title: Enzymatic Characterization of ER Stress-Dependent Kinase, PERK, and Development of a High-Throughput Assay for Identification of PERK Inhibitors.
abstract: PERK is serine/threonine kinase localized to the endoplasmic reticulum (ER) membrane. PERK is activated and contributes to cell survival in response to a variety of physiological stresses that affect protein quality control in the ER, such as hypoxia, glucose depravation, increased lipid biosynthesis, and increased protein translation. Pro-survival functions of PERK are triggered by such stresses, suggesting that development of small-molecule inhibitors of PERK may be efficacious in a variety of disease scenarios. Hence, we have conducted a detailed enzymatic characterization of the PERK kinase to develop a high-throughput-screening assay (HTS) that will permit the identification of small-molecule PERK inhibitors. In addition to establishing the K(m) of PERK for both its primary substrate, eIF2α, and for adenosine triphosphate, further mechanistic studies revealed that PERK targets its substrate via either a random/steady-state ordered mechanism. For HTS, we developed a time-resolved fluorescence resonance energy transfer-based assay that yielded a robust Z' factor and percent coefficient of variation value, enabling the successful screening of 79,552 compounds. This approach yielded one compound that exhibited good in vitro and cellular activity. These results demonstrate the validity of this screen and represent starting points for drug discovery efforts.
authors.affiliation: The Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, USA Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA, USA Abramson Cancer Center and Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA.
authors.forename: Dariusz
authors.initials: D
authors.lastname: Pytel
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Seyb
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA.
authors.forename: Soumya S
authors.initials: SS
authors.lastname: Ray
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA.
authors.forename: John
authors.initials: J
authors.lastname: Concannon
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA.
authors.forename: Mickey
authors.initials: M
authors.lastname: Huang
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Cuny
authors.affiliation: The Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, USA Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA, USA Abramson Cancer Center and Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA adiehl@mail.med.upenn.edu mglicksman@rics.bwh.harvard.edu.
authors.forename: J Alan
authors.initials: JA
authors.lastname: Diehl
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA.
authors.forename: Marcie A
authors.initials: MA
authors.lastname: Glicksman
mesh_terms: D000818:Animals; D003198:Computer Simulation; D015195:Drug Design; D055808:Drug Discovery / Q000379:methods*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D059865:Endoplasmic Reticulum Stress; D004791:Enzyme Inhibitors / Q000737:chemistry*; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry; D005347:Fibroblasts / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D051379:Mice; D010641:Phenotype; D010766:Phosphorylation; D015398:Signal Transduction; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry*
chemical_list: D004791:Enzyme Inhibitors; D015852:Eukaryotic Initiation Factor-2; D019892:eIF-2 Kinase; C460640:EIF2AK3 protein, human
keywords: HTS; PERK; small-molecule inhibitors