Structure-enabled discovery of carboxylate esters prodrugs as potential potent inhibitors targeting Glutaminase C

The role of glutaminase C (GAC) in regulating glutaminolysis has established it as a promising therapeutic target for cancer treatment, driving increased interest in GAC inhibitors in recent years. The mitochondrial localization of GAC presents a particular challenge for balancing high target binding affinity with effective delivery to its subcellular site. Our previous study identified a basic hotspot within the allosteric pocket of GAC, which could be leveraged to enhance binding affinity. Herein, a series of acidic derivatives targeting the basic hotspot were designed based on the scaffold of CB839. This was followed by the development of prodrugs based on carboxylic acid inhibitors 9 and 11 to improve their lipophilicity. Prodrug 16 showed pronounced antiproliferative activity in A549 cells (IC₅₀ = 2.45 nM) alongside favorable stability, and additionally regulated cellular metabolites and increased reactive oxygen species by blocking glutamine metabolism. Ultimately, prodrug 16 exhibits strong in vivo antitumor activity in an A549 xenograft model, establishing it as a highly potent GAC inhibitor and suggesting a novel approach for the development of next-generation GAC-targeting therapeutics.