New insights into the molecular mechanisms of glutaminase C inhibitors in cancer cells using serial room temperature crystallography

Shawn K Milano,Qingqiu Huang,Thuy-Tien T Nguyen,Sekar Ramachandran,Aaron Finke,Irina Kriksunov,David J Schuller,D Marian Szebenyi,Elke Arenholz,Lee A Mcdermott,N Sukumar,Richard A Cerione,William P Katt

doi:10.1016/j.jbc.2021.101535

Shawn K Milano, Qingqiu Huang + Show 11 more

Open Access

https://doi.org/10.1016/j.jbc.2021.101535

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Abstract

Cancer cells frequently exhibit uncoupling of the glycolytic pathway from the TCA cycle (i.e., the “Warburg effect”) and as a result, often become dependent on their ability to increase glutamine catabolism. The mitochondrial enzyme Glutaminase C (GAC) helps to satisfy this ‘glutamine addiction’ of cancer cells by catalyzing the hydrolysis of glutamine to glutamate, which is then converted to the TCA-cycle intermediate α-ketoglutarate. This makes GAC an intriguing drug target and spurred the molecules derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (the so-called BPTES class of allosteric GAC inhibitors), including CB-839, which is currently in clinical trials. However, none of the drugs targeting GAC are yet approved for cancer treatment and their mechanism of action is not well understood. Here, we shed new light on the underlying basis for the differential potencies exhibited by members of the BPTES/CB-839 family of compounds, which could not previously be explained with standard cryo-cooled X-ray crystal structures of GAC bound to CB-839 or its analogs. Using an emerging technique known as serial room temperature crystallography, we were able to observe clear differences between the binding conformations of inhibitors with significantly different potencies. We also developed a computational model to further elucidate the molecular basis of differential inhibitor potency. We then corroborated the results from our modeling efforts using recently established fluorescence assays that directly read out inhibitor binding to GAC. Together, these findings should aid in future design of more potent GAC inhibitors with better clinical outlook.

Highlights

One of the most recognized phenotypes of many cancer cells is a metabolic shift from oxidative phosphorylation to aerobic glycolysis, commonly described as the Warburg effect [1]
Members of the UPGL series engage in a 4 conserved hydrogen bonding network via their thiadiazole rings to the backbone atoms of Lys 320, Phe 322 and Leu 323 of glutaminase 2C (GAC), and/or the hydroxyl hydrogen of Tyr 394, similar to what has been observed for BPTES and CB-839 [14]
Journal Pre-proof To test the suggestion from our modeling efforts that the terminal groups of the BPTES/CB-839 class of inhibitors contribute to their ability to bind and affect GAC catalytic activity, we examined a subset of the UPGL series with identical molecular structure at the centers, but which differ in the number of their terminal groups

Summary

Introduction

One of the most recognized phenotypes of many cancer cells is a metabolic shift from oxidative phosphorylation to aerobic glycolysis, commonly described as the Warburg effect [1]. Cells undergoing aerobic (Warburg) glycolysis make use of additional sources of carbon, such as glutamine, which exists in high concentrations in blood plasma [2]. High levels of GAC have been observed in aggressive cancers and the inhibition of its enzymatic activity has been shown to reduce the proliferative capability of a variety of different cancer cells, and often their survival, both in vitro and in mouse models [4,5,6]. GAC inhibitors have been shown to improve sensitivity to different clinical drug candidates, including the recent demonstration that their combination with antibodies targeting the immune checkpoint protein PD-L1 offers exciting therapeutic

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