The Evolution of Malerial Antifiolate Resistance

描述（由申请人提供）：自1990年以来，主要由于耐药寄生虫的传播，恶性疟原虫疟疾的死亡率有所增加。虽然抗叶酸药价格低廉，临床上仍然有效，但由于耐药性的演变，它们的使用受到了影响。抗性来自于两种叶酸合成酶的少量氨基酸替换：二氢叶酸还原酶（DHFR）和二氢叶酸合成酶（DHPS）。了解这些突变被选择的可能顺序，以及中间体的抗性表型和抗性的生物物理基础，对于延长抗叶酸药物的有效治疗寿命和开发新的更有效的抗叶酸药物非常重要。我们已经开发并验证了一种方法来推断在耐药性进化过程中可能发生的事件顺序。该方法还揭示了耐药的分子机制。我们构建了所有可能的相关突变组合，并分析了每个等位基因对生物体耐药表型的影响及其生化和生物物理特性。这些数据揭示了所有可能的耐药谱系中每一步的概率，从而揭示了每条进化途径的总体概率。我们之前在另一个实验系统中的工作表明，大多数可能性来自少数进化途径。我们的实验方法非常适合于剖析DHFR中多重抗叶酸突变的进化途径和生物物理基础。因此，我们建议构建临床重要的恶性疟原虫DHFR突变的所有可能组合。每个突变组合的抗性表型将使用酿酒酵母的表达系统进行分析，并确定每个蛋白质的生化和生物物理特性。基于这些数据，将开发一个系统级模型，将增加的耐药性与进化中间体的生化和生物物理特性联系起来。在未来的工作中，我们还将采取措施开发类似的DHPS系统。本研究有可能改进该领域的耐药性监测方法，为改进流行病学建模和药物部署方案提供所需的基础信息，并为合理设计新型抗叶酸药物提供基础见解。耐药疟疾是一个日益严重的问题。今年大约有150万非洲儿童将死于这种疾病。该研究项目将揭示为什么特定基因的某些突变有助于对一种关键的可负担得起的抗疟疾药物抗叶酸药物产生耐药性。这项研究可以延长现有抗叶酸药物的有效性，并有助于开发新的抗叶酸药物。

DESCRIPTION (provided by applicant): Since 1990 mortality from Plasmodium falciparum malaria has increased, due primarily to the spread of drug-resistant parasites. Although antifolates are affordable and still clinically effective, their use has been compromised by the evolution of drug resistance. Resistance results from a small number of amino acid replacements in two enzymes for folate synthesis: dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS). An understanding of the likely order in which these mutations were selected, as well as the resistance phenotypes of the intermediates and the biophysical basis of the resistance, would be important in prolonging the useful therapetic lifetime of antifolates and in developing novel and more effective antifolates. We have developed and verified a method for inferring the likely sequence of events in the evolution of drug resistance. The method also reveals the molecular mechanisms of resistance. We construct all possible combinations of relevant mutations, and assay each allele for its effect on the drug resistance phenotype of the organism and for its biochemical and biophysical properties. These data reveal the probability of each individual step in all possible genealogies of drug resistance, and hence the overall probability of each evolutionary pathway. Our previous work in another experimental system indicates that most of the probability comes from a small number of evolutionary pathways. Our experimental approach is ideally suited for dissecting the evolutionary pathways and biophysical basis' of the multiple antifolate mutations in DHFR. We therefore propose to construct all possible combinations of clinically important P. falciparum DHFR mutations. The resistance phenotype of each mutant combination will be assayed using the expression system in S. cerevisiae, and the biochemical and biophysical properties of each protein determined. Based on these data, a systems-level model will be developed that relates increased drug resistance to the biochemical and biophysical properties of the evolutionary intermediates. For future work, we will also take steps to develop an analogous DHPS system. This research has the potential to improve methods of resistance surveillance in the field, to provide basic information required for improved epidemiological modeling and drug deployment protocols, and to contribute fundamental insights1- into the rational design of novel antifolate drugs. LAY LANGUAGE SUMMARY: Drug-resistant malaria is an ever increasing problem. This year about 1.5 million African children will die from the disease. This research project will uncover why certain mutations in a particular gene contribute to resistance to a key affordable class of antimalaria drugs, the antifolates. The research may prolong the effectiveness of present antifolates, and help in the development of new ones.