A Novel MAPK family in T. gondii

描述（由申请人提供）：我们最近克隆了第一个T。弓形虫MAP激酶，MAPK 1-Tg，其作为应激MAPK，与人p38 MAPK具有40%的同源性。MAPK 1-Tg在速殖子中表达为全长58 kDa的蛋白，是一个新的MAPK家族的第一个成员。T.表达显性阴性MAPK 1-Tg的弓形虫速殖子在体外的复制速度明显慢于亲本寄生虫，并表达明显更多的缓殖子抗原。最引人注目的是，这些速殖子在小鼠中的毒力显著减弱。 这些数据暗示MAPK 1-Tg在控制速殖子增殖，阶段分化和毒力。 我们以前发现，设计用于阻断人p38 MAPK活化的吡啶基咪唑类药物也能阻断T。弓形虫体外复制、治愈T. gondii感染的小鼠，并在体外诱导阶段分化。人p38 MAPK抑制剂在体外阻断MAPK 1-Tg活性。我们假设p38 MAPK抑制剂治疗T.通过抑制MAPK 1-Tg抑制弓形虫感染。本研究将从两个方面对MAPK 1-Tg进行研究：i）将MAPK 1-Tg作为一种新的药物发现靶点，ii）将MAPK 1-Tg作为一种工具来增加我们对T的理解。弓形虫生物学67657是使用的原型p38 MAPK抑制剂。它在关节炎的人体I期试验中是安全的，并且可以作为抗寄生虫剂迅速转化为人体临床试验。在疟原虫、利什曼原虫和锥虫的基因组数据库中鉴定了具有MAPK 1-Tg结构特征的MAPK同源物。我们假设，寄生虫MAPK代表新的，广谱的药物开发目标。最后，T。弓形虫是B类生物恐怖剂。因此，这些发现可能会导致新的治疗方法来打击生物恐怖。我们的具体目标是：1检验关于MAPK 1-Tg如何调节复制和阶段分化的假设。开发的试剂，以检测内源性产生的总的和活性MAPK 1-Tg在野生型寄生虫，和显性负性MAPK 1-Tg的重组寄生虫的发展，现在允许明确的测试假说，关于如何MAPK 1-Tg激活调节速殖子复制和阶段分化，和细胞周期调控的影响。检验67657阻断MAPK 1-Tg活化的假设。这些研究有助于阐明控制寄生虫毒力的因素。2验证阻断MAPK 1-Tg活化是p38 MAPK抑制剂治疗作用机制的假设。3检验阻断MAPK 1-Tg增强体内抗弓形虫疗法功效的假设。

DESCRIPTION (provided by applicant): We recently cloned the first T. gondii MAP kinase, MAPK1-Tg, which acts as a stress MAPK and has 40% homology to human p38 MAPK. MAPK1-Tg is expressed as the full-length 58 kDa protein in tachyzoites, and is the first member of a novel MAPK family. T. gondii tachyzoites expressing a dominant-negative MAPK1-Tg replicated significantly more slowly than parental parasites in vitro, and expressed significantly more bradyzoite antigens. Most strikingly, these tachyzoites were remarkably attenuated in mouse virulence. These data implicate MAPK1-Tg in control of tachyzoite proliferation, stage differentiation and virulence. We previously showed that pyridinylimidazole drugs designed to block human p38 MAPK activation also blocked T. gondii replication in vitro, cured T. gondii-infected mice, and induced stage differentiation in vitro. Human p38 MAPK inhibitors block MAPK1-Tg activity in vitro. We hypothesize that p38 MAPK inhibitors treat T. gondii infection through inhibition of MAPK1-Tg. This proposal will study MAPK1-Tg with two fundamental objectives: i) to exploit MAPK1-Tg as a novel drug discovery target, and ii) to use MAPK1-Tg as a tool to increase our understanding of T. gondii biology. 67657 is the prototypical p38 MAPK inhibitor used. It was safe in human Phase I trials in arthritis, and could be translated into a human clinical trial as an anti-parasitic agent rapidly. MAPK homologues with structural features of MAPK1-Tg were identified in genomic databases for Plasmodium, Leishmania and Trypanosoma. We hypothesize that parasite MAPKs represent novel, broad spectrum drug development target. Finally, T. gondii is a category B bioterror agent. Thus, these discoveries could lead to novel treatment approaches to combating bioterror. Our Specific aims are: 1 Test hypotheses regarding how MAPK1-Tg regulates replication and stage differentiation. The development of reagents to detect endogenously produced total and active MAPK1-Tg in wild-type parasites, and the development of recombinant parasites with dominant-negative MAPK1-Tg now permit definitive testing of hypotheses regarding how MAPK1-Tg activation regulates tachyzoite replication and stage differentiation, and effects on the cell cycle regulation. Test the hypothesis that 67657 blocks MAPK1-Tg activation. These studies help elucidate factors controlling parasite virulence. 2 Test the hypothesis that blocking MAPK1-Tg activation is a mechanism of therapeutic action of p38 MAPK inhibitors. 3 Test the hypothesis that blocking MAPK1-Tg augments efficacy of anti-Toxoplasma therapies in vivo.