The Evolution of Malerial Antifiolate Resistance

描述（申请人提供）：自1990年以来，恶性疟原虫疟疾的死亡率有所增加，主要是由于抗药性寄生虫的传播。虽然抗叶酸剂是负担得起的，仍然临床有效，他们的使用已受到损害的耐药性的演变。耐药性是由叶酸合成的两种酶中的少量氨基酸取代引起的：二氢叶酸还原酶（DHFR）和二氢蝶酸合酶（DHPS）。了解这些突变被选择的可能顺序，以及中间体的耐药表型和耐药的生物物理基础，对于延长抗叶酸剂的有效治疗寿命和开发新的更有效的抗叶酸剂将是重要的。我们已经开发并验证了一种方法，用于推断耐药性演变中可能的事件顺序。该方法还揭示了抗性的分子机制。我们构建所有可能的相关突变组合，并测定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性谱系中每个步骤的概率，从而揭示了每个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，大部分概率来自少数进化途径。我们的实验方法非常适合于解剖DHFR中多种抗叶酸突变的进化途径和生物物理基础。因此，我们建议构建临床上重要的恶性疟原虫DHFR突变的所有可能组合。将使用S中的表达系统测定每个突变体组合的抗性表型。酿酒酵母，并确定每种蛋白质的生物化学和生物物理性质。基于这些数据，将开发一个系统级模型，该模型将耐药性增加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一个类似的DHPS系统。这项研究有可能改善该领域的耐药性监测方法，提供改进流行病学建模和药物部署方案所需的基本信息，并为新型抗叶酸药物的合理设计提供基本见解。耐药性疟疾是一个日益严重的问题。今年将有大约150万非洲儿童死于这种疾病。该研究项目将揭示为什么特定基因中的某些突变会导致对一种关键的负担得起的抗疟药物抗叶酸剂的耐药性。这项研究可能会延长目前抗叶酸剂的有效性，并有助于开发新的抗叶酸剂。

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.