Abstract B171: Anti-oncogenic effects of RP10107, a novel and potent glutaminase inhibitor, in solid cancer cell lines
Authors: Viswanadha Srikant , Bhavar Prashant , Merikapudi Gayatriswaroop , Govindrajulu Babu , Eleswarapu Satyanarayana , Veeraraghavan Sridhar , Vakkalanka Swaroop 
Published: May 2016

Abstract
Background: Cancer cells preferentially channel glucose towards lactate production even when oxygen is plentiful, a process termed “aerobic glycolysis” or the Warburg effect. The entire metabolic machinery is reprogrammed in cancer cells wherein glutamine utilization is increased via elevation of glutaminase activity thereby generating the necessary substrates required for eventual ATP synthesis and energy production. Targeting glutaminase therefore represents a potential therapeutic strategy to curb malignant transformation of cells and combat cancer progression. Herein, we describe the biological and pharmacokinetic properties of RP10107, a novel small molecule glutaminase inhibitor, with scope to be further developed as a clinical candidate for solid tumors.
Methods: Inhibition of glutaminase (GLS1) activity was determined in rodent brain lysates and recombinant human enzyme preparations using a colorimetric or fluorometric procedure. Selectivity over liver glutaminase (GLS2) was confirmed using mouse liver mitochondrial lysates. Viability was assessed using the colorimetric MTT reagent after incubation of RP10107 with cell lines representative of breast, lung, and colorectal cancer. Glutamate concentrations in cell lines following treatment with RP10107 was estimated by LC-MS/MS. Expression of GLS-1, cMyc, pS6, and p-mTOR were estimated by Western Blotting and bands were quantified using ImageJ after normalization with Actin. Pharmacokinetic profile of RP10107 in plasma after single dose oral administration (10 mg/kg) or IV injection (1 mg/kg) was determined in mice.
Results: RP10107 inhibited glutaminase (GLS1) activity in mouse brain lysate, rat brain lysate, and recombinant human enzyme with IC50 of 21.2, 18.2, and 26.4 nM, respectively. In contrast, activity in liver mitochondrial preparations (GLS2) was minimal (&lt;10% inhibition) even at 10 μM concentration. RP10107 caused a dose-dependent inhibition in proliferation of breast, lung, and colorectal cell lines. Response was the highest in MDA-MB-231 (GI50 = 27 nM) with half-maximal growth inhibition for ZR.75.1, MCF-7, A549, H441, HCT-8, and HCT-116 observed at &lt;2 μM RP10107. Reduction in cell viability was accompanied by a dose-dependent increase in the ratio of glutamine to glutamate with &gt;50% response noticed at 100 nM in MDA-MB-231 cells. While p-mTOR expression levels were not affected, addition of RP10107 caused a reduction in cMyc and pS6 (&gt;50% at 100-1000 nM) in lung and breast cancer cell lines. Pharmacokinetic studies of RP10107 in mice indicated high oral bioavailability (62%) with maximum plasma concentrations (Cmax) exceeding 10 μM.
Conclusions: RP10107 is a potent and selective GLS1 inhibitor that inhibited growth of solid tumor cell lines with a concomitant increase in glutamine to glutamate ratio. Reduction in glutaminase activity resulted in downregulation of cMyc and pS6 in breast and lung cancer cells. Based on the in vitro efficacy and excellent pharmacokinetic profile, RP10107 is current being tested in various mouse xenograft models.
Citation Format: Srikant Viswanadha, Prashant Bhavar, Gayatriswaroop Merikapudi, Babu Govindrajulu, Satyanarayana Eleswarapu, Sridhar Veeraraghavan, Swaroop Vakkalanka. Anti-oncogenic effects of RP10107, a novel and potent glutaminase inhibitor, in solid cancer cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B171.