Abstract 2242: Development of selective irreversible antagonists for sigma-2 receptors.
Authors: Nicholson Hilary , Comeau Anthony , Mesangeau Christophe , McCurdy Christopher R. , Bowen Wayne D. 
Published: August 2014

Sigma-1 and sigma-2 receptors are potential targets for therapeutic and diagnostic agents for various types of cancer. Sigma-1 receptors promote proliferation and survival, whereas activation of sigma-2 receptors induces apoptosis. Thus, there has been particular focus on sigma-2 receptors, which may be related to the PGRMC1 complex. While several selective ligands for sigma-2 receptors have been developed, there is a scarcity of tools for studying structure and function, particularly antagonists. SN-79 is a high affinity ligand for sigma-2 receptors, with 10-fold selectivity for sigma-2 over sigma-1 (Ki = 30 nM vs. 290 nM, respectively). Four isothiocyanate derivatives of SN-79 were characterized in rat liver membranes using [3H](+)-pentazocine to label sigma-1 and [3H]DTG (in presence of unlabeled (+)-pentazocine) for sigma-2. The isothiocyanate derivatives all exhibited lower affinity for sigma-2 receptors compared to SN-79. Sigma-2 Ki values were 320, 102, 395, and 271 nM for CM-572, CM-617, CM-621, and CM-769, respectively. Irreversible binding was examined by pretreating membranes for 60 min with the compound, extensively washing the membranes, and then determining recovery of radioligand binding. Loss of radioligand binding upon pretreatment results from “wash-resistant binding” of the compound and indicates covalent attachment to the receptor. Pretreatment with 100 nM CM-572 or CM-617 resulted in recovery of only 25% and 40% of sigma-2 binding, respectively. Treatment with 100 nM CM-621 or CM-769 had no effect. Therefore, while all four compounds bind to sigma-2 receptors with roughly comparable affinities, only CM-572 and CM-617 bind irreversibly. None of the compounds had an effect on sigma-1 receptor binding, indicating selective acylation of the sigma-2 receptor. In dose studies, CM-572 exhibited an IC50 ∼30 nM. Human SK-N-SH neuroblastoma cells were used to examine the biological activity of CM-572. Cells were pretreated for 1 h with CM-572 at concentrations up to 100 uM, then washed free of ligand, normal media added, and monitored by MTT assay after 24 h. There was no effect on cell viability up to a concentration of 10 uM, where ∼20% cytotoxicity was observed. About 80% cytotoxicity was observed at 100 uM. This suggests that CM-572 may have an irreversible partial agonist effect. However, pretreatment with CM-572 was unable to attenuate cell death induced by the sigma-2 agonist, CB-64D. A component of the signaling cascade of sigma-2 receptor activation is calcium release from the ER. In Fura-2 loaded cells, CM-572 produced no calcium signal at 3 uM and a small signal at 10 uM, though much smaller than the robust signal induced by CB-64D. Larger signals were produced at 30 uM and 100 uM CM-572. Interestingly, 3 uM and 10 uM CM-572 attenuated the calcium signal induced by 30 uM CB-64D. Taken together, these data indicate that CM-572 may be an irreversible partial agonist at sigma-2 receptors.

Citation Format: Hilary Nicholson, Anthony Comeau, Christophe Mesangeau, Christopher R. McCurdy, Wayne D. Bowen. Development of selective irreversible antagonists for sigma-2 receptors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2242. doi:10.1158/1538-7445.AM2013-2242