Mechanisms of Dinitroaniline Action, Selectivity and Resistance

二硝基苯胺的作用、选择性和耐药性机制

• 批准号: 7027921
• 负责人: NAOMI S MORRISSETTE
• 金额: $ 34.79万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7027921
• 关键词: Saccharomyces cerevisiae; Toxoplasma gondii; alleles; aniline; bacterial genetics; binding sites; biochemistry; chemical kinetics; computational biology; dimer; drug resistance; gene expression; gene mutation; intermolecular interaction; microtubules; pharmacogenetics; pharmacokinetics; protein structure function; tubulin

DESCRIPTION (provided by applicant): Microtubules (MTs) are essential components of all eukaryotic cells. Compounds that affect MT function are used to treat medical conditions including cancer, gout and helminth infection. Dinitroanilines (oryzalin, ethafluralin and trifluralin) disrupt MTs in plants and protozoa but are ineffective against vertebrate or fungal MTs. These compounds inhibit growth of diverse protozoan parasites (Trypanosoma spp., Leishmania spp., Entamoeba spp., Plasmodium falciparum, Cryptosporidium parvum and Toxoplasma gondii). Our previous studies used a combination of genetic analysis and computational biology to conclude that the dinitroanilines have a novel mechanism of action: they bind to a-tubulin and disrupt protofilament contacts. These conclusions evoke additional questions that we propose to address in this research. To begin with, our proposed dinitroaniline binding site is defined by amino acids that are not restricted to sensitive organisms and cannot explain why dinitroanilines only bind to plant or protozoan tubulin. We hypothesize that other residues that are restricted to plant/protozoan lineages influence the capacity of the binding site amino acids to interact with dinitroanilines. We will use directed changes to the Toxoplasma gondii and Saccharomyces cerevisiae a-tubulin genes to identify the basis of tubulin susceptibility and mechanisms of a-tubulin-based resistance. These data will be integrated with refined computational studies. Secondly, in previous work, we isolated a large number of dinitroaniline-resistant Toxoplasma lines which harbor point mutations to a-tubulin. We hypothesize that the a-tubulin mutations fall into two categories based on their underlying mechanism of action. Some of the mutations localize to regions of tubulin that are essential for dimer contacts within the MT lattice. We predict that these mutant tubulins continue to bind dinitroanilines but that the substitutions increase the dimer affinity within the MT lattice to compensate for the destabilizing effect of bound dinitroaniline. Other a-tubulin mutations localize to the region of our proposed binding site and we predict that these mutant a-tubulins have decreased affinity for dinitroanilines. We will measure dinitroaniline binding by isolated wild type and mutant Toxoplasma a-¿ tubulin dimers to assess whether the observed dinitroaniline affinities reflect our predicted resistance mechanisms. Lastly, all of the dinitroaniline resistant Toxoplasma lines that we have characterized to date have mutations to a-tubulin that confer resistance to 1-5 uM oryzalin. Parasites that have resistance to higher dinitroaniline concentrations (5 to >50 ¿M) harbor an additional non- tubulin mutation(s) in the mutant a-tubulin genetic background. We hypothesize that these other resistance alleles will have diverse activities including mutations to regulatory proteins that increase MT stability and mutations that decrease dinitroaniline concentration such as drug efflux pumps. We will use a "step-up" selection strategy to isolate these non- tubulin resistance alleles. In summary, we believe that further study of the dinitroanilines will elucidate new aspects of tubulin function and define novel ways to specifically disrupt parasite tubulins.

描述（由申请人提供）：微管（MT）是所有真核细胞的基本组成部分。影响MT功能的化合物用于治疗包括癌症、痛风和蠕虫感染在内的医学病症。二硝基苯胺类（安磺灵、乙氟乐灵和氟乐灵）破坏植物和原生动物中的MT，但对脊椎动物或真菌MT无效。这些化合物抑制多种原生动物寄生虫（锥虫属，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）。我们之前的研究结合遗传分析和计算生物学得出结论，二硝基苯胺具有一种新型的作用机制：它们与α-微管蛋白结合并破坏原丝接触。这些结论引发了我们在本研究中提出的其他问题。开始，我们提出的二硝基苯胺结合位点是由不限于敏感生物体的氨基酸定义的，并且不能解释为什么二硝基苯胺只与植物或原生动物微管蛋白结合。我们假设，其他残基，仅限于植物/原生动物谱系的影响的结合位点氨基酸与二硝基苯胺相互作用的能力。我们将使用弓形虫和酿酒酵母α-微管蛋白基因的定向改变来鉴定微管蛋白易感性的基础和基于α-微管蛋白的抗性机制。这些数据将与精确的计算研究相结合。其次，在前期的工作中，我们分离到了大量的抗二硝基苯胺的弓形虫株，这些株含有α-微管蛋白的点突变。我们假设α-微管蛋白突变根据其潜在的作用机制分为两类。一些突变定位于微管蛋白的区域，这些区域对于MT晶格内的二聚体接触是必需的。我们预测，这些突变的微管蛋白继续结合二硝基苯胺，但取代增加MT晶格内的二聚体亲和力，以补偿结合二硝基苯胺的不稳定作用。其他α-微管蛋白突变定位于我们提出的结合位点的区域，并且我们预测这些突变的α-微管蛋白对二硝基苯胺的亲和力降低。我们将通过分离的野生型和突变型弓形虫a-1微管蛋白二聚体测量二硝基苯胺结合，以评估观察到的二硝基苯胺亲和力是否反映了我们预测的耐药机制。最后，迄今为止我们已经表征的所有二硝基苯胺抗性弓形虫系都具有α-微管蛋白突变，其赋予对1-5 μ M安磺灵的抗性。对较高二硝基苯胺浓度（5至>50 μ M）具有抗性的寄生虫在突变体α-微管蛋白遗传背景中具有额外的非微管蛋白突变。我们假设这些其他耐药等位基因将具有不同的活性，包括增加MT稳定性的调节蛋白突变和降低二硝基苯胺浓度的突变，如药物外排泵。我们将使用“逐步”选择策略来分离这些非微管蛋白抗性等位基因。总之，我们相信对二硝基苯胺的进一步研究将阐明微管蛋白功能的新方面，并确定特异性破坏寄生虫微管蛋白的新方法。