New Insights into the Mechanisms Used by Inhibitors Targeting Glutamine Metabolism in Cancer Cells

Abstract

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.

Room-temperature serial synchrotron crystallography of Drosophila cryptochrome

“…Serial crystallography determines the structural and dynamic properties of proteins with synchrotron radiation at near-physiologically relevant temperatures. Compared with cryogenic conditions, higher temperature allows conformational motions needed to access catalytic processes, photocycles and binding modes for interaction partners (Milano et al, 2022;Zhu et al, 2020). In this study, the structure of D. melanogaster CRY was determined at room temperature to provide this information for a protein that undergoes lightdriven conformational changes as part of its normal function.…”

“…The crystal slurry was gently pipetted onto Kapton chips (sample supports) in a humidified glovebox (humidity ! 97%) provided by MiTeGen (Ithaca, New York, USA; Illava et al, 2021;Milano et al, 2022). 10-20 ml crystal slurry was pipetted onto each chip, representing 25-50 mg protein per chip.…”

“…Notably, sample consumption was low, with the entire experiment only requiring $0.5 mg of protein. Given the crystal variability, the low signal to noise and the sheer number of observations recorded, SSX experiments produce data sets with large merging R factors and low correlation factors among symmetry-related reflections (Illava et al, 2021;Milano et al, 2022;Weinert et al, 2019). As such, we investigated several straightforward methods to remove outlier observations from the data.…”

“…On the other hand, fixed-target delivery systems place crystals onto sample supports that are then rastered through an X-ray beam (Hunter et al, 2014;Wierman et al, 2019;Illava et al, 2021;Ren et al, 2020;Stellato et al, 2014;Gati et al, 2014;Coquelle et al, 2015). A recent example of a fixed-target system was used to observe differences in the binding conformations of inhibitors to glutaminase C, which is a commonly upregulated enzyme in several different cancers (Milano et al, 2022). Although issues may arise with having to maintain sample stability during data collection, fixed-target systems are generally considered to be high throughput and require low sample consumption (Cheng, 2020).…”

Room-temperature serial synchrotron crystallography of the human phosphatase PTP1B

“…X-ray crystallography offers detailed insights into protein structure. Although most X-ray crystallography is performed with the sample at cryogenic temperature (cryo), data collection at elevated temperatures including room temperature (RT) offers unique insights into protein conformational heterogeneity and function (Fraser et al, 2009(Fraser et al, , 2011Keedy et al, 2014Keedy et al, , 2015Fischer et al, 2015;Milano et al, 2022). Previously, a barrier to RT crystallography was growing sufficiently large crystals to counteract the increased sensitivity to radiation damage at RT relative to cryo (Garman & Nave, 2009;Warkentin et al, 2013).…”