Structure-Enabled Discovery of Novel Macrocyclic Inhibitors Targeting Glutaminase 1 Allosteric Binding Site

Abstract

The inhibition of glutaminase 1 (GLS1) represents a potential treatment of malignant tumors. Structural analysis led to the design of a novel series of macrocyclic GLS1 allosteric inhibitors. Through extensive structure-activity relationship studies, a promising candidate molecule <b>13b</b> (<b>LL202</b>) was identified with robust GLS1 inhibitory activity (IC₅₀ = 6 nM) and high GLS1 binding affinity (SPR, <i>K</i>_d = 24 nM; ITC, <i>K</i>_d = 37 nM). The X-ray crystal structure of the <b>13b</b>-GLS1 complex was resolved, revealing a unique binding mode and providing a novel structural scaffold for GLS1 allosteric inhibitors. Importantly, <b>13b</b> clearly adjusted the cellular metabolites and induced an increase in the ROS level by blocking glutamine metabolism. Furthermore, <b>13b</b> exhibited a similar <i>in vivo</i> antitumor activity as <b>CB839</b>. This study adds to the growing body of evidence that macrocyclization provides an alternative and complementary approach for the design of small-molecule inhibitors, with the potential to improve the binding affinity to the targets.