Identification and Characterization of PERK Activators by Phenotypic Screening and Their Effects on NRF2 Activation

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 number of hits were identified and validated. The molecular mechanisms for several selected hits were further characterized in terms of PERK activation and effects on PERK downstream components. Specificity of these compounds in activating PERK was demonstrated with a PERK specific inhibitor and in PERK knockout mouse embryonic fibroblast (MEF) cells. In addition, these hits showed NRF2-dependent anti-oxidant gene induction. In summary, our phenotypic screening assay is demonstrated to be able to identify PERK specific activators. The identified PERK activators could potentially be used as chemical probes to further investigate this pathway as well as the link between PERK activation and NRF2 pathway activation.

[1]  Rakesh Kumar,et al.  Conversion of 2-deoxyglucose-induced growth inhibition to cell death in normoxic tumor cells , 2013, Cancer Chemotherapy and Pharmacology.

[2]  A. Schönthal.  Pharmacological targeting of endoplasmic reticulum stress signaling in cancer. , 2013, Biochemical pharmacology.

[3]  S. Shapiro,et al.  Cigarette smoking affects oxidative protein folding in endoplasmic reticulum by modifying protein disulfide isomerase , 2013, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[4]  D. Slebos,et al.  Cigarette smoke induces endoplasmic reticulum stress response and proteasomal dysfunction in human alveolar epithelial cells , 2013, Experimental physiology.

[5]  R. Ames,et al.  Development of a cell-based high throughput luciferase enzyme fragment complementation assay to identify nuclear-factor-e2-related transcription factor 2 activators. , 2012, Assay and drug development technologies.

[6]  S. Kelsen.  Respiratory epithelial cell responses to cigarette smoke: the unfolded protein response. , 2012, Pulmonary pharmacology & therapeutics.

[7]  L. Shewchuk,et al.  Discovery of 7-methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), a potent and selective first-in-class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK). , 2012, Journal of medicinal chemistry.

[8]  C. M. Ribeiro,et al.  Endoplasmic reticulum stress in chronic obstructive lung diseases. , 2012, Current molecular medicine.

[9]  A. Blumental-Perry.  Unfolded protein response in chronic obstructive pulmonary disease: smoking, aging and disease: a SAD trifecta. , 2012, Current molecular medicine.

[10]  C. Rodrigues,et al.  Endoplasmic Reticulum Enrollment in Alzheimer’s Disease , 2012, Molecular Neurobiology.

[11]  N. Vij,et al.  Critical role of proteostasis-imbalance in pathogenesis of COPD and severe emphysema , 2011, Journal of Molecular Medicine.

[12]  Masayuki Yamamoto,et al.  Molecular mechanisms of the Keap1–Nrf2 pathway in stress response and cancer evolution , 2011, Genes to cells : devoted to molecular & cellular mechanisms.

[13]  R. Yu,et al.  Regulation of NF-E2-related factor 2 signaling for cancer chemoprevention: antioxidant coupled with antiinflammatory. , 2010, Antioxidants & redox signaling.

[14]  Bernd R. Binder,et al.  Oxidized Phospholipids Regulate Expression of ATF4 and VEGF in Endothelial Cells via NRF2-Dependent Mechanism: Novel Point of Convergence Between Electrophilic and Unfolded Protein Stress Pathways , 2010, Arteriosclerosis, thrombosis, and vascular biology.

[15]  F. Khodagholi,et al.  Stabilization of transcription factor Nrf2 by tBHQ prevents oxidative stress-induced amyloid beta formation in NT2N neurons. , 2010, Biochimie.

[16]  S. Biswal,et al.  Nrf2-dependent sulfiredoxin-1 expression protects against cigarette smoke-induced oxidative stress in lungs. , 2009, Free radical biology & medicine.

[17]  M. Sporn,et al.  Targeting Nrf2 with the triterpenoid CDDO- imidazolide attenuates cigarette smoke-induced emphysema and cardiac dysfunction in mice , 2009, Proceedings of the National Academy of Sciences.

[18]  John Calvin Reed,et al.  Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities , 2008, Nature Reviews Drug Discovery.

[19]  Shyam Biswal,et al.  Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. , 2007, Annual review of pharmacology and toxicology.

[20]  M. Sporn,et al.  Pharmacodynamic characterization of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-regulated genes , 2006, Molecular Cancer Therapeutics.

[21]  R. Ames,et al.  Gene expression in mammalian cells using BacMam, a modified baculovirus system. , 2007, Methods in molecular biology.

[22]  T. Iwawaki,et al.  Analysis of the XBP1 splicing mechanism using endoplasmic reticulum stress-indicators. , 2006, Biochemical and biophysical research communications.

[23]  F. Michelangeli,et al.  The Effects of the Phenylalanine 256 to Valine Mutation on the Sensitivity of Sarcoplasmic/Endoplasmic Reticulum Ca2+ ATPase (SERCA) Ca2+ Pump Isoforms 1, 2, and 3 to Thapsigargin and Other Inhibitors* , 2006, Journal of Biological Chemistry.

[24]  J. Diehl,et al.  Coordination of ER and oxidative stress signaling: the PERK/Nrf2 signaling pathway. , 2006, The international journal of biochemistry & cell biology.

[25]  R. Hamanaka,et al.  PERK and GCN2 contribute to eIF2alpha phosphorylation and cell cycle arrest after activation of the unfolded protein response pathway. , 2005, Molecular biology of the cell.

[26]  Makoto Kobayashi,et al.  Molecular mechanisms activating the Nrf2-Keap1 pathway of antioxidant gene regulation. , 2005, Antioxidants & redox signaling.

[27]  J. Alan Diehl,et al.  PERK-dependent Activation of Nrf2 Contributes to Redox Homeostasis and Cell Survival following Endoplasmic Reticulum Stress* , 2004, Journal of Biological Chemistry.

[28]  Randal J. Kaufman,et al.  Nrf2 Is a Direct PERK Substrate and Effector of PERK-Dependent Cell Survival , 2003, Molecular and Cellular Biology.

[29]  A. Choi,et al.  Identification of Activating Transcription Factor 4 (ATF4) as an Nrf2-interacting Protein , 2001, The Journal of Biological Chemistry.