surrogate of a mammalian protein of interest, where the fusion protein has the function of the natural protein. A vector is provided comprising a regulatory region functional in a mammalian host cell, a sequence encoding the ED joined to a multiple cloning site, an enzyme acceptor (“EA”) protein or enzyme acceptor sequence encoding such protein, and substrate for the enzyme formed by ED and EA.Type: GrantFiled: August 27, 2002Date of Patent: January 20, 2009Assignee: DiscoveRx CorporationInventors: Sharon Zhao, Inna Vainshtein, Richard M. Eglen
-
Patent number: 7452690Abstract: Methods and reagents are provided for measuring protease activity. The reagent comprises a surface to which is linked an enzyme donor fragment through a protease recognition sequence, where the enzyme donor fragment complexes with an enzyme acceptor fragment to form an active indicator enzyme when the enzyme donor fragment is cleaved from said surface. Conveniently, the surface is a cell membrane surface where the reagent is expressed in the cell. The method comprises bringing together the reagent, the protease, the enzyme acceptor and substrate for the indicator enzyme and measuring the indicator enzyme activity as a measure of the protease activity. The method finds application in screening for compounds modulating the activity of the protease.Type: GrantFiled: January 28, 2003Date of Patent: November 18, 2008Assignee: DiscoveRx CorporationInventors: Pyare Khanna, Joseph L. Horecka
-
Patent number: 7371534Abstract: A sensitive intracellular calcium assay is disclosed comprising conveniently a reagent comprised of a dye precursor capable of entering cells and being hydrolyzed to a dye, whereby the dye complexes with calcium in the cells and provides a luminescent signal, an antibody specific for the dye and conjugated with a quencher, and a cellular anion exchange enzyme inhibitor. In performing the assay, the reagent is combined with cells expressing a receptor responsive to a ligand resulting in a change in cytosolic calcium. After incubation for the dye precursor to permeate the cells, the calcium may be determined by exciting the dye precursor and determining the peak fluorescence over a time course. The method can be used for measuring the effect of an agent on cytosolic calcium by binding to a cell surface membrane receptor.Type: GrantFiled: May 23, 2005Date of Patent: May 13, 2008Assignee: Discoverx CorporationInventors: Linda Kauffman, Rajendra Singh, Edwin F. Ullman