FUNGAL MICROTUBULES--GENETICS AND DRUG TARGET

广泛用作驱虫剂的苯并咪唑类化合物在体外具有抑制作用 与艾滋病相关的病原体卡氏肺孢子虫的生长。这个 阿苯达唑衍生物对急性髓细胞白血病具有治疗和预防作用 卡氏肺孢子虫感染的小鼠模型，尽管需要高剂量。在……里面 苯并咪唑的体外活性延伸到新生隐球菌和 另外两种与艾滋病相关的真菌--组织胞浆菌。 重要的是，这种活性是杀伤性的，就像新生葡萄球菌所证明的那样。 了解这种活动的分子基础可能会导致 临床上更有活性的衍生物的开发或发现 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被鉴定 来自卡氏假单胞菌、新生革兰氏杆菌、囊状隐孢子虫和其他生物。 分离到一株耐阿苯达唑的新生隐球菌突变体，并对其进行了测序 分析证实，β-微管蛋白是目标。涉及的氨基酸 在苯并咪唑的活性已经开始被鉴定；作用之一 利用酵母菌的定点突变直接检测残留物 酿酒用β-微管蛋白。 这项提议的长期目标是开发艾滋病的新疗法-- 以微管为靶点的相关真菌感染。而当 苯并咪唑代表着一个很有希望的先导，显然更多的 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 以微管为基础的有丝分裂纺锤体。我们的具体目标是：(I) 通过遗传分析定义苯并咪唑与微管的相互作用。 新生链球菌和酿酒酵母/PC(编码杂交链霉菌)突变株 Cerevisiae/P.cariniiβ-微管蛋白)对特定的衍生品产生抗药性 被选中并具有特征。沙门氏菌的定点突变研究 然后将使用酿酒酵母和酿酒酵母/PC来定义该角色 特定的氨基酸；这一信息应该允许对 苯并咪唑-微管蛋白相互作用。(2)体内活性检测 苯并咪唑类药物在卡氏肺孢子虫和包膜螺旋体感染小鼠模型中的作用。 水溶性衍生物的非肠道给药将是 探索，因为肝脏的口服吸收和新陈代谢不良代表 治疗活动的主要障碍。抗药性的发展 在活体内也将进行检查。(3)表征细胞因素和 微管蛋白多肽对酿酒酵母微管聚合的调控。 用携带α-或β-微管蛋白基因的载体转染 致命性的，可能是由于微管-微管平衡的破坏。 这一效应将被用来确定一种最小抑制微管蛋白 多肽。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的孵化而产生的抑制- 在其限制性温度下敏感的β-微管蛋白突变体。这 将检验微管靶向药物模拟这种作用的假设 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.