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弓形虫MAP激酶MAPK1-Tg，它作为应激MAPK，与人类p38 MAPK具有40%的同源性。MAPK1-Tg在速殖子中表达为全长58 kDa蛋白，是MAPK家族的第一个成员。表达显性阴性MAPK1-Tg的弓形虫速殖子在体外的复制速度明显低于亲本寄生虫，并表达更多的慢殖子抗原。最引人注目的是，这些速殖子对小鼠的毒力显著减弱。这些数据提示MAPK1-Tg在速殖子增殖、阶段分化和毒力控制中起作用。我们之前的研究表明，设计用于阻断人类p38 MAPK激活的吡啶酰基咪唑药物也能在体外阻断弓形虫的复制，治愈弓形虫感染的小鼠，并诱导体外阶段分化。人p38 MAPK抑制剂体外阻断MAPK1-Tg活性。我们假设p38 MAPK抑制剂通过抑制MAPK1-Tg治疗弓形虫感染。本研究将以两个基本目标来研究MAPK1-Tg: i)利用MAPK1-Tg作为新的药物发现靶点，ii)利用MAPK1-Tg作为工具来增加我们对弓形虫生物学的理解。67657是典型的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、利什曼原虫和锥虫基因组数据库中鉴定出具有MAPK1-Tg结构特征的MAPK同源物。我们假设寄生虫MAPKs代表了新的广谱药物开发靶点。最后，弓形虫是B类生物恐怖剂。因此，这些发现可能会导致对抗生物恐怖的新治疗方法。我们的具体目标是：1 .验证关于MAPK1-Tg如何调节复制和阶段分化的假设。检测野生型寄生虫内源性总MAPK1-Tg和活性MAPK1-Tg的试剂的开发，以及MAPK1-Tg显性阴性的重组寄生虫的开发，现在允许对MAPK1-Tg激活如何调节速殖子复制和阶段分化以及对细胞周期调节的影响的假设进行明确的测试。验证67657阻断MAPK1-Tg激活的假设。这些研究有助于阐明控制寄生虫毒力的因素。2 .验证阻断MAPK1-Tg活化是p38 MAPK抑制剂治疗作用机制的假设。3在体内验证阻断MAPK1-Tg增强抗弓形虫治疗效果的假设。

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.