Traditionally known for its toxicity, hydrogen sulfide (H₂S) also possesses physiological roles as an endogenous gaseous signaling molecule in multiple biological processes. Previous research has demonstrated changes in levels of H₂S-producing enzymes during oxidative stress, hypoxia, and inflammation in various tissues including the liver and heart. H₂S protects cells from cytotoxicity in part by promoting the synthesis of glutathione, neutralizing reactive oxygen species, and inhibiting apoptosis signaling pathways. Thiol methyltransferases TMT1A and TMT1B can methylate endogenous H₂S to methanethiol. TMT1B has been shown to have a potential role in mediating the toxic effects of methanethiol. Gene silencing of TMT1B was found to significantly alleviate the observed cytotoxicity induced by methanethiol in human bronchial epithelial cells (16HBE). Methanethiol may induce harm to human respiratory tract cells, and understanding the mechanisms involved, including the role of TMT1B, could potentially lead to insights for mitigating these harmful effects. Doxorubicin (Dox) is a widely used chemotherapy drug for the treatment of various cancers but can induce oxidative stress in cells. In my preliminary experiments, I assessed the cell viability of HepG2 liver cells that were supplemented with various concentrations of NaSH (H₂S donor) and sodium methanethiolate (NaSMe, MeSH donor), followed by Dox treatment. Supplementing the cells with H₂S significantly increased cell viability in the presence of doxorubicin, while the methanethiol had no effect. The goal of my project is to identify H₂S-dependent protective pathways during cellular stress in HepG2 versus cardiomyocytes. My preliminary data indicates that both H₂S and its metabolite, methanethiol, may alter cellular responses following treatment of exogenous compounds that induce cellular stress such as CoCl₂, hydrogen peroxide and Dox. My goal is to pinpoint genes altered during the stress response. This understanding of H₂S-dependent pathways may pave the way for designing novel therapeutics that maintain or enhance H₂S levels.

There are three enzymes that S-methylate thiols: thiopurine methyltransferase (TPMT) and two enzymes that our lab has recently identified, alkyl thiol methyltransferase 1A and 1B (TMT1A and TMT1B). These enzymes have been studied predominantly because of their role in drug metabolism. Thiopurines, potent chemotherapeutics, are inactivated through thiol methylation by TPMT. While there are not many drugs that contain alkyl thiols, nearly every alkyl thiol-containing drug is metabolized via S-methylation, presumably mediated by TMT1A or TMT1B. With any drug used there is a possibility of drug-drug interactions (DDI) that can lead to a drug concentration in the cell that is above the therapeutic index, leading to toxicity. Thus, it is important to have a method that allows for quick determination of possible DDIs in the body. In this work, using ligation cloning and nickel affinity chromatography, I recombinantly expressed and purified TPMT from bacteria. I then developed an absorbance-based high throughput assay to compare the substrate specificity of TPMT with that of recombinant TMT1A and TMT1B. I determined that TMT1A and TMT1B preferentially methylate alkyl thiols, while TPMT exclusively methylates thiols involved in a conjugated electron system. Although these enzymes serve a crucial role as drug metabolizing enzymes, it is not known if these enzymes have a function beyond drug metabolism. To study the endogenous role of these enzymes beyond drug metabolism, I am utilizing my developed assay to screen for compounds that can specifically inhibit TMT1A, TMT1B, and TPMT. In addition, the assay is optimized in order to screen for potential drug-drug interactions that might result due to interactions with TPMT. Based on literature, I expect most benzoic acid derivatives and similar structured compounds to result in DDIs.