Stimuli-Responsive Small Molecule Persulfidating Agents

Abstract

Hydrogen sulfide (H2S) plays an integral role in several intracellular signaling processes, modulating an array of physiological functions including vasodilation, neurotransmission, antioxidant response, inflammation, angiogenesis, hypoxia sensing to list a few. One of the widely accepted mechanisms of H2S signaling is the oxidative post translational modification of cysteine residues in proteins, known as protein persulfidation. While H2S can persulfidate proteins only under oxidizing conditions, persulfides and polysulfides, collectively termed as sulfane sulfurs can modify proteins directly and are more efficient than H2S. Additionally, low molecular weight (LMW) persulfides are better reductants and multiple evidences suggest that persulfides are far superior at sequestering oxidants compared to its congeners, thiols and H2S. Hence, small molecule persulfides have emerged as important intermediates in combating oxidative and electrophilic stress. This has fueled the development of strategies to enhance cellular persulfides and modulate the levels of intracellular protein persulfidation. LMW persulfides are unstable in aqueous solution due to its propensity to undergo disproportionation, therefore, generating these species in cells has been a major challenge. In this regard, we designed two distinct strategies for the generation of persulfides. The first strategy involved design and synthesis of prodrugs of persulfides, wherein the persulfide moiety is masked by a protecting group which can be cleaved in response to an external stimulus. Any diseased condition is often accompanied by an overproduction of free radicals and reactive oxygen species (ROS). So, we synthesized and evaluated a persulfide prodrug responsive to hydrogen peroxide (H2O2), a stable ROS. The compound upon activation by H2O2 was found to generate persulfides and exhibit cytoprotective effects in cells against oxidative stress induced damage. The next class of prodrugs were designed to be activated by the enzymes b-glycosidase, which are upregulated in cells exposed to stress such an inflammation and cancer. Two series of compounds were synthesized, responsive to b-glucosidase and b-galactosidase respectively. The compounds were found to be cleaved by the aforementioned enzymes to generate sulfane sulfurs along with H2S. Although these compounds were helpful in providing insights into the physicochemical properties of persulfides and its therapeutic potential, this strategy is limited by the complexity of synthesis and low shelf-stability of the compounds. Our second approach was to leverage the biosynthetic machinery of the cell to generate persulfides. We developed compounds that does not contain the persulfide functional group but can generate persulfides upon entry into cells. Artificial substrates for 3-mercaptopyruvate sulfurtransferase (3-MST), an enzyme involved in sulfur trafficking was synthesized and evaluated. This compound was found to generate persulfides/polysulfides upon turnover by 3-MST and generate H2S in cells. Further, antioxidative and anti-inflammatory effects of this compound was demonstrated in- vitro and in-vivo. Lastly, a prodrug of the artificial substrate for 3-MST, responsive to b- galactosidase, a biomarker for senescent cells was synthesized, as a tool to interrogate the functional consequences of persulfidation in senescence and aging. The compound was selective towards activation by b-galactosidase. Generation of H2S in the presence of 3-MST and persulfidation of proteins in senescent cells was observed with the compound. Taken together, we have developed strategies for efficient generation of intracellular persulfides with benign byproducts, that should help us delineate the role of persulfides in cell signaling while harnessing its therapeutic potential.