Characterization of CM572, a Selective Irreversible Partial Agonist of the Sigma-2 Receptor with Antitumor Activity

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-(4-14.6 6 6.9 nM), a novel isothiocyanate derivative of the putative sigma-2 antagonist, SN79 [6-acetyl-3-(4-(4-(4-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 EC 50 5 7.6 6 1.7 mM. 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 [(1)-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.

Sigma-2 Receptors Play a Role in Cellular Metabolism: Stimulation of Glycolytic Hallmarks by CM764 in Human SK-N-SH Neuroblastoma

“…A 24-hour treatment of 30 mM SN79 alone had no significant effect on MTT reduction in SK-N-SH neuroblastoma compared with an untreated control. Similarly, 0.3 mM CM572, a sigma-2 receptor partial agonist (K i 5 15 nM at sigma-2 receptors and K i $ 10 mM at sigma-1 receptors), which we have previously shown has antagonist properties at this concentration in SK-N-SH neuroblastoma (Nicholson et al, 2015), eliminated CM764-induced MTT hyper-reduction when dosed in combination with 10 mM CM764. The sigma-1/sigma-2 antagonists BD1047 [N9-[2-(3,4-dichlorophenyl)ethyl]-N,N,N9-trimethylethane-1,2-diamine] (K i 5 47 nM at sigma-2 receptors and K i 5 0.93 nM at sigma-1 receptors) (Matsumoto et al, 1995) and BD1063 [1-2-(3,4-dichlorophenyl)ethyl-4-methylpiperazine] (K i 5 449 nM at sigma-2 receptors and K i 5 9.15 nM at sigma-1 receptors) (Matsumoto et al, 1995) were both able to eliminate the increase in MTT reduction induced by CM764 treatment alone.…”

“…The induction of apoptosis upon activation combined with endogenous cancer cell selectivity makes the sigma-2 receptor an attractive target for chemotherapeutic intervention. We have recently shown that the irreversible sigma-2 receptor par tial agonist CM572 [3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)-6-isothiocyanatobenzo [d]oxazol-2(3H)-one] selectively induces cell death in neuroblastoma, pancreatic, and breast cancer cell lines compared with effects in normal epithelial melanocytes or normal breast epithelial cells (Nicholson et al, 2015). However, the wide variety of apoptotic mechanisms induced by sigma-2 receptor activation combined with a disparity among criteria for defining functional classes of sigma-2 receptor ligands makes studying the efficacy of such drugs difficult.…”

BRD2 regulation of sigma-2 receptor expression upon cytosolic cholesterol deprivation

“…haloperidol) 6 . Moreover, high S2R abundance was found in tumor tissues and cells, as detected with labeled S2R ligands 7,22 or unknowingly as TMEM97/ MAC30 23 . As such, S2R is often targeted for cancer imaging (e.g.…”

“…(2) S2R was implicated (via pharmacology) in neurological diseases 19 (e.g. Alzheimer's) 20 , psychiatric disorders 6,21 , and cancers 22 . In fact, S2R ligands have long been clinically used as antidepressants (e.g.…”

Divergent Cytotoxic and Metabolically Stimulative Functions of Sigma-2 Receptors: Structure-Activity Relationships of 6-Acetyl-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one (SN79) Derivatives

“…Generally, agonists of the sigma-2 receptor are considered to induce programmed cell death. Cleavage of Bid, caspase-3 activation, induction of an immediate rise in cytosolic calcium, and positive terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining have been proposed as markers of sigma-2 receptor agonist activity (Vilner and Bowen, 2000;Crawford and Bowen, 2002;Hazelwood and Bowen, 2006;Wang and Bowen, 2006;Zeng et al, 2014;Nicholson et al, 2015). Recently, we described a novel function of sigma-2 receptors, where a sigma-2 receptor ligand, CM764, induced an increase in various markers of glycolytic cellular metabolism as initially assessed by an increase in MTT reduction, without inducing changes in cell viability or cell proliferation (Nicholson et al, 2016).…”