A Novel MAPK family in T. gondii

描述(申请人提供)：我们最近克隆了第一个弓形虫MAPK，MAPK1-Tg，它作为一个应激MAPK，与人p38MAPK有40%的同源性。MAPK1-TG在速殖子中表达为58 kDa的全长蛋白，是一个新的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慢于亲本寄生虫，并且表达的缓殖子抗原明显多于亲本。最引人注目的是，这些速殖子对小鼠的毒力显著减弱。这些数据表明MAPK1-TG在控制速殖子的增殖、阶段分化和毒力方面起着重要作用。我们之前的研究表明，设计用于阻断人p38 MAPK激活的吡啶并咪唑类药物在体外也可以阻断弓形虫的复制，治愈弓形虫感染的小鼠，并在体外诱导阶段分化。人p38MAPK抑制剂体外阻断MAPK1-TG活性。我们推测p38 MAPK抑制剂通过抑制MAPK1-TG来治疗弓形虫感染。研究MAPK1-TG有两个基本目的：一是开发MAPK1-TG作为新的药物发现靶点；二是利用MAPK1-TG作为工具增加我们对弓形虫生物学的了解。67657是典型的p38MAPK抑制剂。在人类关节炎的I期试验中，它是安全的，并可以作为一种抗寄生虫剂迅速转化为人类临床试验。在疟原虫、利什曼原虫和锥虫基因组数据库中发现了具有MAPK1-TG结构特征的MAPK同源物。我们假设寄生虫MAPK代表了新的、广谱的药物开发靶点。最后，弓形虫是B类生物恐怖因子。因此，这些发现可能导致对抗生物恐怖的新治疗方法。我们的具体目标是：1关于MAPK1-TG如何调控复制和阶段分化的假设。检测野生型寄生虫内源性产生的总MAPK1-Tg和活性MAPK1-Tg的试剂的发展，以及显性阴性MAPK1-Tg重组寄生虫的发展，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说进行明确的检验。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激活是p38 MAPK抑制剂治疗作用机制的假说。3在体内验证阻断MAPK1-TG增强弓形虫治疗效果的假说。

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.