Enzymatic Characterization of ER Stress-Dependent Kinase, PERK, and Development of a High-Throughput Assay for Identification of PERK Inhibitors


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 Km of PERK for both its primary substrate, eIF2?, 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.



PERK, HTS, small-molecule inhibitors


Copyright 2014 Journal of Biomolecular Screening. This is a post-print versoin of a published paper that is available at: https://journals.sagepub.com/doi/full/10.1177/1087057114525853. Recommended citation: Pytel, Dariusz, Kathleen Seyb, Min Liu, Soumya S. Ray, John Concannon, Mickey Huang, Gregory D. Cuny, J. Alan Diehl, and Marcie A. Glicksman. "Enzymatic characterization of ER stress-dependent kinase, PERK, and development of a high-throughput assay for identification of PERK inhibitors." Journal of biomolecular screening 19, no. 7 (2014): 1024-1034.. doi: 10.1177/1087057114525853. This item has been depositied in accordance with publisher copyright and licensing terms and with the author's permission.