Mechanism by which a recently discovered allosteric inhibitor blocks glutamine metabolism in transformed cells
Clint A. Stalnecker, a, Scott M. Ulrich, b, Yunxing Li, a, Sekar Ramachandran, a, Mary Kate McBrayer, c, Ralph J. DeBerardinis, d, Richard A. Cerione, a, e, 1, and Jon W. Erickson, a
Departments of
a Chemistry and Chemical Biology andeMolecular Medicine, Cornell University, Ithaca, NY 14853;
b Department of Chemistry, Ithaca College, Ithaca, NY 14850;
c Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, NY 10962; and
d Childrens Medical Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390
Edited by Joseph Schlessinger, Yale University School of Medicine, New Haven, CT, and approved December 5, 2014 (received for review July 23, 2014)
Significance
The work described here was motivated by our previous discovery of a connection between Rho GTPase activation and the up-regulation of mitochondrial glutaminase C (GAC), which is responsible for satisfying the glutamine addiction of cancer cells. This connection was originally established by our identification of a lead compound, 968, for a new class of inhibitors of oncogenic transformation. Although GAC was identified as the putative target for 968, how it regulated GAC was poorly understood. Here we provide important insights into the actions of 968, through the development of novel assays for its direct binding to GAC and its effects on enzyme activity. These findings offer exciting new strategies for interfering with the metabolic reprogramming critical for malignant transformation.
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 13C-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.
http://www.pnas.org/content/112/2/394.abstract.html?etoc |
