Targeting the Subpocket Enables the Discovery of Thiadiazole-Pyridazine Derivatives as Glutaminase C Inhibitors

As Glutaminase C (GAC) has become an attractive target for Cancer treatment by regulating glutaminolysis, thus, interest in GAC inhibitors has risen in recent years. Herein, a potential binding subpocket comprising basic residues was identified, and through extensive structure-activity relationship studies, promising inhibitors 11 and 39 were identified with robust GAC inhibitory activity and A549 cell antiproliferative activity. X-ray crystallography of the 11-GAC and 27-GAC complexes revealed a novel binding mode against GAC. The potency of 11 and 27 against GAC^K320A further highlighted the importance of the binding. Notably, compounds 11 and 39 regulated the cellular metabolite, thereby increasing Reactive Oxygen Species by blocking glutamine metabolism. Compound 11 also exhibited excellent antiproliferative activity in the A549 cell xenograft model. We further proved that 11 is a safe GAC allosteric inhibitor. A basic subpocket is proposed that might provide new strategies for the development of novel GAC inhibitors in the future.