title: Identification and characterization of PERK activators by phenotypic screening and their effects on NRF2 activation.
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 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.
authors.affiliation: Department of Biological Sciences, Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania, United States of America.
authors.forename: Wensheng
authors.initials: W
authors.lastname: Xie
authors.affiliation: Stress & Repair Discovery Performance Unit, Respiratory Therapy Area, GlaxoSmithKline, King of Prussia, Pennsylvania, United States of America.
authors.forename: Marie
authors.initials: M
authors.lastname: Pariollaud
authors.affiliation: Stress & Repair Discovery Performance Unit, Respiratory Therapy Area, GlaxoSmithKline, King of Prussia, Pennsylvania, United States of America.
authors.forename: William E
authors.initials: WE
authors.lastname: Wixted
authors.affiliation: Hollings Cancer Center, Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, United States of America.
authors.forename: Nilesh
authors.initials: N
authors.lastname: Chitnis
authors.affiliation: Department of Biological Sciences, Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania, United States of America.
authors.forename: James
authors.initials: J
authors.lastname: Fornwald
authors.affiliation: Department of Biological Sciences, Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania, United States of America.
authors.forename: Maggie
authors.initials: M
authors.lastname: Truong
authors.affiliation: Department of Biological Sciences, Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania, United States of America.
authors.forename: Christina
authors.initials: C
authors.lastname: Pao
authors.affiliation: Department of Sample Management Technologies, Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania, United States of America.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Biological Sciences, Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania, United States of America.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Ames
authors.affiliation: Stress & Repair Discovery Performance Unit, Respiratory Therapy Area, GlaxoSmithKline, King of Prussia, Pennsylvania, United States of America.
authors.forename: James
authors.initials: J
authors.lastname: Callahan
authors.affiliation: Stress & Repair Discovery Performance Unit, Respiratory Therapy Area, GlaxoSmithKline, Stevenage, Hertfordshire, United Kingdom.
authors.forename: Roberto
authors.initials: R
authors.lastname: Solari
authors.affiliation: Stress & Repair Discovery Performance Unit, Respiratory Therapy Area, GlaxoSmithKline, King of Prussia, Pennsylvania, United States of America.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Sanchez
authors.affiliation: Hollings Cancer Center, Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, United States of America.
authors.forename: Alan
authors.initials: A
authors.lastname: Diehl
authors.affiliation: Department of Biological Sciences, Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania, United States of America.
authors.forename: Hu
authors.initials: H
authors.lastname: Li
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D059865:Endoplasmic Reticulum Stress; D015852:Eukaryotic Initiation Factor-2 / Q000032:analysis / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis; D057166:High-Throughput Screening Assays / Q000379:methods*; D006706:Homeostasis; D051379:Mice; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D010641:Phenotype; D010766:Phosphorylation; D014176:Protein Biosynthesis; D015398:Signal Transduction; D054852:Small Molecule Libraries; D019284:Thapsigargin / Q000737:chemistry; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*
chemical_list: D015852:Eukaryotic Initiation Factor-2; D049452:Green Fluorescent Proteins; D051267:NF-E2-Related Factor 2; D054852:Small Molecule Libraries; D019284:Thapsigargin; D019892:eIF-2 Kinase; C495636:Nfe2l2 protein, mouse