P50 Mitochondria-targeted hydrogen sulfide donors: A novel twist to an old “tail”?

Abstract

Hydrogen sulfide (H 2 S) has recently been identified as an endogenous redox active mediator and proposed as an endogenous regulator of nitric oxide bioavailability, vascular tone and inflammation. Novel slow release H 2 S donor (SRHD) molecules (e.g. GYY4137) have been shown to regulate blood pressure in experimental and genetically induced hypertension, and to inhibit tissue damage, oedema and inflammatory signalling in sepsis and arthritis; conditions involving perturbed mitochondrial function and oxidative stress. In this study, we have synthesized a series of compounds containing a mitochondria-targeting moiety (triphenylphosphonium), a C 2 -C 18 linker and H 2 S releasing moieties (dithiolethione or thiohydroxybenzamide). Human brain microvascular endothelial cells (HMEC) were exposed to oxidative stress agents (SIN-1/H 2 O 2 /4-HNE), the complex I inhibitor rotenone and the apoptosis-inducers staurosporine or etoposide, in the presence/absence of mitochondria-targeted compounds or non-targeted SRHDs (GYY4137/AP72) for comparison. Initial experiments on the mitochondria-targeting SRHD showed a C 10 linker to be optimal; therefore AP39 and AP123 were chosen. Oxidative stress-induced toxicity was assessed using alamarBlue®, and cytosolic/mitochondrial reactive oxygen species (“ROS”) generation was estimated using CM–H 2 DCFDA , MitoSOX™ Red and Dihydroethidium. Mitochondrial membrane potential (ΔΨm) was assessed using TMRM. Fluorescence activity assay was used to determine caspase3/7 activity. Mitochondrial ATP synthesis was finally assessed with the Luciferase assay. Cell viability was significantly preserved by GYY4137 and AP72 (100–500 μM) after treatment with oxidative stress agents. However, the potency was substantially increased by targeting mitochondria with AP39 and AP123 (<100 nM). The collapse of ΔΨm normally observed in oxidative stress conditions was inhibited by all SRHDs. Overall “ROS” generation was markedly decreased after treatment with SRHDs. Only AP72 was able to inhibit the activation of caspase 3 after treatment with staurosporine/etoposide, but all compounds inhibited caspase-3/7 activity. Finally, pre-treatment of HMEC with SRHDs preserved ATP generation after treatment with rotenone. These data suggest that SRHDs can inhibit/reverse oxidative stress-mediated cellular injury, and highlight the increased potency of the mitochondria- targeting H 2 S donors AP39 and AP123 compared to GYY4137 and AP72. Strategies increasing H 2 S bioavailability, in particular targeting mitochondria, may represent a new therapeutic opportunity to limit mitochondrial dysfunction.