Dinitroaniline Specificity for Tubulin

描述(申请人提供)：二硝基苯胺化合物抑制不同植物和原生动物中的微管聚合，但不破坏脊椎动物或真菌的微管。这些化合物破坏原生动物寄生虫包括锥虫、利什曼原虫、内阿米巴、恶性疟原虫、微小隐孢子虫和弓形虫的微管。不幸的是，二硝基苯胺较差的水溶性阻碍了其作为抗寄生虫剂的发展。与所有以前描述的破坏微管的药物不同，二硝基苯胺作用于α-微管蛋白，后者结合β-微管蛋白。这项建议中的实验试图确定二硝基苯胺对α-微管蛋白的影响，并鉴定二硝基苯胺的可溶性衍生物。酿酒酵母通常对二硝基苯胺不敏感，但据预测，微管蛋白的表达改变会增强二硝基苯胺的敏感性。敏感和耐药的酵母菌株将被用来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。该筛选将利用敏感和不敏感酿酒酵母菌株之间的生长竞争，以区分对这两个菌株非特异性毒性的化合物和那些专门破坏植物/原生动物样微管蛋白的化合物。二硝基苯胺衍生物将在酵母生长竞争试验中进行评估，以确定具有更高的溶解度、亚微摩尔活性和特定破坏植物/原生动物样微管的化合物。通过酵母筛选鉴定的活性化合物将利用细胞内的弓形虫对寄生虫微管的特定破坏进行测试。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部位、表征二硝基苯胺与微管蛋白二聚体结合的动力学、识别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.