title: Mechanism by which a recently discovered allosteric inhibitor blocks glutamine metabolism in transformed cells.
abstract: The mitochondrial enzyme glutaminase C (GAC) catalyzes the hydrolysis of glutamine to glutamate plus ammonia, a key step in the metabolism of glutamine by cancer cells. Recently, we discovered a class of allosteric inhibitors of GAC that inhibit cancer cell growth without affecting their normal cellular counterparts, with the lead compound being the bromo-benzophenanthridinone 968. Here, we take advantage of mouse embryonic fibroblasts transformed by oncogenic Dbl, which hyperactivates Rho GTPases, together with (13)C-labeled glutamine and stable-isotope tracing methods, to establish that 968 selectively blocks the enhancement in glutaminolysis necessary for satisfying the glutamine addiction of cancer cells. We then determine how 968 inhibits the catalytic activity of GAC. 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 demonstrate 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 with newly developed 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 of 968 to GAC monomers directly matches its dose-dependent inhibition of enzyme activity and cellular transformation. Together, these findings highlight the requirement of tetramer formation as the mechanism of GAC activation and shed new light on how a distinct class of allosteric GAC inhibitors impacts the metabolic program of transformed cells.
authors.affiliation: Departments of Chemistry and Chemical Biology and.
authors.forename: Clint A
authors.initials: CA
authors.lastname: Stalnecker
authors.affiliation: Department of Chemistry, Ithaca College, Ithaca, NY 14850;
authors.forename: Scott M
authors.initials: SM
authors.lastname: Ulrich
authors.affiliation: Departments of Chemistry and Chemical Biology and.
authors.forename: Yunxing
authors.initials: Y
authors.lastname: Li
authors.affiliation: Departments of Chemistry and Chemical Biology and.
authors.forename: Sekar
authors.initials: S
authors.lastname: Ramachandran
authors.affiliation: Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, NY 10962; and.
authors.forename: Mary Kate
authors.initials: MK
authors.lastname: McBrayer
authors.affiliation: Childrens Medical Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390.
authors.forename: Ralph J
authors.initials: RJ
authors.lastname: DeBerardinis
authors.affiliation: Departments of Chemistry and Chemical Biology and Molecular Medicine, Cornell University, Ithaca, NY 14853; rac1@cornell.edu.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Cerione
authors.affiliation: Departments of Chemistry and Chemical Biology and.
authors.forename: Jon W
authors.initials: JW
authors.lastname: Erickson
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D000818:Animals; D053119:Benzophenanthridines / Q000494:pharmacology; D002471:Cell Transformation, Neoplastic / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D005973:Glutamine / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D000637:Transaminases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics
chemical_list: D053119:Benzophenanthridines; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D005973:Glutamine; D000637:Transaminases; C020656:glutamine-pyruvate aminotransferase
keywords: FRET; Rho GTPases; benzophenanthridines; glutaminase; glutaminolysis