Abstract

Prexasertib (Prex) is a selective checkpoint kinase 1 and 2 (CHK1/2) inhibitor reported to induce the phosphorylation of AMP-activated protein kinase (AMPK) at threonine 172. However, the mechanism by which Prex regulates AMPK and whether this regulation contributes to Prex sensitivity is unknown. Here, we provide data that suggest that Prex directly binds to the cystathionine β-synthase (CBS) pockets of AMPK complex, leading to increased AMPKα Thr172 phosphorylation via CAMKK2 and LKB1. Intriguingly, Prex also functions as an ATP-competitive inhibitor of AMPK, and prolonged Prex treatment significantly inhibited AMPK in vivo . AMPK depletion enhanced cellular sensitivity to CHK1 inhibition, suggesting that Prex-induced cytotoxicity results from inhibition of CHK1 and AMPK. Exonuclease 1 (Exo1) hyperactivation following combined AMPK and CHK1 inhibition may represent a critical mechanism underlying Prex sensitivity. Our findings revealed an additional role of Prex in AMPK regulation and elucidated the functional significance of AMPK inhibition in CHK1 inhibitor-induced cell death.