1 A novel hydrogen sulfide-releasing NMDA receptor antagonist prevents ischemic neuronal death Eizo Marutani 1 , Shizuko Kosugi 1 , Kentaro Tokuda 1 , Ashok Khatri 2 , Rebecca Nguyen 1 , Dmitriy N. Atochin 3 , Kotaro Kida 1 , Klaus Van Leyen 4 , Ken Arai 4 , and Fumito Ichinose 1 From 1 the Anesthesia Center for Critical Care Research of the Department of Anesthesia, Critical Care and Pain Medicine, 2 the Endocrine Unit and 3 the Cardiovascular Research Center and Cardiology Division of the Department of Medicine, and 4 the Department of Radiology at the Massachusetts General Hospital and Harvard Medical School, Charlestown, MA02129, USA *Running title: H 2 S-releasing NMDA receptor antagonist and neuronal ischemia 1 Address correspondence to: Fumito Ichinose, MD, PhD, Department of Anesthesia and Critical Care, Massachusetts General Hospital, 149 13 th Street, 4315, Charlestown, Massachusetts 02129, Phone: 617- 643-0987, Fax: 617-643-4490, E-mail: fichinose@partners.org Background: Hydrogen sulfide (H 2 neuroprotective effects, whereas H S) exerts 2 neurotoxicity via N-methyl-D-aspartate receptor S may cause (NMDAR) activation. Result: A newly-synthesized H 2 NMDAR antagonist S-memantine exerted lower S-releasing neurotoxicity and prevented ischemic neuronal death more markedly than conventional H 2 Conclusion: S-memantine prevents ischemic brain injury without neurotoxicity. S- releasing compounds or memantine alone. Significance: H 2 may prevent neurodegeneration of various causes. S-releasing NMDAR antagonists SUMMARY Physiological levels of H 2 S exert neuroprotective effects, whereas high concentrations of H 2 S may cause neurotoxicity in part via activation of NMDAR. To characterize the neuroprotective effects of combination of exogenous H 2 S and NMDAR antagonism, we synthesized a novel H 2 S- releasing NMDAR antagonist N-((1r,3R,5S,7r)- 3,5-dimethyladamantan-1-yl)-4-(3-thioxo-3H- 1,2-dithiol-4-yl)-benzamide (S-memantine) and examined its effects in vitro and in vivo. S- memantine was synthesized by chemically combining a slow releasing H 2 S donor 4-(3- thioxo-3H-1,2-dithiol-4-yl)-benzoic acid (ACS48) with a NMDAR antagonist memantine. S-memantine increased intracellular sulfide levels in human neuroblastoma cells (SH- SY5Y) ten fold as high as that was achieved by ACS48. Incubation with S-memantine after reoxygenation following oxygen and glucose deprivation (OGD) protected SH-SY5Y cells and murine primary cortical neurons more markedly than did ACS48 or memantine. Glutamate-induced intracellular calcium accumulation in primary cortical neurons were aggravated by Sodium sulfide (Na 2 S) or ACS48, but suppressed by memantine and S- memantine. S-memantine prevented glutamate- induced glutathione depletion in SH-SY5Y cells more markedly than did Na 2 S or ACS48. Administration of S-memantine after global cerebral ischemia and reperfusion more robustly decreased cerebral infarct volume and improved survival and neurological function of mice than did ACS48 or memantine. These results suggest that a novel H 2 S-releasing NMDAR antagonist derivative S-memantine prevents ischemic neuronal death, providing a novel therapeutic strategy for ischemic brain injury. Hydrogen sulfide has been proposed as a gaseous signaling molecule along with nitric oxide and carbon monoxide (1). A number of studies http://www.jbc.org/cgi/doi/10.1074/jbc.M112.374124 The latest version is at JBC Papers in Press. Published on July 19, 2012 as Manuscript M112.374124 Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc. at Harvard Libraries, on August 21, 2012 www.jbc.org Downloaded from