Abstract
Endoplasmic reticulum stress plays a critical role to restore the homeostasis of protein production in eukaryotic cells. This vital process is hence involved in many types of diseases including COPD. PERK, one branch in the ER stress signaling pathways, has been reported to activate NRF2 signaling pathway, a known protective response to COPD. Based on this scientific rationale, we aimed to identify PERK activators as a mechanism to achieve NRF2 activation. In this report, we describe a phenotypic screening assay to identify PERK activators. This assay measures phosphorylation of GFP-tagged eIF2α upon PERK activation via a cell-based LanthaScreen technology. To obtain a robust assay with sufficient signal to background and low variation, multiple parameters were optimized including GFP-tagged eIF2α BacMam concentration, cell density and serum concentration. The assay was validated by a tool compound, Thapsigargin, which induces phosphorylation of eIF2α. In our assay, this compound showed maximal signal window of approximately 2.5-fold with a pEC50 of 8.0, consistent with literature reports. To identify novel PERK activators through phosphorylation of eIF2α, a focused set of 8,400 compounds was screened in this assay at 10 µM. A ...Continue Reading
References
Mar 29, 2001·The Journal of Biological Chemistry·C H HeJ Alam
Oct 1, 2003·Molecular and Cellular Biology·Sara B CullinanJ Alan Diehl
Feb 24, 2004·The Journal of Biological Chemistry·Sara B Cullinan, J Alan Diehl
Feb 12, 2005·Antioxidants & Redox Signaling·Makoto Kobayashi, Masayuki Yamamoto
Sep 24, 2005·Molecular Biology of the Cell·Robert B HamanakaJ Alan Diehl
Nov 18, 2005·The International Journal of Biochemistry & Cell Biology·Sara B Cullinan, J Alan Diehl
Jan 18, 2006·The Journal of Biological Chemistry·Laura L Wootton, Francesco Michelangeli
Sep 14, 2006·Annual Review of Pharmacology and Toxicology·Thomas W KenslerShyam Biswal
Oct 10, 2006·Biochemical and Biophysical Research Communications·Takao Iwawaki, Ryoko Akai
Jan 24, 2007·Molecular Cancer Therapeutics·Melinda S YatesThomas W Kensler
Jul 10, 2007·RNA·Jing ZhangDemin Zhou
Oct 24, 2007·Methods in Molecular Biology·James A FornwaldRobert S Ames
Nov 26, 2008·Free Radical Biology & Medicine·Anju SinghShyam Biswal
Dec 2, 2008·Nature Reviews. Drug Discovery·Inki KimJohn C Reed
Dec 24, 2008·Proceedings of the National Academy of Sciences of the United States of America·Thomas E SussanShyam Biswal
Dec 23, 2009·Biochimie·Bahareh EftekharzadehFariba Khodagholi
Feb 27, 2010·Arteriosclerosis, Thrombosis, and Vascular Biology·Taras AfonyushkinValery N Bochkov
May 22, 2010·Antioxidants & Redox Signaling·Rong HuAh-Ng Tony Kong
Jan 22, 2011·Genes to Cells : Devoted to Molecular & Cellular Mechanisms·Keiko TaguchiMasayuki Yamamoto
Feb 15, 2011·Journal of Molecular Medicine : Official Organ of the Gesellschaft Deutscher Naturforscher Und Ärzte·Taehong MinNeeraj Vij
May 12, 2012·Assay and Drug Development Technologies·Wensheng XieHu Li
Jun 16, 2012·Current Molecular Medicine·A Blumental-Perry
Jun 16, 2012·Current Molecular Medicine·C M P Ribeiro, W K O'Neal
Jul 11, 2012·Atherosclerosis·Taras AfonyushkinValery N Bochkov
Jul 21, 2012·Molecular Neurobiology·Ricardo J S VianaCecília M P Rodrigues
Jul 26, 2012·Journal of Medicinal Chemistry·Jeffrey M AxtenRobert T Gampe
Aug 1, 2012·Pulmonary Pharmacology & Therapeutics·Steven G Kelsen
Aug 1, 2012·Experimental Physiology·Anita Somborac-BacuraAntoon J M van Oosterhout
Sep 25, 2012·Biochemical Pharmacology·Axel H Schönthal
Nov 22, 2012·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Harshavardhan KencheAnna Blumental-Perry
May 24, 2013·Cancer Chemotherapy and Pharmacology·Huaping LiuTheodore J Lampidis
Citations
Mar 30, 2017·Pulmonary Pharmacology & Therapeutics·Li LinQiuyue Wang
May 11, 2016·Human Molecular Genetics·Gali MaorMia Horowitz
Sep 27, 2018·Cancers·Eoghan P McGrathAfshin Samali
Oct 23, 2019·Neural Regeneration Research·Wensheng Lin, Sarrabeth Stone
Sep 21, 2016·Expert Opinion on Therapeutic Patents·Jeffrey M Axten
Nov 1, 2016·IUBMB Life·Hector VegaMarek Michalak
Sep 6, 2020·The Journal of Biological Chemistry·Julia M D Grandjean, R Luke Wiseman
Jan 16, 2019·Journal of Medicinal Chemistry·Sabine DepauwMarie-Hélène David-Cordonnier