P CARINII MICROTUBULES--GENETICS & BENZIMIDAZOLE TARGET

微管通过以下途径在真核细胞分裂中发挥关键作用 有丝分裂纺锤体的形成。在许多情况下，它们也是主要组成部分 细胞骨架和鞭毛的细胞类型。与他们的 重要的是，微管是多种药物的靶标， 毒素。然而，苯并咪唑类化合物值得注意的是它们对 哺乳动物细胞，但对蠕虫和真菌病原体具有很高的活性。 最近，我们已经展示了精选的临床有用活性。 苯并咪唑对原生动物蓝氏贾第鞭毛虫的影响。卡氏肺孢子虫 肺炎是艾滋病的主要感染性并发症，对它来说，新的 化疗方法是必要的。卡氏P.carinii似乎是一种真菌 具有原生动物的药物敏感性；因此，抗P。脊椎动物 苯并咪唑类化合物的活性值得检查。一项初步研究 培养模型表明，卡氏肺孢子虫的生长对所选择的 苯并咪唑类药物在1微克/毫升以下。然而，要实现充分的 这一药物群的潜力，重要的是了解分子 它们的选择性毒性的基础。要做到这一点，我们将致力于 具体目标如下：(1)卡氏肺孢子虫微管蛋白的克隆和鉴定 基因。微管蛋白是微管的主要组成部分，比较 敏感和耐药生物体的微管蛋白序列可以识别 对苯并咪唑活性很重要的部位。(2)表征微管蛋白- 苯并咪唑相互作用的突变分析。酵母菌 酿酒酵母将被用来在遗传学上定义微管蛋白-苯并咪唑 结合部位。然后将使用部分基因替换来允许 卡氏肺孢子虫微管蛋白在酵母宿主中的突变分析。(三)制定 苯并咪唑结合部位模型及其定点评价 诱变。酿酒酵母P。卡里尼氏微管蛋白 将测试构建物对苯并咪唑敏感性的影响。(4) 评估抗P。苯并咪唑类化合物的体内外卡氏活性。 苯并咪唑-微管蛋白结合位点的模型应该允许我们 合理选择或设计具有增强选择性毒性的衍生物。 这些将在培养和老鼠模型中进行测试。对……的影响 将检查依附、形态和分化，并尝试 将被用来分离抗药性突变体。

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.