Determination of the mechanism of the immunosuppressive effects of thiopurines

描述（由申请人提供）：该研究将探讨6-硫嘌呤（6-TP）药物（如6-硫胍（6-TG））及其异构体（如8-硫嘌呤（8-TG））对Rac1 GTPase的抑制作用及其在t细胞失活中的意义。6-TP类似物是治疗自身免疫性疾病和各种肿瘤（包括急性白血病和腺癌）的重要化疗药物。6-TP药物对急性白血病的治疗作用之一是激活DNA错配修复系统，从而诱导细胞凋亡。最近的研究表明，6-TP药物对某些免疫疾病的治疗作用是通过阻断T淋巴细胞中Rac1的激活诱导免疫抑制。然而，与Rac1相关的6-TP药物作为免疫疾病化疗药物的作用机制尚不明确。我们假设6-或8-TG在细胞中转化为6-或8-硫鸟嘌呤核苷酸（6-或8-TGNP），该核苷酸靶向Rac1，在6-或8-TGNP和Rac1的氧化还原敏感的GXXXXGK(S/T)C基序之间形成二硫加合物。这种Rac1-6-或-8- tgnp加合物使T细胞失活；并且需要氧化还原剂来形成Rac1-6-或-8- tgnp加合物。虽然我们的初步研究结果提供了关于6-TP和8-TG药物在T细胞中的作用的关键信息，但我们缺乏一些必要的重要元素来更好地理解6-TP和8-TG药物对Rac1和T细胞失活的影响。例如，对于氧化还原剂通过形成Rac1-6-或-8- tgnp二硫加合物对6-或8- tp介导的Rho蛋白抑制作用的详细分子机制，目前还没有明确的解释。为了更好地理解tp通过与氧化还原剂相关的Rac1-6-或-8- tgnp加合物的形成介导的Rac1失活及其在细胞中的意义，我们提出了使用动力学和质谱分析以及分子和细胞生物学方法进行详细的机制研究。本研究的预期结果将为开发更好的靶向和灭活Rac1的免疫抑制剂提供基础。考虑到与6-TP相比，8-TG处理的细胞凋亡率最低，6-TP的类似物（包括8-TG）在靶向Rac1的药物中可能优于6-TP，而细胞毒性比6-TP小。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.