BPTES inhibition of hGA_124–551, a truncated form of human kidney-type glutaminase

Abstract

The initial transcript of the GLS1 gene undergoes alternative splicing to produce two glutaminase variants (KGA and GAC) that contain unique C-terminal sequences. A truncated form of human glutaminase (hGA(124-551)) that lacks either C-terminal sequence was expressed in E.Coli and purified. This construct exhibits a hyperbolic glutamine saturation profile (K(m) of 1.6 mM). BPTES, bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide, functions as a potent uncompetitive inhibitor of this construct (K(i) of 0.2 µM). The hGA(124-551) is inactive in the absence of phosphate, but exhibits a hyperbolic phosphate-dependent activation profile that is also inhibited by BPTES. Gel filtration studies indicate that hGA(124-551) forms a dimer in the absence or presence of 100 mM phosphate, whereas addition of BPTES causes the formation of an inactive tetramer. The combined data indicate that BPTES inhibits human glutaminase by a novel mechanism and that BPTES is a potential lead compound for development of an effective cancer chemotherapeutic agent.

Antitumor Activity of the Glutaminase Inhibitor CB-839 in Triple-Negative Breast Cancer

“…S1A) and a shift toward greater potency with increasing glutamine concentrations ( Supplementary Fig. S1C), consistent with previous reports (18,21). In contrast, CB-839 behaved as a primarily noncompetitive inhibitor impacting V max but with minimal effect on K m ( Supplementary Fig.…”

“…The small-molecule BPTES was previously described as an allosteric glutaminase inhibitor active against both splice variants of the GLS gene, GAC and KGA (reported Ki between 0.2 and 3 mmol/L), but not the liver form of glutaminase, encoded by the GLS2 gene (18,19,21,22). CB-839 (Fig.…”

“…Clinical studies with these compounds showed limited antitumor activity but their development was restricted by severe toxicities, potentially due to the broad antagonism of multiple glutamine-utilizing enzymes and transporters (17). More recently, two allosteric inhibitors of glutaminase have been reported, compound 968 and BPTES (bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide), that act by distinct mechanisms (6,18,21). The allosteric mechanism for BPTES (and BPTES analogs) entails formation of an inactive glutaminase homotetramer through the binding of two inhibitor molecules at the interface between a pair of homodimers (18,22,23).…”

Role of Glutamate Dehydrogenase in Cancer Growth and Homeostasis

“…In addition NHE mediated acid extrusion is up-regulated in cancer cells [19,20] importing a Na + load requiring Na+/K+ ATPase -ATP expenditure and ATP regeneration associated with acidogenic aerobic glycolysis(Warburg effect) and by substrate level phosphorylation. Because PIG (GGT/GGTP), NHE, glutamine transporter and glutaminase activities are all up-regulated in rapidly growing tumors, tagging molecular target inhibitors [21][22][23][24] with a ϒ-glutamyl moiety offers a tumor specific vehicle specific for limiting anaplerosis and preventing elevated cell pH, prerequisites for rapid tumor growth. …”

Glucose-Independent Glutamine-Driven TCA Cycle in Cancer Cells

“…6.7 , one primitive test of this concept involves BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfi de]. This uncompetitive inhibitor has been elucidated to inhibit human glutaminase [ 12 ]. Studies have revealed that BPTES, a small molecule, binds to an allosteric site at the dimer interface of KGA-a kidney-type glutaminase isoform (GLS1)-and leads to a conformational change near the catalytic site, which inactivates the enzyme [ 13 ].…”