Characterization of the interactions of potent allosteric inhibitors with glutaminase C, a key enzyme in cancer cell glutamine metabolism

Abstract

Altered glycolytic flux in cancer cells (the "Warburg effect") causes their proliferation to rely upon elevated glutamine metabolism ("glutamine addiction"). This requirement is met by the overexpression of glutaminase C (GAC), which catalyzes the first step in glutamine metabolism and therefore represents a potential therapeutic target. The small molecule CB-839 was reported to be more potent than other allosteric GAC inhibitors, including the parent compound BPTES, and is in clinical trials. Recently, we described the synthesis of BPTES analogs having distinct saturated heterocyclic cores as a replacement for the flexible chain moiety, with improved microsomal stability relative to CB-839 and BPTES. Here, we show that one of these new compounds, UPGL00004, like CB-839, more potently inhibits the enzymatic activity of GAC, compared to BPTES. We also compare the abilities of UPGL00004, CB-839, and BPTES to directly bind to recombinant GAC, and demonstrate that UPGL00004 has a similar binding affinity as CB-839 for GAC. We go on to show that UPGL00004 potently inhibits the growth of triplenegative breast cancer cells, as well as tumor growth when combined with the anti-VEGF antibody bevacizumab. Finally, we compare the Xray crystal structures for UPGL00004 and CB-839 bound to GAC, verifying that UPGL00004 occupies the same binding site as CB-839 or BPTES, and that all three inhibitors regulate the enzymatic activity of GAC via a similar allosteric mechanism.These results provide insights regarding the potency of these inhibitors that will be useful in designing novel small-molecules that target a key enzyme in cancer cell metabolism.The Warburg effect in cancer cells refers to the significant alteration of the glycolytic pathway that results in the increased generation of lactate, decreased mitochondrial metabolism of pyruvate, and an accompanying reduction in oxidative phosphorylation (1-3). This altered glucose flux is thought to be advantageous for rapidly proliferating cells, such as cancer cells, by providing the building blocks for biosynthetic processes, at the expense of ATP synthesis. A significant consequence of the Warburg effect is that cancer cells need to develop alternative mechanisms that provide inputs into the citric acid cycle. One of the most common of these mechanisms results in an addiction to glutamine, an amino acid that is abundant in the bloodstream and can enter the citric acid cycle through an anaplerotic pathway initiated by the catalytic activity of the enzyme glutaminase (4,5).Two isozymes of mammalian glutaminase have been identified: kidney-type glutaminase encoded by the GLS gene, and liver-type glutaminase encoded by GLS2. Each gene expresses two major A new potent glutaminase allosteric inhibitor 2 splice variants, with the GLS gene expressing the KGA (kidney-type glutaminase) and the C-terminal truncated splice variant GAC (glutaminase C) isoforms, while the GLS2 gene also expresses one longer and one shorter isoform, collectively referred to here as GLS2 (6,7). Of the...

Emerging roles of Myc in stem cell biology and novel tumor therapies

“…BPTES inhibits the dimer-to-tetramer transition of GLS in the allosteric manner, which is essential for the activation of the enzyme [ 169 ]. A large number of derivatives of BPTES such as CB-839 have been designed [ 170 – 172 ]. BPTES effectively inhibits the proliferation of numerous types of malignancies, including c-Myc-dependent hepatocellular carcinoma, malignant lymphoma and renal cell carcinoma [ 172 ].…”

Pyruvate anaplerosis is a mechanism of resistance to pharmacological glutaminase inhibition in Triple-receptor Negative Breast Cancer

“…Whilst lung and brain cancers appear to fuel the TCA cycle via pyruvate dehydrogenase complex-dependent glucose metabolism, the results from studies in clear cell renal cell carcinoma (ccRCC) display supressed glucose oxidation in the TCA cycle, more reflective of the classic "Warburg Effect" observed in most in vitro studies [17,32]. In agreement, [1][2][3][4][5][6][7][8][9][10][11][12][13] C-glutamine studies in VHL-deficient ccRCC tumour…”

Metabolism of glucose and glutamine is critical for skeletal muscle stem cell activation

“…Glutamine is another major metabolic substrate that is oxidized by the mitochondria via the TCA cycle. To examine the contribution of glutamine metabolism in MuSC activation, we treated cells with Bis-2-(5-phenylacetamido-1, 3, 4-thiadiazol-2-yl)ethyl sulfide (BPTES), a small molecule which inhibits glutaminase, which converts glutamine to glutamate 14 . Glutamate is a precursor for the TCA cycle intermediate α -ketoglutarate; therefore, we expected BPTES treatment to suppress mitochondrial oxygen consumption in activating MuSCs 15 .…”