Abstract 3237: Characterization of CM-572 and CM-769: Novel irreversible modulators of sigma-2 receptor function (2014)

Abstract Sigma-2 receptors are highly upregulated in a wide variety of cancer cells compared to normal cells, and increase by at least ten-fold in rapidly proliferating cancer cells compared to quiescent. Activation of the sigma-2 receptor induces apoptosis. Thus, there has been increasing interest in sigma-2 receptors as targets for cancer therapeutic and diagnostic agents and a need for small molecule tools to study function. We previously reported analogs of the sigma-2 antagonist, SN-79, that incorporate an isothiocyanate (ITC) group in order to impart irreversible binding to the receptor (Nicholson et al., Proc. Amer. Assoc. Cancer Res. 54: #2242, 2013). Here we further characterize CM-572 (sigma-2 Ki = 320nM; ITC in heterocyclic ring) and CM-769 (sigma-2 Ki = 271nM; ITC in fluorophenyl ring) for their effects in SK-N-SH neuroblastoma, MCF-7 breast, and PANC-1 pancreatic cancer cell lines. Previous data showed that CM-572 potently and selectively acylated sigma-2 receptors over sigma-1, while CM-769 was less potent at irreversible binding, presumably due to non-optimal placement of the ITC group. CM-572 induced dose-dependent cell death in SK-N-SH neuroblastoma cells with ED50 about 5uM. CM-572 was also cytotoxic in MCF-7 breast and PANC-1 pancreatic cancer cells, however with reduced potency and efficacy (40% and 50% maximal cell death, respectively, at 30uM). The cytotoxic effect of CM-572 in SK-N-SH cells was retained after a 60min pretreatment of cells, washout of compound, and continued incubation for 24h, indicating irreversible agonist activity. Interestingly, CM-769 produced an apparent dose-dependent proliferative effect on SK-N-SH neuroblastoma cells. This effect was also retained after 60min pretreatment and washout, again indicating an irreversible affect. A subtoxic dose of CM-572 (300nM) was able to partially attenuate CM-769-induced cell proliferation, indicating that these two compounds act at the same receptor site. Cytosolic BID cleavage is a hallmark of sigma-2 receptor induced apoptosis in SK-N-SH cells. CM-572 (30uM) induced rapid BID cleavage beginning at 30min, with near completion at 6h. By contrast, CM-769 (30uM) did not induce BID cleavage, but rather appeared to increase cytosolic levels of full-length BID relative to control, consistent with the observed proliferative or protective effect. Together, the data indicate that CM-572 is an irreversible partial agonist at sigma-2 receptors. This is consistent with previously demonstrated effects of CM-572 on CB-64D (an agonist)-induced calcium release and cytotoxicity, where low-dose CM-572 attenuated CB-64D effects, and high-dose CM-572 mimicked CB-64D. CM-769 is an irreversible inverse agonist at the sigma-2 receptor, a novel effect not shown by any sigma-2 ligand to date. These two compounds represent novel tools for further study of sigma-2 receptor function. Citation Format: Hilary E. Nicholson, Pei Ling Chia, Anthony Comeau, Christophe Mesangeau, Christopher McCurdy, Wayne D. Bowen. Characterization of CM-572 and CM-769: Novel irreversible modulators of sigma-2 receptor function. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3237. doi:10.1158/1538-7445.AM2014-3237