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

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 13b ( LL202 ) was identified with robust GLS1 inhibitory activity (IC ₅₀ = 6 nM) and high GLS1 binding affinity (SPR, K _d = 24 nM; ITC, K _d = 37 nM). The X-ray crystal structure of the 13b -GLS1 complex was resolved, revealing a unique binding mode and providing a novel structural scaffold for GLS1 allosteric inhibitors. Importantly, 13b clearly adjusted the cellular metabolites and induced an increase in the ROS level by blocking glutamine metabolism. Furthermore, 13b exhibited a similar in vivo antitumor activity as CB839 . 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.