title: A FRET-based assay platform for ultra-high density drug screening of protein kinases and phosphatases.
abstract: Protein phosphorylation is one of the major regulatory mechanisms involved in signal-induced cellular events, including cell proliferation, apoptosis, and metabolism. Because many facets of biology are regulated by protein phosphorylation, aberrant kinase and/or phosphatase activity forms the basis for many different types of pathology. The disease relevance of protein kinases and phosphatases has led many pharmaceutical and biotechnology companies to expend significant resources in lead discovery programs for these two target classes. The existence of >500 kinases and phosphatases encoded by the human genome necessitates development of methodologies for the rapid screening for novel and specific compound inhibitors. We describe here a fluorescence-based, molecular assay platform that is compatible with robotic, ultra-high throughput screening systems and can be applied to virtually all tyrosine and serine/threonine protein kinases and phosphatases. The assay has a coupled-enzyme format, utilizing the differential protease sensitivity of phosphorylated versus nonphosphorylated peptide substrates. In addition to screening individual kinases, the assay can be formatted such that kinase pathways are re-created in vitro to identify compounds that specifically interact with inactive kinases. Miniaturization of this assay format to the 1-microl scale allows for the rapid and accurate compound screening of a host of kinase and phosphatase targets, thereby facilitating the hunt for new leads for these target classes.
authors.affiliation: Aurora Biosciences Corp, and Vertex Pharmaceuticals, Inc, San Diego, CA, USA.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Rodems
authors.forename: Brian D
authors.initials: BD
authors.lastname: Hamman
authors.forename: Christina
authors.initials: C
authors.lastname: Lin
authors.forename: Jane
authors.initials: J
authors.lastname: Zhao
authors.forename: Sundeep
authors.initials: S
authors.lastname: Shah
authors.forename: David
authors.initials: D
authors.lastname: Heidary
authors.forename: Lew
authors.initials: L
authors.lastname: Makings
authors.forename: Jeffrey H
authors.initials: JH
authors.lastname: Stack
authors.forename: Brian A
authors.initials: BA
authors.lastname: Pollok
mesh_terms: D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D001681:Biological Assay / Q000295:instrumentation*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D008387:Marine Toxins; D052998:Microcystins; D019151:Peptide Library; D010456:Peptides, Cyclic / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000737:chemistry* / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D019311:Staurosporine / Q000494:pharmacology; D013449:Sulfonamides; D014638:Vanadates / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D007546:Isoquinolines; D008387:Marine Toxins; D052998:Microcystins; D019151:Peptide Library; D010456:Peptides, Cyclic; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D014638:Vanadates; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; C057862:cyanoginosin LR; D019311:Staurosporine; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide