Differential sigma-2 receptor-mediated metabolic stimulative and apoptotic effects of monovalent CM571 and its homo-bivalent counterpart, MAM03055A

Sigma-2 receptors, recently identified as TMEM97, are highly expressed in various types of cancer cells. Sigma-2 receptor agonists have traditionally been characterized as ligands that induce programmed cell death in various cancer cell types. Sigma-2 receptor-mediated cell death involves a number of mechanisms including caspase activation, mitochondrial depolarization, and autophagy. Recently, we reported a novel metabolically stimulative function of the sigma-2 receptor with stimulation of glycolytic hallmarks, including increase in cellular ATP level, reduced ROS, and stabilization of HIF-1α. We have investigated a family of 6-substituted analogs of the canonical sigma-2 receptor antagonist, SN79, members of which can produce both the metabolic stimulative and the cytotoxic effects. Here we compare the activities of two closely related compounds. CM571, the 6-amino derivative, bound with high affinity to both sigma-1 and sigma-2 receptors (sigma-1 Ki = 15.5 nM; sigma-2 Ki = 21.7 nM). MAM03055A (MAM) is essentially a homo-bivalent dimer of CM571, linked by a thiourea moiety. MAM exhibited high affinity and selectivity for sigma-2 receptors (sigma-1 Ki = 3,371 nM; sigma-2 Ki = 55.9 nM). The functional effects of the two compounds were compared in human SK-N-SH neuroblastoma cells. CM571 caused a dose-dependent stimulation of MTT reduction, indicating the metabolic stimulative-effect, producing 40% stimulation at 30 µM. By contrast, MAM induced dose-dependent cell death with an EC50 = 4.24 µM. MAM induced time-dependent cleavage of proapoptotic Bid, a key component of the programmed cell death mechanism of sigma-2 ligands in these cells. Previous studies had shown that only irreversibly binding, isothiocyanate analogs of SN79 induced programmed cell death, while other analogs produced metabolic stimulation. Rat liver membranes were treated with compound, washed to remove unbound ligand, and then subjected to radioligand binding to determine recovery of sigma-1 receptors ([3H](+)-pentazocine) and sigma-2 receptors ([3H]DTG + (+)-pentazocine). Treatment with CM571 resulted in full recovery of both sigma-1 and sigma-2 binding. However, MAM treatment resulted in loss of sigma-2 binding and full recovery of sigma-1 activity, indicating irreversible binding to the sigma-2 receptor. Since there is no chemically reactive moiety in MAM to react covalently, MAM binds “pseudo-irreversibly”. Treatment of cells with MAM for 60 min, followed by washout produced nearly comparable levels of cell death 24 h later as continuous exposure of cells for 24 hours, consistent with pseudo-irreversible action at the sigma-2 receptor. The results show that mono-valent and bivalent sigma-2 ligands in this series show different modes of interaction with the receptor, resulting in divergent effects on cell viability. Citation Format: Cheri Z. Liu, Bridget M. McVeigh, Marco Mottinelli, Hilary E. Nicholson, Christopher R. McCurdy, Wayne D. Bowen. Differential sigma-2 receptor-mediated metabolic stimulative and apoptotic effects of monovalent CM571 and its homo-bivalent counterpart, MAM03055A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1294.