Design and evaluation of novel glutaminase inhibitors

McDermott, Lee A.; Iyer, Prema; Vernetti, Lawrence; Rimer, Shawn; Sun, Jiangman; Boby, Melissa; Yang, Tianyi; Fioravanti, Michael; O’Neill, Jason; Wang, Liwei; Drakes, Dylan J.; Katt, William P.; Huang, Qingqiu; Cerione, Richard A.

doi:10.1016/j.bmc.2016.03.009

Cited by 55 publications

(78 citation statements)

References 27 publications

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“…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…”

Section: Discussion 13mentioning

confidence: 86%

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

Singleton

Dechaume

Murray

et al. 2020

Preprint

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Background Glutamine serves as an important nutrient with many cancer types displaying glutamine dependence. Following cellular uptake glutamine is converted to glutamate in a reaction catalysed by mitochondrial glutaminase. This glutamate has many uses, including acting as an anaplerotic substrate (via alpha-ketoglutarate) to replenish TCA cycle intermediates. CB-839 is a potent, selective, orally bioavailable inhibitor of glutaminase that has activity in Triple receptor-Negative Breast Cancer (TNBC) cell lines and evidence of efficacy in advanced TNBC patients. Methods A panel of eleven breast cancer cell lines was used to investigate the anti-proliferative effects of the glutaminase inhibitors CB-839 and BPTES in different types of culture medium, with or without additional pyruvate supplementation. The abundance of the TCA cycle intermediate fumarate was quantified as a measure if TCA cycle anaplerosis. Pyruvate secretion by TNBC cultures was then assessed with or without AZD3965, a monocarboxylate transporter 1 (MCT1) inhibitor. Finally, two dimensional (2D) monolayer and three dimensional (3D) spheroid assays were used to compare the effect of microenvironmental growth conditions on CB-839 activity.Results The anti-proliferative activity of CB-839 in a panel of breast cancer cell lines was similar to published reports, but with a major caveat; growth inhibition by CB-839 was strongly attenuated in culture medium containing pyruvate. This pyruvate-dependent attenuation was also observed with a related glutaminase inhibitor, BPTES. Studies demonstrated that exogenous pyruvate acted as an anaplerotic substrate preventing the decrease of fumarate in CB-839-treated conditions. Furthermore, endogenously produced pyruvate secreted by TNBC cell lines was able to act in a paracrine manner to significantly decrease the sensitivity of recipient cells to glutaminase inhibition. Suppression of pyruvate secretion using the MCT1 inhibitor AZD3965, antagonised this paracrine effect and increased CB-839 activity. Finally, CB-839 activity was significantly compromised in 3D compared with 2D TNBC culture models, suggesting that 3D microenvironmental features impair glutaminase inhibitor responsiveness. Conclusion This study highlights the potential influence that both circulating and tumour-derived pyruvate can have on glutaminase inhibitor efficacy. Furthermore, it highlights the benefits of 3D spheroid cultures to model the features of the tumour microenvironment and improve the in vitro investigation of cancer metabolism-targeted therapeutics.

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Section: Discussion 13mentioning

confidence: 86%

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

Singleton

Dechaume

Murray

et al. 2020

Preprint

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“…Recently, there has been a concerted effort to find small molecule inhibitors of the glutaminase enzymes using medicinal chemistry approaches (22,31,36,37). Here we first compared the differences between the allosteric activators inorganic phosphate and sulfate and the two most commonly used allosteric inhibitors, CB-839 and BPTES, in fluorescence assays that directly monitor GAC tetramer formation.…”

Section: Discussionmentioning

confidence: 99%

Conformational changes in the activation loop of mitochondrial glutaminase C: A direct fluorescence readout that distinguishes the binding of allosteric inhibitors from activators

Stalnecker

Erickson

Cerione

2017

Journal of Biological Chemistry

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The first step in glutamine catabolism is catalysis by the mitochondrial enzyme glutaminase, with a specific isoform, glutaminase C (GAC), being highly expressed in cancer cells. GAC activation requires the formation of homotetramers, promoted by anionic allosteric activators such as inorganic phosphate. This leads to the proper orientation of a flexible loop proximal to the dimer-dimer interface that is essential for catalysis ( the "activation loop"). A major class of allosteric inhibitors of GAC, with the prototype being bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and the related molecule CB-839, binds to the activation loop and induces the formation of an inactive tetramer (two inhibitors bound per active tetramer). Here we describe a direct readout for monitoring the dynamics of the activation loop of GAC in response to these allosteric inhibitors, as well as allosteric activators, through the substitution of phenylalanine at position 327 with tryptophan (F327W). The tryptophan fluorescence of the GAC(F327W) mutant undergoes a marked quenching upon the binding of BPTES or CB-839, yielding titration profiles that make it possible to measure the binding affinities of these inhibitors for the enzyme. Allosteric activators like phosphate induce the opposite effect ( fluorescence enhancement). These results describe direct readouts for the binding of the BPTES class of allosteric inhibitors as well as for inorganic phosphate and related activators of GAC, which should facilitate screening for additional modulators of this important metabolic enzyme.

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“…One of our most advanced compounds is UPGL00004, which shows better microsomal stability when compared to either BPTES or CB-839 (28). In the present study, we performed comparisons of UPGL00004 and CB-839, including a comparison of their X-ray structures bound to the mature form of human GAC (residues 73-598), in order to learn more about their mode of binding, and to gain insights for the design of novel and improved inhibitors.…”

Section: Discussionmentioning

confidence: 99%

“…CB-839 and UPGL00004 were prepared as previously described (13,28). BPTES was a kind gift of Dr. Scott Ulrich (Ithaca College).…”

Section: Methodsmentioning

confidence: 99%

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

Huang

Stalnecker

Zhang

et al. 2018

Journal of Biological Chemistry

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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...

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Design and evaluation of novel glutaminase inhibitors

Cited by 55 publications

References 27 publications

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

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

Conformational changes in the activation loop of mitochondrial glutaminase C: A direct fluorescence readout that distinguishes the binding of allosteric inhibitors from activators

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

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