TARGETS AND MECHANISMS OF ACTION FOR PARASITICAL AGENTS

我们的合作小组作为一个整体采取了多方面的方法 发展新的艾滋病化疗策略 弓形虫病除了努力改善 现有的药物和寄生虫特异性靶点的验证，我们 还研究了新型杀寄生虫剂活性 抗犬弓刚地。项目一着眼于后一种化合物， 发现现有药物的相关靶点， 有效性，利用分子工具（许多是在 这使得弓形虫成为唯一 可以进行基因操作 大环内酯类和林可酰胺类抗生素对T. 弓形虫，但体外研究揭示了不寻常的药代动力学： 寄生虫仅在进入下一个寄生虫液泡后死亡， 治疗后数小时甚至数天。交叉耐药谱 突变寄生虫支持蛋白质抑制机制，但分子 完整寄生虫的序列分析和生化研究， 分离的核糖体表明，无论是细胞质， 线粒体核糖体可能参与其中。我们已经确定 然而，替代的核糖体靶点，包括相关基因， 新的类质体染色体外基因组。除了 研究合适的核糖体基因在突变寄生虫，我们建议 通过遗传学方法鉴定大环内酯类药物耐药位点。返回 到类质体基因组，我们将确定亚细胞位置 这种寄生虫特有的结构，并研究相关的目标 具有治疗潜力 在筛选杀寄生虫活性的许多化合物中， 二硝基苯胺除草剂是一个特别有趣的组， 抑制T.通过破坏寄生虫的 核内有丝分裂纺锤体它们对植物微管的破坏 似乎是一种间接效应，而精确的分子靶点 (and抵抗的基础尚不清楚。我们已经分离出除草剂 抗性T.弓形虫突变体的遗传特性，并将 测试原始先导化合物的合适类似物用于体内 抗弓形虫活性。在其他实验中， 也被发现是有效的杀寄生虫药物，大概是通过 与寄生虫亲环素的相互作用。两种亲环素 从T.我们会调查 其功能的生物学基础是利用生物化学， 免疫学和分子遗传学技术。两者 二硝基苯胺和亲环素显示出产生新的 深入了解蛋白质分泌途径的细胞生物学， 可能会提供额外的干预策略。 进一步的药理学筛选（和有希望的基因分析） 化合物）将集中在已知的除草剂和抗球虫药， 研究艾美耳球虫和疟原虫。

Our collaborative group as a whole has taken a multifaceted approach to the development of new chemotherapeutic strategies for AIDS- toxoplasmosis. In addition to working towards the improvement of existing drugs and the validation of parasite-specific targets, we have also examined novel classes of parasiticidal agents active against T. gondii. Focusing on these latter compounds, Project I seeks to discover the relevant targets for existing drugs of proven efficacy, taking advantage of molecular tools (many developed under the auspices of this grant) which now render Toxoplasma uniquely accessible to genetic manipulation. Macrolide and lincosamide antibiotics are highly effective against T. gondii, but in vitro studies reveal unusual pharmacokinetics: parasites die only after entry into the next parasitophorous vacuole, hours or even days after treatment. Cross-resistance profiles of mutant parasites support a protein-inhibitory mechanism, but molecular sequence analysis and biochemical studies on intact parasites and isolated ribosomes indicates that neither cytoplasmic nor mitochondrial ribosomes is likely to be involved. We have identified alternative ribosomal targets, however, including genes associated with the novel plastid-like extrachromosomal genome. In addition to examining appropriate ribosomal genes in mutant parasites, we propose to identify the macrolide-resistance locus by genetic means. Returning to the plastid-like genome, we will determine the subcellular location of this parasite-specific structure and investigate associated targets with therapeutic potential. Among the many compounds screened for parasiticidal activity, the dinitroaniline herbicides are a particularly intriguing group, inhibiting T. gondii replication via disruption of the parasite's intranuclear mitotic spindle. Their disruption of plant microtubules appears to be an indirect effect, and the precise molecular target (and basis for resistance) remains unclear. We have isolated herbicide resistant T. gondii mutants for genetic characterization, and will test suitable analogs of the original lead compounds for in vivo activity against Toxoplasma. In other experiments, cyclosporins were also found to be potent parasiticidal drugs, presumably through interaction with parasite cyclophilins. Two species of cyclophilin cDNAs have been cloned from T. gondii, and we will investigate the biological basis of their function using a combination of biochemical, immunological, and molecular genetic techniques. Both the dinitroanilines and cyclophilins exhibit features which yield new insights into the cell biology of the protein secretory pathway, which may be expected to provide additional strategies for intervention. Further pharmacological screening (and genetic analysis of promising compounds) will focus on herbicides and coccidiostats known from studies on Eimeria and Plasmodium species.