A Novel MAPK family in T. gondii

弓形虫中一个新的 MAPK 家族

• 批准号: 7408234
• 负责人: Tyler J. Curiel
• 金额: $ 6.14万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7408234
• 关键词: RNA interference; SDS polyacrylamide gel electrophoresis; Toxoplasma gondii; autoradiography; drug discovery /isolation; drug resistance; enzyme activity; enzyme inhibitors; gene expression; gene targeting; genetically modified animals; laboratory mouse; microorganism growth; mitogen activated protein kinase; parasite infection mechanism; pharmacology; polymerase chain reaction; virulence; western blottings

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.

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，它充当应激 MAPK，与人类 p38 MAPK 具有 40% 同源性。 MAPK1-Tg 在速殖子中表达为全长 58 kDa 蛋白，是新型 MAPK 家族的第一个成员。表达显性失活 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，并且表达明显更多的缓殖子抗原。最引人注目的是，这些速殖子对小鼠的毒力显着减弱。 这些数据表明 MAPK1-Tg 控制速殖子增殖、阶段分化和毒力。 我们之前表明，旨在阻断人 p38 MAPK 激活的吡啶基咪唑药物也能在体外阻断弓形虫复制，治愈弓形虫感染的小鼠，并在体外诱导阶段分化。人类 p38 MAPK 抑制剂在体外阻断 MAPK1-Tg 活性。我们假设 p38 MAPK 抑制剂通过抑制 MAPK1-Tg 来治疗弓形虫感染。该提案将研究 MAPK1-Tg，有两个基本目标：i）利用 MAPK1-Tg 作为新的药物发现靶点，ii）使用 MAPK1-Tg 作为工具来增加我们对弓形虫生物学的了解。 67657 是所使用的原型 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药物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 MAPK 代表新颖、广谱的药物开发靶点。最后，弓形虫是 B 类生物恐怖制剂。因此，这些发现可能会带来对抗生物恐怖的新治疗方法。我们的具体目标是： 1 测试有关 MAPK1-Tg 如何调节复制和阶段分化的假设。用于检测野生型寄生虫中内源产生的总和活性 MAPK1-Tg 的试剂的开发，以及具有显性失活 MAPK1-Tg 的重组寄生虫的开发，现在允许对有关 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。检验 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 检验以下假设：阻断 MAPK1-Tg 激活是 p38 MAPK 抑制剂的治疗作用机制。 3 检验阻断 MAPK1-Tg 可增强体内抗弓形虫疗法疗效的假设。