Dinitroaniline Specificity for Tubulin

二硝基苯胺对微管蛋白的特异性

• 批准号: 6672840
• 负责人: NAOMI S MORRISSETTE
• 金额: $ 15.97万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6672840
• 关键词: Chordata; Saccharomyces cerevisiae; Toxoplasma gondii; analog; aniline; chemical binding; drug detection; flow cytometry; fungal genetics; gene expression; gene mutation; high throughput technology; host organism interaction; method development; microorganism growth; microtubules; nitro compounds; pharmacokinetics; plants; postdoctoral investigator; protein structure function; protozoa; solubility; species difference; tubulin

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.

描述（由申请人提供）：二硝基苯胺化合物抑制多种植物和原生动物中的微管聚合，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的微管，包括锥虫、利什曼原虫、内阿米巴原虫、恶性疟原虫、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。不像所有先前描述的微管破坏药物，它们结合-微管蛋白，二硝基苯胺作用于-微管蛋白。本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。酿酒酵母通常对二硝基苯胺不敏感，但微管蛋白的表达改变预计会赋予二硝基苯胺敏感性。敏感和耐药酵母菌株将用于开发一种廉价的高通量筛选有效和可溶性二硝基苯胺衍生物。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来区分对两种菌株都具有非特异性毒性的化合物和那些特异性破坏植物/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，以确定具有更高溶解度、亚微摩尔活性和对植物/原生动物样微管的特异性破坏的化合物。通过酵母筛选鉴定的活性化合物将在细胞内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、识别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力学行为的影响来表征。