Abstract 1155: Regulation of glutamine metabolism: Allosteric activation and inhibition of mitochondrial glutaminase

Abstract

Abstract The hydrolysis of glutamine to glutamate plus ammonia, catalyzed in the mitochondria by the enzyme glutaminase (Gls), plays a crucial role in the metabolism of glutamine by cancer cells. Recently, we discovered a novel class of allosteric inhibitors of a splice variant of Gls, known as glutaminase C (GAC), which inhibits the growth of Rho-GTPase transformed cells without affecting their normal cellular counterparts, with the lead compound being the bromo-benzophenanthridinone 968. Here, we took advantage of mouse embryonic fibroblasts (MEFs) transformed by the induced expression of an oncogenic guanine nucleotide exchange factor (GEF) named Dbl, which hyper-activates the Rho GTPases Rac, Rho, and CDC42, together with 13C-labeled glutamine and stable-isotope tracing methods, to establish that 968 selectively blocks the enhancement in glutaminolysis. We then determined how 968 inhibits the catalytic activity of GAC using biophysical approaches on purified GAC enzyme. First, we developed a FRET assay to examine the effects of 968 on the ability of GAC to undergo the dimer-to-tetramer transition necessary for enzyme activation. We next demonstrated how the fluorescence of a reporter group attached to GAC provides a direct read-out of the binding of 968 and related compounds to the enzyme. By combining these fluorescence assays, together with novel GAC mutants trapped in either the monomeric or dimeric state, we show that 968 has the highest affinity for monomeric GAC, and that the dose-dependent binding directly matches its dose-dependent inhibition of enzyme activity and cellular transformation. We then investigated the 968 structure-activity relationship, and to this end, characterized a robust real-time direct binding and inhibition assay for testing a panel of 968 derivatives. Additionally, we have developed novel 968-derivatives with unique functional moieties, such as fluorescent and photo-cross linking groups, to further elucidate the mechanism of GAC inhibition using recombinant GAC in vitro as well as in cancer cells. Together, these findings represent recent advances in our understanding of the regulation of GAC activity both in vitro and in cells, and highlight the potential future of targeting glutamine metabolism with novel small molecule therapeutics. Citation Format: Clint Stalnecker, Scott Ulrich, Jon Erickson, Sekar Ramachandran, Ralph DeBerardinis, Rick Cerione. Regulation of glutamine metabolism: Allosteric activation and inhibition of mitochondrial glutaminase. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1155. doi:10.1158/1538-7445.AM2015-1155