Mechanisms of Dinitroaniline Action, Selectivity and Resistance

微管是所有真核细胞的重要组成部分。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疾病。二硝基苯胺(稻草灵、乙基氟尿灵和 氟乐灵)破坏植物和原生动物的MTS，但对脊椎动物或真菌MTS无效。这些化合物 抑制多种原生动物寄生虫(锥虫、利什曼原虫、内阿米巴、疟原虫)的生长 镰刀菌胺、微小隐孢子虫和弓形虫)。我们之前的研究结合了基因 分析和计算生物学得出结论，二硝基苯胺具有一种新的作用机制：它们结合到 A-微管蛋白和破坏原细丝接触。这些结论引发了我们建议解决的其他问题 在这项研究中。首先，我们提出的二硝基苯胺结合位点是由不受限制的氨基酸定义的。 并不能解释为什么二硝基苯胺只与植物或原生动物的微管蛋白结合。我们假设 限制在植物/原生动物谱系中的其他残基会影响结合位点氨基酸的能力 与二硝基苯胺相互作用。我们将对弓形虫和酿酒酵母进行定向改变 A-微管蛋白基因，以确定微管蛋白敏感性的基础和基于a-微管蛋白的耐药机制。这些数据 将与精细化的计算研究相结合。其次，在以前的工作中，我们分离出了大量的 对α-微管蛋白有点突变的抗二硝基苯胺弓形虫品系。我们假设α-微管蛋白 根据突变的基本作用机制，突变可分为两类。一些突变定位于 微管蛋白的区域，对于MT晶格中的二聚体接触是必不可少的。我们预测这些突变的微管蛋白 继续结合二硝基苯胺，但取代增加了MT晶格内的二聚体亲和力以补偿 对于结合的二硝基苯胺的不稳定作用。其他a-微管蛋白突变定位于我们建议的 我们预测这些突变的α-微管蛋白与二硝基苯胺的亲和力降低。我们将衡量 用分离的野生型和突变型弓形虫a-(3微管蛋白二聚体)与二硝基苯胺结合来评估观察到的 二硝基苯胺的亲和力反映了我们预测的抗性机制。最后，所有对二硝基苯胺都有抗性 到目前为止，我们已鉴定的弓形虫株具有突变的α-微管蛋白，使其对1-5um产生抗性 奥里扎林。对较高的二硝基苯胺浓度(5至50微米)有抵抗力的寄生虫有一个额外的非 微管蛋白突变(S)突变的α-微管蛋白遗传背景。我们假设这些其他抗性等位基因 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MT稳定性的突变 二硝基苯胺浓缩泵等药物外排泵。我们将使用一种“逐步”的选择策略来分离这些非 微管蛋白抗性等位基因。综上所述，我们相信，对二硝基苯胺的进一步研究将阐明 微管蛋白的功能，并定义了专门破坏寄生虫微管蛋白的新方法。

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 falcipamm, 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-(3 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 uM) 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.