Abstract 1671: Identification and characterization of MAM03055A, a novel bivalent sigma-2 receptor agonist

Abstract

Abstract Sigma-2 receptors are highly expressed in various types of cancer and mediate dual effects on cancer cell viability. Agonists induce programmed cell death by a number of pathways including apoptosis and autophagy. Other classes of sigma-2 receptor ligands activate pathways related to glycolytic metabolism and hypoxia. CM572 is a 6-isothiocyanato benzoxazolone derivative that binds irreversibly and selectively to sigma-2 receptors, presumably through attack of the isothiocyanate moiety by a receptor nucleophile. It is a partial agonist, inducing dose-dependent cell death. We observed by LC/MS analysis, that upon storage in DMSO solution at -20oC over several months, CM572 degrades into a mixture of compounds that appear to retain both receptor binding activity and ability to induce cell death. The degradation was eventually convergent to one main compound (CM572-HN). CM572-HN was identified as a dimeric molecule based on the isotopic fingerprint of the high-resolution mass spectrum. Similarly, a degradation study by 1H NMR of a solution of CM572 in deuterated DMSO showed convergence to one major compound. The degradation site was narrowed down to the isothiocyanate group, as only the signals for its neighboring protons were shifted in the degradation product. We suspected hydrolysis, the final degradation step thus being the corresponding amine. It was then hypothesized that the kinetics of degradation of CM572 towards the corresponding 6-substituted amine (CM571) was slower than that of the nucleophilic attack of CM571 on the isothiocyanate group of CM572. Hence, CM571 and CM572 were synthesized and then reacted together to produce, in fact, a single compound (MAM03055A), with mass identical to one originally identified in the stock solution and 1H NMR spectra fitting those of the compound originally obtained during the NMR degradation study. Thus the novel compound is essentially a thiourea-linked dimer of the amine, CM571. Receptor binding assays revealed that MAM03055A (MAM) exhibited selectivity for sigma-2 receptors over sigma-1, with sigma-1 Ki = 2,829 nM and sigma-2 Ki = 56.7 nM, resulting in 50-fold selectivity for sigma-2 receptors. MAM induced dose-dependent cell death in human SK-N-SH neuroblastoma cells, with an EC50 = 9.24 ± 1.15 µM for a 24h incubation. Interestingly, unlike other sigma-2 agonists that give 100% cell kill, MAM reached a maximum of only 80%, even at a concentration of 100 µM. The true EC50 in this case was therefore around 2 µM. Thus, MAM behaved like a partial agonist. The binding affinities and cytotoxic activity of the dimer resembles that of CM572 (isothiocyanate; EC50 = 7.6 µM), but is very different from CM571 (amine), which has high affinity at both subtypes and stimulates metabolism. Bivalent sigma-2 ligands may thus have interesting properties that deserve further exploration. (*C. L. and M. M. contributed equally as first authors) Citation Format: Cheri Z. Liu, Marco Mottinelli, Hilary E. Nicholson, Eric Zhong, Christopher R. McCurdy, Wayne D. Bowen. Identification and characterization of MAM03055A, a novel bivalent sigma-2 receptor agonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1671.