Many cancer cells become dependent on glutamine metabolism to compensate for glycolysis being uncoupled from the TCA cycle. The mitochondrial enzyme Glutaminase C (GAC) satisfies this ‘glutamine addiction’ by catalyzing the first step in glutamine metabolism, making it an attractive drug target. Despite one of the allosteric inhibitors (CB-839) being in clinical trials, none of the drugs targeting GAC are approved for cancer treatment and their mechanism of action is not well understood. A major challenge has been the rational design of better drug candidates: standard cryo-cooled X-ray crystal structures of GAC bound to CB-839 and its analogs fail to explain their potency differences. Here, we address this problem by using an emerging technique, serial room temperature crystallography, which enabled us to observe clear differences between the binding conformations of inhibitors with significantly different potencies. A computational model was developed to further elucidate the molecular basis of inhibitor potency. We then corroborated the results from our modeling efforts by using recently established fluorescence assays that directly read-out inhibitor binding to GAC. Together, these findings provide new insights into the mechanisms used by a major class of allosteric GAC inhibitors and for the future rational design of more potent drug candidates.

中文翻译：

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对靶向癌细胞中谷氨酰胺代谢的抑制剂所使用机制的新见解

许多癌细胞变得依赖谷氨酰胺代谢来补偿与 TCA 循环脱钩的糖酵解。线粒体酶谷氨酰胺酶 C (GAC) 通过催化谷氨酰胺代谢的第一步来满足这种“谷氨酰胺成瘾”，使其成为有吸引力的药物靶点。尽管其中一种变构抑制剂 (CB-839) 正在进行临床试验，但没有一种靶向 GAC 的药物被批准用于癌症治疗，其作用机制尚不清楚。一个主要挑战是合理设计更好的候选药物：与 CB-839 及其类似物结合的 GAC 的标准低温冷却 X 射线晶体结构无法解释它们的效力差异。在这里，我们通过使用一种新兴技术，串行室温晶体学来解决这个问题，这使我们能够观察到具有显着不同效力的抑制剂的结合构象之间的明显差异。开发了一个计算模型以进一步阐明抑制剂效力的分子基础。然后，我们通过使用最近建立的直接读出与 GAC 结合的抑制剂的荧光测定来证实我们建模工作的结果。总之，这些发现为一类主要变构 GAC 抑制剂所使用的机制以及未来更有效候选药物的合理设计提供了新的见解。然后，我们通过使用最近建立的直接读出与 GAC 结合的抑制剂的荧光测定来证实我们建模工作的结果。总之，这些发现为一类主要变构 GAC 抑制剂所使用的机制以及未来更有效候选药物的合理设计提供了新的见解。然后，我们通过使用最近建立的直接读出与 GAC 结合的抑制剂的荧光测定来证实我们建模工作的结果。总之，这些发现为一类主要变构 GAC 抑制剂所使用的机制以及未来更有效候选药物的合理设计提供了新的见解。