title: Dual activators of protein kinase R (PKR) and protein kinase R-like kinase PERK identify common and divergent catalytic targets.
abstract: Chemical genetics has evolved into a powerful tool for studying gene function in normal and pathobiology. PKR and PERK, two eukaryotic translation initiation factor 2 alpha (eIF2α) kinases, play critical roles in the maintenance of cellular hemostasis, metabolic stability, and anti-viral defenses. Both kinases interact with and phosphorylate additional substrates including tumor suppressor p53 and nuclear protein 90. Loss of function of both kinases has been studied by reverse genetics and with recently identified inhibitors. In contrast, no activating probes for studying the catalytic activity of these kinases are available. We identified 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5,7-dihydroxy-4H-chromen-4-one (DHBDC) as a specific dual activator of PKR and PERK by screening a chemical library of 20 000 small molecules in a dual luciferase surrogate eIF2α phosphorylation assay. We present here extensive biological characterization and a preliminary structure-activity relationship of DHBDC, which phosphorylates eIF2α by activating PKR and PERK but no other eIF2α kinases. These agents also activate downstream effectors of eIF2α phosphorylation by inducing CEBP homologue protein, suppressing cyclin D1 expression, and inhibiting cancer cell proliferation, all in a manner dependent on PKR and PERK. Consistent with the role of eIF2α phosphorylation in viral infection, DHBDC inhibits the proliferation of human hepatitis C virus. Finally, DHBDC induces the phosphorylation of IκBα and activates the NF-κB pathway. Surprisingly, activation of the NF-κB pathway is dependent on PERK but independent of PKR activity. These data indicate that DHBDC is an invaluable probe for elucidating the role of PKR and PERK in normal and pathobiology.
authors.affiliation: Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, 6 Rong Jing Jie, Beijing 100176, China.|Hematology Laboratory for Translational Research, Department of Medicine. Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA.|Harvard Medical School, 240 Longwood Avenue, Boston MA 02115.
authors.forename: Huijun
authors.initials: H
authors.lastname: Bai
authors.affiliation: Hematology Laboratory for Translational Research, Department of Medicine. Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA.|Harvard Medical School, 240 Longwood Avenue, Boston MA 02115.
authors.forename: Ting
authors.initials: T
authors.lastname: Chen
authors.affiliation: Harvard Medical School, 240 Longwood Avenue, Boston MA 02115.
authors.forename: Jie
authors.initials: J
authors.lastname: Ming
authors.affiliation: Harvard Medical School, 240 Longwood Avenue, Boston MA 02115.|Basic Medical College, Hebei United University, Tangshan, Hebei, 063000, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Sun
authors.affiliation: Harvard Medical School, 240 Longwood Avenue, Boston MA 02115.
authors.forename: Peng
authors.initials: P
authors.lastname: Cao
authors.affiliation: Gastrointestinal Unit, Massachusetts General Hospital Boston MA 02114.
authors.forename: Dahlene N
authors.initials: DN
authors.lastname: Fusco
authors.affiliation: Gastrointestinal Unit, Massachusetts General Hospital Boston MA 02114.
authors.forename: Raymond T
authors.initials: RT
authors.lastname: Chung
authors.affiliation: Hematology Laboratory for Translational Research, Department of Medicine. Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA.|Harvard Medical School, 240 Longwood Avenue, Boston MA 02115.
authors.forename: Michael
authors.initials: M
authors.lastname: Chorev
authors.affiliation: Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, 6 Rong Jing Jie, Beijing 100176, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Jin
authors.affiliation: Hematology Laboratory for Translational Research, Department of Medicine. Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA.|Harvard Medical School, 240 Longwood Avenue, Boston MA 02115.
authors.forename: Bertal H
authors.initials: BH
authors.lastname: Aktas
mesh_terms: D001578:Benzopyrans / Q000494:pharmacology*; D002384:Catalysis; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D013329:Structure-Activity Relationship; D014162:Transfection; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
chemical_list: D001578:Benzopyrans; D016328:NF-kappa B; D019892:eIF-2 Kinase
keywords: NF-κB; chemical genetics; eIF2α; endoplasmic reticulum stress; kinases