FUNGAL MICROTUBULES--GENETICS AND DRUG TARGET

苯并咪唑类化合物广泛用作驱虫剂， 艾滋病相关病原体卡氏肺孢子虫的生长。的 衍生物阿苯达唑具有治疗和预防活性， 卡氏肺孢子虫感染的小鼠模型，尽管需要高剂量。在 体外苯并咪唑活性延伸至新型隐球菌， 荚膜组织胞浆菌，另外两种与艾滋病相关的真菌。 重要的是，如用C.新人类 了解这种活动的分子基础可能会导致 开发临床上更有活性的衍生物或发现 新型微管靶向剂。假定的苯并咪唑目标， 微管亚基β-微管蛋白，已在基因水平上进行了表征 从卡氏原甲（P. carinii）、卡氏原甲C.新形式，H. capsulatum和其他生物。 对阿苯达唑耐药的C.分离并测序新型变形杆菌突变体 分析证实β-微管蛋白是靶点。 涉及的氨基酸 在苯并咪唑活性已开始被确定;一个的作用 通过酵母菌定点突变直接检测残留物 酿酒酵母β-微管蛋白。 这项提案的长期目标是开发艾滋病的新疗法- 相关的真菌感染。而 苯并咪唑代表了一个有前途的铅，很明显， 需要对苯并咪唑-微管相互作用有基本的了解， 以及调节聚合和解聚的细胞机制 有丝分裂纺锤体的结构 我们的具体目标是：（一） 通过遗传分析确定苯并咪唑与微管的相互作用。 突变体C. neoformans和S. cerevisiae/pc（编码杂合S. 酿酒酵母/卡氏毕赤酵母β-微管蛋白）对特定衍生物的抗性将 被挑选出来并加以鉴定。 定点突变S. cerevisiae和S. cerevisiae/pc将被用来定义 特定的氨基酸;这一信息应允许建模 苯并咪唑-微管蛋白相互作用。 (2)检查体内活性 在卡氏肺孢子虫和H.荚膜感染 水溶性衍生物的肠胃外给药将是优选的。 由于口服吸收和肝脏代谢不良， 治疗活动的主要障碍。抗性发展 还将检查体内。 (3)表征细胞因子， 微管蛋白肽调节S.啤酒。 用携带α-或β-微管蛋白基因的质粒转染， 致死，推测是由于微管蛋白-微管平衡的破坏。 这一效应将被利用，以确定一个最小的抑制微管蛋白 肽。S.酿酒酵母基因将被选择用于克服生长 由于苯并咪唑或由于冷或热孵育的抑制作用， 敏感的β-微管蛋白突变体在其限制性温度下。这 将测试微管靶向药物模拟作用的假设， 有丝分裂之前或之后诱导的细胞因子。

Benzimidazoles, widely used as anthelmintic agents, inhibit in vitro growth of the AIDS-associated pathogen Pneumocystis carinii. The derivative albendazole has therapeutic and prophylactic activity in a mouse model of P. carinii infection, although high doses are required. In vitro benzimidazole activity extends to Cryptococcus neoformans and Histoplasma capsulatum, two additional fungi associated with AIDS. Importantly, the activity is cidal, as demonstrated with C. neoformans. Understanding the molecular basis for this activity may lead to the development of clinically more active derivatives or to the discovery of novel microtubule-targeted agents. The presumed benzimidazole target, the microtubule subunit beta-tubulin, has been characterized at the gene level from P. carinii, C. neoformans, H. capsulatum, and additional organisms. An albendazole-resistant C. neoformans mutant was isolated and sequence analysis confirmed that beta-tubulin is the target. Amino acids involved in benzimidazole activity have begun to be identified; the role of one residue was directly tested by site-directed mutagenesis of Saccharomyces cerevisiae beta-tubulin. The long term goal of this proposal is to develop new treatments for AIDS- associated fungal infections by targeting microtubules. While benzimidazoles represent a promising lead, it is apparent that a more basic understanding is needed of benzimidazole-microtubule interactions, and of cellular mechanisms regulating polymerization and depolymerization of the microtubule-based mitotic spindle. Our Specific Aims are to: (I) Define benzimidazole-microtubule interactions by genetic analysis. Mutants of C. neoformans and S. cerevisiae/pc (encoding hybrid S. cerevisiae/P. Carinii beta-tubulin) resistant to specific derivatives will be selected for and characterized. Site-directed mutagenesis of S. cerevisiae and S. cerevisiae/pc will then be employed to define the role of specific amino acids; this information should permit modelling of benzimidazole-tubulin interaction. (2) Examine in vivo activity of benzimidazoles in mouse models of P. carinii and H. capsulatum infection. Parenteral administration of water-solubilized derivatives will be explored, since poor oral absorption and metabolism by the liver represent the primary obstacles to therapeutic activity. Development of resistance in vivo will also be examined. (3) Characterize cellular factors and tubulin peptides regulating microtubule polymerization in S. cerevisiae. Transfection with plasmids carrying alpha- or beta-tubulin genes is lethal, presumably due to disruption of tubulin-microtubule equilibrium. This effect will be exploited to identify a minimal inhibitory tubulin peptide. S. cerevisiae genes will be selected for that overcome growth inhibition due to benzimidazoles or due to incubation of cold- or heat- sensitive beta-tubulin mutants at their restrictive temperatures. This will test the hypothesis that microtubule-targeted drugs mimic the action of cellular factors induced before or after mitosis.