Phosphorylation 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.

翻译起始因子 eIF2α 的磷酸化启动整合应激反应 (ISR) 是一个重要的信号传导事件。激活 ISR 的蛋白激酶（包括 PERK 和 GCN2）已引起药物开发的广泛关注。在这里，我们发现广泛使用的 PERK ATP 竞争性抑制剂 GSK2656157、GSK2606414 和 AMG44 在纳摩尔浓度范围内抑制 PERK，但令人惊讶的是在微摩尔浓度下通过 GCN2 激活 ISR。同样，PKR 抑制剂 C16 也会激活 GCN2。相反，GCN2 抑制剂 A92 沉默其靶标，但通过 PERK 诱导 ISR。这些发现对于理解 ISR 生物学及其治疗操作至关重要，因为大多数临床前研究都以微摩尔浓度使用这些抑制剂。用重组蛋白重建 ISR 激活表明，PERK 和 PKR 抑制剂直接激活二聚体 GCN2，遵循高斯激活-抑制曲线，通过变构增加 GCN2 对 ATP 的亲和力驱动激活。酪氨酸激酶抑制剂 Neratinib 和 Dovitinib 还可通过增加 GCN2 对 ATP 的亲和力来激活 GCN2。因此，这里发现的机制可能与 ATP 竞争性抑制剂以及其他激酶广泛相关。