Acknowledgements: We thank Shashank Rai and LMB Animal Facility for help with animal experiments; Ketan Malhotra for help with protein purification; Stephen McLaughlin for help with SEC-MALS and thermal shift experiments; Alison Inglis, Olga Perisic and Roger Williams for GCN2 and eIF2α plasmids, as well as, recombinant GCN2 pseudokinase and kinase domains; Michel Goedert and Petra Gross for insightful comments on the manuscript; Emily Naden for proof-reading the paper. This work was supported by Medical Research Council (UK) grant MC_U105185860 and Wellcome Trust Principal Investigator Award 206367/Z/ 17/Z to A.B. M.S. was supported by Human Frontier Science Program (HFSP) LT000162/2021-L and European Molecular Biology Organization (EMBO) ALTF 698-2020.Funder: Medical Research Council MC_U105185860 Wellcome Trust 206367/Z/ 17/ZPhosphorylation of the translation initiation factor eIF2α to initiate the integrated stress response (ISR) is a vital signalling event. Protein kinases activating the ISR, including PERK and GCN2, have attracted considerable attention for drug development. Here we find that the widely used ATP-competitive inhibitors of PERK, GSK2656157, GSK2606414 and AMG44, inhibit PERK in the nanomolar range, but surprisingly activate the ISR via GCN2 at micromolar concentrations. Similarly, a PKR inhibitor, C16, also activates GCN2. Conversely, GCN2 inhibitor A92 silences its target but induces the ISR via PERK. These findings are pivotal for understanding ISR biology and its therapeutic manipulations because most preclinical studies used these inhibitors at micromolar concentrations. Reconstitution of ISR activation with recombinant proteins demonstrates that PERK and PKR inhibitors directly activate dimeric GCN2, following a Gaussian activation-inhibition curve, with activation driven by allosterically increasing GCN2 affinity for ATP. The tyrosine kinase inhibitors Neratinib and Dovitinib also activate GCN2 by increasing affinity of GCN2 for ATP. Thus, the mechanism uncovered here might be broadly relevant to ATP-competitive inhibitors and perhaps to other kinases
