Abstract 1877: Anti-tumor activity of novel, potent, selective and orally-bioavailable glutaminase inhibitors.
Authors: Parlati Francesco , Chernov-Rogan Tania , Demo Susan , Gross Matthew , Janes Julie , Kawas Raja , Lewis Evan , Rodriguez Hector , Rodriguez Mirna , Yang Jinfu , Zhao Frances , Richardson Adam , Bennett Mark K. 
Published: August 2014

Abstract
Glutamine is required for the growth of a broad range of tumor cells. An important step in the metabolism of glutamine is its conversion to glutamate, which is catalyzed by the mitochondrial enzyme glutaminase. GLS, the form of the enzyme expressed in most cells, is up-regulated in a sub-set of glutamine-requiring tumor cells. Suppression of GLS by genetic knockdown or with small molecule inhibitors slows the growth of these tumor cells. To further evaluate GLS as an oncology therapeutic target, we have developed a series of novel and potent GLS inhibitors that are orally bioavailable, permitting both in vitro and in vivo analysis of GLS inhibition.
In a reconstituted system with purified enzyme, the inhibitor CB-498 inhibited GLS with an IC50 of &lt;15 nM. Kinetic analyses demonstrated that the mechanism of inhibition is allosteric, time dependent and slowly reversible. In a panel of over 30 tumor cell lines, CB-498 had broad anti-proliferative activity across multiple tumor cell types with IC50’s ranging from 5nM to 300 nM. While the cellular anti-proliferative response was cytostatic in the majority of cell lines, a few cell lines underwent apoptosis. Cellular levels of glutamate decreased and glutamine levels increased in a dose-dependent manner following CB-498 treatment, consistent with GLS inhibition. Flux studies with uniformly 15N, 13C-labeled glutamine demonstrated that CB-498 blocks the incorporation of glutamine-derived carbon or nitrogen into TCA cycle intermediates, pyruvate, lactate, fatty acids, and amino acids coupled to glutamate through transamination. Furthermore, a membrane permeable form of α-ketoglutarate reversed the anti-proliferative effects of CB-498 in most cell lines, suggesting that the anti-tumor activity of CB-498 primarily arises from decreased glutamine flux through the TCA cycle.
CB-839 is an orally bioavailable analog of CB-498 with comparable biochemical and cellular potency but with improved solubility and pharmacokinetic properties. In an H2122 lung adenocarcinoma xenograft model, CB-839 showed single-agent anti-tumor activity. This anti-tumor activity was associated with increased tumor glutamine levels and decreased levels of glutamate and aspartate. Importantly, these glutamate and aspartate decreases were not seen in kidney, liver, or plasma, indicating a tumor specific response to GLS inhibition. Oral administration of CB-839 resulted in significant plasma exposure across multiple species and has been well tolerated in in vivo studies in rodents on either daily or twice-daily dosing schedules. These results motivate a further investigation of these potent and selective GLS inhibitors for clinical utility in oncology.
Citation Format: Francesco Parlati, Tania Chernov-Rogan, Susan Demo, Matthew Gross, Julie Janes, Raja Kawas, Evan Lewis, Hector Rodriguez, Mirna Rodriguez, Jinfu Yang, Frances Zhao, Adam Richardson, Mark K. Bennett. Anti-tumor activity of novel, potent, selective and orally-bioavailable glutaminase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1877. doi:10.1158/1538-7445.AM2013-1877