Abstract
Abstract Background: Store operated calcium channels, namely calcium release activated calcium (CRAC) channels, contribute to calcium influx in nonexcitable cells. Elevation of cytosolic calcium through activation CRAC channels mediates an array of cellular responses including metabolism and gene expression, cell growth, and proliferation. Aberrant CRAC activity has been linked to various autoimmune disorders and certain cancers via the NFAT pathway. Herein, we describe the preclinical profile of RP4010, a novel small-molecule inhibitor of the CRAC channel pathway. Methods: Modulation of CRAC channel activity upon addition of RP4010 was determined in a thapsigargin-induced calcium influx assay using Jurkat cells. Inhibition of downstream NFAT activity in Jurkat cells was measured using a reporter-gene luciferase assay. Effect of RP4010 on viability was determined using an OncoPanel multiplexed cytoxicity assay that measures multiple parallel responses of cancer cell lines to drug treatments through high-content fluorescence imaging. Change in migratory behavior upon addition of RP4010 was studied in a fetal bovine serum (FBS) induced scratch wound assay in A549 cells. Pharmacokinetic behavior of RP4010 in plasma after single dose oral administration or IV injection was determined in mouse and dog. Results: RP4010 demonstrated remarkable potency at inhibiting CRAC channel activity manifested by a reduction in thapsigargin-induced calcium influx into Jurkat cells (IC50 = 60 nM). Subsequent to CRAC channel inhibition, RP4010 dose-dependently attenuated the NFAT-regulated signalling in Jurkat cells with a half-maximal inhibition observed at 100 nM. Data from the OncoPanel testing indicated that RP4010 inhibited growth of cancer cells across diverse tumor sets ranging from breast, lung, lymphomas, skin, colon, prostate, stomach, and kidney with EC50 ranging from 1-10 μM. Interestingly, viability of Wi38, a normal lung cell line, was not affected by RP4010, indicating the specificity of the compound towards cancerous cells. Additionally, increasing concentrations of RP4010 caused a dose-dependent reduction in the migration of FBS induced A549 cells, demonstrating its antimetastatic potential. Pharmacokinetic studies indicated good oral bioavailability of RP4010 with a favorable half-life and peak plasma concentrations in mouse and dog. Conclusions: Results demonstrate the therapeutic potential of RP4010 across various cancer types via inhibition of the CRAC channel pathway and subsequent modulation of the downstream marker, NFAT. Achieving antitumor efficacy through inhibition of CRAC channels represents a novel approach primarily aimed at alleviating the deleterious effects associated with cytotoxic agents. Citation Format: Srikant Viswanadha, Seeta Nyayapathy, Sridhar Veeraraghavan, Swaroop Vakkalanka. Antitumor activity of RP4010, a novel small-molecule inhibitor of the calcium release-activated calcium (CRAC) channel pathway [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B127.
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