Dinitroaniline Specificity for Tubulin

描述（由申请人提供）：二硝基苯胺类化合物抑制多种植物和原生动物中的微管蛋白聚合，但不破坏脊椎动物或真菌微管。这些化合物破坏原生动物寄生虫中的微管，利什曼原虫属，内阿米巴属，恶性疟原虫、微小隐孢子虫和刚地弓形虫。不幸的是，二硝基苯胺的水溶性差，阻碍了它们作为抗寄生虫剂的发展。不同于所有先前表征的微管破坏药物，其结合β-微管蛋白，二硝基苯胺作用于α-微管蛋白。本提案中的实验旨在确定二硝基苯胺对α-微管蛋白的影响，并鉴定二硝基苯胺的可溶性衍生物。酵母S.酿酒酵母通常对二硝基苯胺不敏感，但预测改变的微管蛋白的表达赋予二硝基苯胺敏感性。敏感和耐药的酵母菌株将用于开发一种廉价的高通量筛选有效和可溶的二硝基苯胺衍生物。筛选将利用敏感和不敏感S之间的生长竞争。酿酒酵母菌株以区分对这两种菌株非特异性有毒的化合物和特异性破坏植物/原生动物样微管蛋白的化合物。将在酵母生长竞争试验中评价二硝基苯胺衍生物，以鉴定具有增加的溶解度、亚微摩尔活性和植物/原生动物样微管特异性破坏的化合物。通过酵母筛选鉴定的活性化合物将使用细胞内弓形虫检测寄生虫微管的特异性破坏。二硝基苯胺与α-微管蛋白的相互作用将通过绘制微管蛋白上的二硝基苯胺结合位点、表征二硝基苯胺与微管蛋白二聚体结合的动力学、鉴定与二硝基苯胺结合相关的构象变化以及描述二硝基苯胺对微管动态行为的影响来表征。

DESCRIPTION (provided by applicant): Dinitroanilines compounds inhibit tubulin polymerization in diverse plants and in protozoa but do not disrupt vertebrate or fungal microtubules. These compounds disrupt microtubules in protozoan parasites including Trypanosoma spp., Leishmania spp., Entamoeba spp., Plasmodium falciparum, Cryptosporidium parvum and Toxoplasma gondii. Unfortunately, the poor water solubility of dinitroanilines prevents their development as anti-parasitic agents. Unlike all previously characterized microtubule-disrupting drugs, which bind beta-tubulin, the dinitroanilines act on alpha-tubulin. The experiments in this proposal seek to define the effect of dinitroanilines on alpha-tubulin and to identify soluble derivatives of the dinitroanilines. The yeast S. cerevisiae is normally insensitive to dinitroanilines but expression of altered tubulin is predicted to confer dinitroaniline sensitivity. Sensitive and resistant yeast strains will be used to develop an inexpensive high throughput screen for effective and soluble dinitroaniline derivatives. The screen will exploit growth competition between sensitive and insensitive S. cerevisiae strains to discriminate between compounds that are non-specifically toxic to both strains and those that specifically disrupt plant/protozoan-like tubulin. Dinitroaniline derivatives will be evaluated in the yeast growth competition assay to identify compounds with increased solubility, sub-micromolar activity and specific disruption of plant/protozoan-like microtubules. Active compounds identified by the yeast screen will be tested for specific disruption of parasite microtubules using intracellular Toxoplasma gondii. Dinitroaniline interaction with alpha-tubulin will be characterized by mapping the dinitroaniline binding site on tubulin, characterizing the kinetics of dinitroaniline binding to the tubulin dimer, identifying conformation changes associated with dinitroaniline binding and describing dinitroaniline effects upon the dynamic behavior of microtubules.