TY - JOUR
T1 - Mechanism by which a recently discovered allosteric inhibitor blocks glutamine metabolism in transformed cells
AU - Stalnecker, Clint A.
AU - Ulrich, Scott M.
AU - Li, Yunxing
AU - Ramachandran, Sekar
AU - McBrayer, Mary Kate
AU - De Berardinis, Ralph J.
AU - Cerione, Richard A.
AU - Erickson, Jon W.
PY - 2015/1/13
Y1 - 2015/1/13
N2 - 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 13Clabeled 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 formonomeric GAC and that the dose-dependent binding of 968 to GAC monomers directlymatches 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.
AB - 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 13Clabeled 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 formonomeric GAC and that the dose-dependent binding of 968 to GAC monomers directlymatches 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.
KW - Benzophenanthridines
KW - FRET
KW - Glutaminase
KW - Glutaminolysis
KW - Rho GTPases
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U2 - 10.1073/pnas.1414056112
DO - 10.1073/pnas.1414056112
M3 - Article
C2 - 25548170
AN - SCOPUS:84920990454
SN - 0027-8424
VL - 112
SP - 394
EP - 399
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 2
ER -