Abstract

The sigma-2 receptors are promising therapeutic targets because of their significant upregulation in tumor cells compared with normal tissue. Here, we characterize CM572 [3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)-6-isothiocyanatobenzo[d]oxazol-2(3H)-one] (sigma-1 K_i ≥ 10 µM, sigma-2 K_i = 14.6 ± 6.9 nM), a novel isothiocyanate derivative of the putative sigma-2 antagonist, SN79 [6-acetyl-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one]. CM572 bound irreversibly to sigma-2 receptors by virtue of the isothiocyanate moiety but not to sigma-1. Studies in human SK-N-SH neuroblastoma cells revealed that CM572 induced an immediate dose-dependent increase in cytosolic calcium concentration. A 24-hour treatment of SK-N-SH cells with CM572 induced dose-dependent cell death, with an EC₅₀ = 7.6 ± 1.7 µM. This effect was sustained over 24 hours even after a 60-minute pretreatment with CM572, followed by extensive washing to remove ligand, indicating an irreversible effect consistent with the irreversible binding data. Western blot analysis revealed that CM572 also induced cleavage activation of proapoptotic BH3-interacting domain death agonist. These data suggest irreversible agonist-like activity. Low concentrations of CM572 that were minimally effective were able to attenuate significantly the calcium signal and cell death induced by the sigma-2 agonist CB-64D [(+)-1R,5R-(E)-8-benzylidene-5-(3-hydroxyphenyl)-2-methylmorphan-7-one]. CM572 was also cytotoxic against PANC-1 pancreatic and MCF-7 breast cancer cell lines. The cytotoxic activity of CM572 was selective for cancer cells over normal cells, being much less potent against primary human melanocytes and human mammary epithelial cells. Taken together, these data show that CM572 is a selective, irreversible sigma-2 receptor partial agonist. This novel irreversible ligand may further our understanding of the endogenous role of this receptor, in addition to having potential use in targeted cancer diagnosis and therapy.