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.

Keywords: NF-κB; chemical genetics; eIF2α; endoplasmic reticulum stress; kinases.

Figure 1

Characterization of hit compounds. A) Structure of active compound DHBDC and its inactive analog BDC. B) Dose dependent activity of DHBDC and BDC in the dual luciferase surrogate eIF2α phosphorylation assay. C) Activity of the DHBDC and BDC in dual luciferase Xbp-1 mRNA splicing counter-assay. D) Induction of eIF2α phosphorylation and E) CHOP by DHBDC and BDC, Thapsigargin (TG) is used as a positive control. F/R = F to R luciferase ratio. The experiment was conducted in triplicate and each experiment was independently performed three times. Data are shown as Mean±S.E.M.

Figure 2

Identifying PKR and PERK as the molecular targets of DHBDC. A) Expression of all four eF2α kinases was knocked-down individually or in all combination by transfecting cell with siRNAs specific to all four eIF2α kinases or non-targeted siRNA and the effect of DHBDC on the ternary complex assay was determined. Only PKR and PERK dual knock-down is shown. B) Expression of all four eF2α kinases was knocked-down individually or PKR and PERK expression was knocked down simultaneously by transfecting cells with specific siRNAs. Effect of DHBDC treatment on eIF2α phosphorylation in the transfected cells was determined by western blot analysis with antibodies specific to phosphorylated and total eIF2α. C) and D) Expression of all four eF2α kinases was knocked-down as in B and the effect of DHBC treatment on CHOP mRNA expression was determined by real-time PCR (C) and CHOP protein by western blot analysis (D). NTC = Non-Target siRNA Control. F/R = F to R luciferase ratio. The experiment was conducted in triplicate and each experiment was independently performed three times. Data are shown as Mean±S.E.M.

Figure 3

Dependence of anti-proliferative activity of DHBDC on PKR and PERK. CRL-2813 human melanoma or MCF-7 cells were transfected with siRNAs targeting PKR, PERK, or both and treated with various doses of DHBDC for three days and cell proliferation was quantified by SRB assay. A) Growth inhibition curve for Non-Target Control and both PERK and PKR siRNAs in MCF-7 cells. B) Calculated IC₅₀ for Non-Target Control, PERK, PKR, and both PERK and PKR siRNAs in CRL-2813 and MCF-7 cells. NTC = Non-Target Control. The experiment was conducted in triplicate and each experiment was independently performed three times. Data are shown as Mean±S.E.M.

Figure 4

DHBDC inhibits expression of cyclin D1 and cyclin E. A) and B) CRL-2813 cells were treated with 10 or 20 μM DHBDC, 20 μM BDC or 50 nM TG and expression of Cyclin D1 protein was determined by Western blot (A) and cyclin D1 mRNA was quantified by real-time PCR (B). C) and D) CRL-2813 cells were treated with 20 μM DHBDC, 20 μM BDC or 50 nM TG and expression of Cyclin E protein was determined by Western blot (C) and cyclin E mRNA was quantified by real-time PCR (D). β-actin was utilized as a loading control for Western blot analysis. The experiment was conducted in triplicate and each experiment was independently performed three times. Data are shown as Mean±S.E.M.

Figure 5

Choromone ring and electron withdrawing substitution of phenyl ring is necessary but Michael-type acceptor is dispensable for the activity of DHBDC. A) Structure of various DHBDC analogs obtained for the preliminary SAR. B) and C). Activity of these analogs in the surrogate eIF2α phosphorylation assay. Reporter cell lines described in Figure S1 were treated with the indicated concentration of each compound and F/R ratio was determined. F/R = ratio of F to R luciferase. The experiment was conducted in triplicate and each experiment was independently performed three times. Data are shown as Mean±S.E.M.

Figure 6

DHBDC induces phosphorylation of IκBα and activates NF-κB promoters. A) CRL-2813 cells were treated for the indicated times with vehicle, DHBDC, TG or BDC, cell lysates were separated by SDS-PAGE and probed with antibodies specific to phosphorylated and total IκBα. B) Cells were transfected with a NF-κB responsive promoter driven F luciferase reporter plasmid and TK-promoter driven R luciferase reporter, treated with vehicle, TNF, DHBDC, BDC or BTdCPU and F/R ratio was determined. Almost all the observed change in F/R was accounted for by increased expression of F luciferase. C) Cells were co-transfected with non-targeted siRNA or siRNAs targeting PKR, PERK, or both and NF-κB promoter driven F luciferase and TK-promoter driven R luciferase plasmids used in A and B above. Cells were treated with DHBDC and F/R ratio was determined by dual luciferase assay. F/R = ratio of F to R luciferase. The experiment was conducted in triplicate and each experiment was independently performed three times. Data are shown as Mean±S.E.M.

Figure 7

DHBDC inhibits HCV infection. Huh7.5.1 human hepatoma cells were treated with 5 μM DHBDC or BDC, interferon alpha (PBL) 25IU/ml, or DMSO for 24 hours fallowed by addition of fully infectious JFH1 HCV. After 48h additional culture cells were fixed in 4% PFA, permeabilized and stained with anti-HCV primary and Alexa-488 tagged secondary antibody then Hoechst DNA stain. Cells were then imaged on an automated microscope at 4X for percent infected cells. Percent infection for all groups was normalized to that of DMSO treated cells. The experiment was conducted in triplicate and each experiment was independently performed three times. Data are shown as Mean±S.E.M.