An inhibitor of GCN2 and the integrated stress response directly targets ZAK protein kinase to limit cytotoxicity

The integrated stress response (ISR) is a major mechanism protecting cells against environmental and physiological stresses. Central to the ISR is a collection of stress-sensing kinases, such as GCN2 (EIF2AK4). When nutrients are limiting or translating ribosomes stall or collide, activated GCN2 phosphorylates eIF2, lowering global protein synthesis, which conserves resources and confers targeted expression of stress-adaptive genes, such as the transcription factor ATF4. While beneficial during acute stress, chronic GCN2 activation can promote Cancer progression and Neurological Disease, spurring the development of GCN2 inhibitors. However, achieving therapeutic specificity and understanding the pathological context of ISR modulation remains challenging and requires careful evaluation. One of the earliest and most widely used GCN2 inhibitors is GCN2iB. In this study, we report that GCN2iB is a direct inhibitor of the ZAK protein kinase, a critical upstream regulator of stress-activated MAPK signaling that functions in the ribotoxic stress response. Using biochemical measurements, cell-based assays, and structural modeling, we demonstrate that inhibition of ZAK by GCN2iB dampens stress-induced JNK and p38 activation, thereby masking the cytotoxic consequences normally associated with GCN2 inhibition. While suppression of GCN2 activity may be beneficial in specific disease models, concurrent inhibition of ZAK can negate these effects, obscure its therapeutic benefits, and lead to unanticipated phenotypes. These findings highlight the importance of assessing kinase selectivity in pharmacological studies of ISR modulation and emphasize that dual inhibition of GCN2 and ZAK can yield complex and context-dependent cellular responses.