The research proposed will examine the chemistry-based cellular mechanism behind the inhibitory action of 6-thiopurine (6-TP) drugs such as 6-thioguaine (6-TG) and its isomers such as 8-thiogunaine (8-TG) on Rac1 GTPase and its implication in T-cell inactivation. 6-TP analogs are important chemotherapeutic agents in the treatment of autoimmune disorders and various tumors, including acute leukemia and adenocarcinomas. One of therapeutic actions of 6-TP drugs for acute leukemia is to activate the DNA mismatch repair system, which results in induction of cell apoptosis. Recent studies show that the therapeutic action of 6-TP drugs in certain immune disorders is to induce immunosuppression by blockage of Rac1 activation in T lymphocytes. However, a clear mechanism of action of 6-TP drugs associated with Rac1 as an immune disorder chemotherapeutic is yet to be determined. We hypothesize that 6- or 8-TG is cellularly converted into a 6- or 8-thioguanine nucleotide (6- or 8-TGNP) that targets Rac1 to form a disulfide adduct between 6- or 8-TGNP and the redox-sensitive GXXXXGK(S/T)C motif of Rac1. This Rac1-6- or -8-TGNP adduct inactivates T cells;and a redox agent is required for the formation of the Rac1-6- or -8-TGNP adduct. Although our preliminary study results have provided critical information about the action of 6-TP and 8-TG drugs in T cells, we lack several important elements necessary to a better understanding of the effect(s) of 6- TP and 8-TG drugs on Rac1 and T cell inactivation. For example, no clear explanation has been elucidated for the detailed molecular mechanism that governs the role of the redox agent on the 6- or 8-TP-mediated inhibition of Rho protein via formation of the Rac1-6- or -8-TGNP disulfide adduct. To better understand the TP-mediated inactivation of Rac1 via formation of the Rac1-6- or -8-TGNP adduct associated with a redox agent and its implication in cells, we propose a detailed mechanistic study using kinetic and mass spectrometric analyses as well as molecular and cell biology approaches. The anticipated results of our proposed study will provide the basis for development of a better immunosuppressive agent(s) to target and inactivate Rac1. Given that the rate of apoptosis of cells treated with 8-TG was minimal compared with that of 6-TP, analogs of 6-TP - including 8-TG - may be superior to those of 6-TP for drugs that target Rac1 with less cytotoxicity than what is encountered with 6-TP.
This proposed research seeks to understand the Rac GTPase-targeting action of thiopurine drugs in vitro and in T cells. This action may suppress T-cell activation, thereby suppressing certain immune responses. The anticipated results of our proposed study may lead to a molecular and mechanistic basis for developing therapies for immune disorders, such as Inflammatory Bowel Disease, that are associated, at least in part, with abnormal regulation of Rac proteins.
|Shin, Jin-Young; Wey, Michael; Umutesi, Hope G et al. (2016) Thiopurine Prodrugs Mediate Immunosuppressive Effects by Interfering with Rac1 Protein Function. J Biol Chem 291:13699-714|
|Wey, Michael; Phan, Vinh; Yepez, Gerardo et al. (2014) Superoxide inhibits guanine nucleotide exchange factor (GEF) action on Ras, but not on Rho, through desensitization of Ras to GEF. Biochemistry 53:518-32|
|Wey, Michael; Lee, Jungwoon; Jeong, Soon Seog et al. (2013) Kinetic mechanisms of mutation-dependent Harvey Ras activation and their relevance for the development of Costello syndrome. Biochemistry 52:8465-79|