Determination of the mechanism of the immunosuppressive effects of thiopurines

描述(申请人提供)：这项研究将探讨6-硫代嘌呤(6-TP)类药物(如6-Tg)及其异构体(如8-Tg)对rac1 GTP酶的抑制作用背后的化学细胞机制及其在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多种肿瘤的重要化疗药物，包括急性白血病和腺癌。6-TP类药物治疗急性白血病的作用之一是激活DNA错配修复系统，诱导细胞凋亡。最近的研究表明，6-TP类药物对某些免疫疾病的治疗作用是通过阻断T淋巴细胞中的rac1活性来诱导免疫抑制。然而，与rac1相关的6-TP药物作为一种免疫紊乱化疗药物的明确作用机制尚未确定。我们假设6-或8-TG在细胞内转化为6-或8-硫代鸟嘌呤核苷酸(6-或8-TGNP)，靶向RAC1，在6-或8-TGNP与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；而形成rac1-6-或-8-TGNP加合物需要氧化还原剂。虽然我们的初步研究结果提供了有关6-TP和8-TG药物在T细胞中作用的关键信息，但我们缺乏几个重要的元素来更好地了解6-TP和8-TG药物对Rac1和T细胞失活的影响(S)。例如，氧化还原剂通过形成rac1-6-或-8-TGNP二硫化物抑制6-或8-TP介导的Rho蛋白抑制的详细分子机制尚未阐明。为了更好地了解TP通过与氧化还原剂相关的rac1-6-或-8-TGNP加合物的形成而介导的rac1失活及其在细胞中的意义，我们提出了一项详细的机制研究，使用动力学和质谱分析以及分子和细胞生物学方法。我们的预期研究结果将为开发一种更好的免疫抑制剂(S)来靶向并灭活Rac1奠定基础。考虑到8-TG处理的细胞凋亡率比6-TP微乎其微，6-TP的类似物--包括8-TG--对于靶向rac1的药物来说可能比6-TP好，细胞毒性比6-TP小。 公共卫生相关性：这项拟议的研究试图了解硫代嘌呤类药物在体外和T细胞中的RAC GTPase靶向作用。这种作用可能会抑制T细胞的激活，从而抑制某些免疫反应。我们拟议的研究的预期结果可能会为开发免疫疾病的治疗方法提供分子和机制基础，例如炎症性肠病，这些疾病至少部分与RAC蛋白的异常调节有关。

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.