P CARINII MICROTUBULES--GENETICS & BENZIMIDAZOLE TARGET

微管在真核细胞分裂中起着至关重要的作用， 有丝分裂纺锤体的形成。 它们也是许多国家的主要组成部分， 细胞类型，细胞骨架和鞭毛。 符合其 重要的是，微管是多种药物的靶点， 毒素 然而，苯并咪唑是值得注意的，因为它们对人的毒性低。 哺乳动物细胞，但对蠕虫和真菌病原体具有高活性。 最近，我们已经证明了临床上有用的活性， 苯并咪唑对抗原生动物蓝氏贾第虫 卡氏肺孢子虫 肺炎是艾滋病的主要传染性并发症， 需要化疗方法。 卡氏疟原虫似乎是一种真菌 具有原生动物的药物敏感性;因此，抗卡氏疟原虫 苯并咪唑的活性值得研究。 初步研究， 培养模型显示，卡氏肺孢子虫的生长对选择的 苯并咪唑，小于1微克/毫升。 然而，要实现全面 这类药物的潜力，重要的是要了解分子 其选择性毒性的基础。 为了实现这一目标，我们将继续努力， 具体目的如下：（1）卡氏肺孢子虫微管蛋白的克隆与鉴定 基因. 微管蛋白是微管的主要成分， 敏感和耐药生物体的微管蛋白序列可以识别 对苯并咪唑活性重要的位点。 (2)表征微管蛋白- 苯并咪唑相互作用的突变分析。 Saccharomyces 酿酒酵母将用于遗传定义微管蛋白苯并咪唑 结合位点 部分基因替换将被用来允许 酵母宿主中卡氏毕赤酵母微管蛋白的突变分析。 (3)制定 苯并咪唑结合位点的模型，并通过定点 诱变 在S.卡氏肺孢子虫微管蛋白 测试构建体对苯并咪唑敏感性的影响。 （四） 评价苯并咪唑类药物的体外和体内抗卡氏肺孢子虫活性。 苯并咪唑-微管蛋白结合位点的模型应该允许我们 合理选择或设计选择性毒性增强的衍生物。 这些将在培养物和大鼠模型中进行测试。 影响 附着，形态学和分化将被检查，并尝试 将用来分离抗性突变体

Microtubules play a crucial role in eukaryotic cell division through formation of the mitotic spindle. They are also major components, in many cell types, of the cytoskeleton and flagella. Consistent with their importance, microtubules are targets for a wide variety of drugs and toxins. The benzimidazoles are notable, however, for their low toxicity to mammalian cells but high activity against helminth and fungal pathogens. Recently, we have demonstrated clinically useful activity of selected benzimidazoles against a protozoan, Giardia lamblia. Pneumocystis carinii pneumonia is a major infectious complication of AIDS for which new chemotherapeutic approaches are needed. P. carinii appears to be a fungus with the drug susceptibility of a protozoan; thus, the anti-P. carinii activity of benzimidazoles warrants examination. Initial studies in a culture model revealed that P. carinii growth is sensitive to selected benzimidazoles at less than 1 microgram/ml. However, to realize the full potential of this drug group, it is important to understand the molecular basis for their selective toxicity. To accomplish this, we will pursue the following specific aims: (1) Clone and characterize P. carinii tubulin genes. Tubulins are the major components of microtubules, and comparisons of tubulin sequences from sensitive and resistant organisms may identify sites important to benzimidazole activity. (2) Characterize tubulin- benzimidazole interactions by mutational analysis. Saccharomyces cerevisiae will be used to genetically define the tubulin-benzimidazole binding site. Partial gene replacement will then be used to permit mutational analysis of P. carinii tubulin in the yeast host. (3) Formulate models for the benzimidazole binding site and evaluate by site-directed mutagenesis. Specific alterations in S. cerevisiae-P. carinii tubulin constructs will be tested for effects on benzimidazole sensitivity. (4) Evaluate anti-P. carinii activity of benzimidazoles in vitro and in vivo. Models for the benzimidazole-tubulin binding site should permit us to rationally select or design derivatives with enhanced selective toxicity. These will be tested in culture and in the rat model. Effects on attachment, morphology, and differentiation will be examined, and attempts will be made to isolate resistant mutants.