Defining the resistome in P. falciparum: evolution and mechanism

摘要 尽管在过去十年中，抗疟疾药物发现流水线已经有所改进，但 由于新药的迅速出现，许多候选新药可能会在临床试验中失败，这一风险仍然很大 抗药性寄生虫。这项研究的长期目标是设计更好的临床前候选药物 疟疾和了解为什么治疗可能会失败。在过去的十年里，我们的调查团队 建立了可靠的方法来发现和表征参与多药耐药的基因和 已经收集了一个庞大的基因和等位基因数据集，这些基因和等位基因与多药耐药有关。 此应用程序的总体目标是扩展和利用这些数据，以确定何时、如何和为什么 出现抗疟药耐药性或持久性。我们的中心假设是临床上出现的 可以使用体外进化分析来预测耐药性。我们还假设阻力参数可以 暴露于现代一线药物和其他选择性药物的当前领域分离株之间存在显著差异 压力，与几十年前分离的参考实验室菌株相比。我们的假设将得到检验 通过追求三个具体目标。在目标1中，我们将使用适应性实验室进化和深度全基因组 测序以获得耐药获得的高分辨率视图。要实现这一点，我们将定义 寄生虫的遗传背景在多大程度上发挥作用，通过比较最近来自非洲、亚洲和 南美临床分离株与40年前用实验室分离株获得的分离株不同。我们还将 测试这些菌株在突变路径、抗药性水平和时间方面是否存在根本差异， 抗药性的最小接种量及抗药性对寄生虫适合度的影响。我们还将回答关键的 抗性倾向更多地是目标的函数还是化学类型的函数的问题，参数 导致抗药性出现的因素，如基因组复制事件的数量和不同 等位基因以及不同的化学化学类型是否与给定的药物靶点相互作用产生不同的结果。在……里面 目标2，我们将试图在一个知之甚少的调解人小组中了解耐药性的机制。这些 研究将结合有条件调节的遗传、蛋白质组、细胞和结构方法来研究 赋予抗药性的基因变化对寄生虫生物学的影响。在目标3中，我们将探讨 恶性疟原虫基因作为抵抗疟原虫压力的一种手段，在调节药物耐受性方面发挥了作用。创新 包括表征地理上不同的现代野外隔离株的抗性演变，而不是 完全依赖于历史上的实验室菌株。新颖性包括评估耐药性风险是否被驱动 通过靶点或化学类型，并定义耐受性在抗疟疾暴露中存活的作用。这 研究意义重大，因为它将改变药物候选的选择方式，而不是广泛 临床和临床前研究，最好是在早期引导阶段。

SUMMARY Although improvements have been made to the antimalarial drug discovery pipeline over the past decade a substantial risk remains that many new drug candidates may fail in clinical trials due to the rapid emergence of drug resistant parasites. The longterm goal of this research is to design better preclinical drug candidates for both malaria and to understand why treatments may fail. Over the past decade, our investigative team has established robust methodologies for discovering and characterizing genes involved in multidrug resistance and has assembled a large dataset of genes and alleles that mediate or are associated with multidrug resistance. The overall objective of this application is to extend and leverage these data to determine when, how and why antimalarial drug resistance or persistence emerges. Our central hypothesis is that the emergence of clinical drug resistance can be predicted using in vitro evolution assays. We also posit that resistance parameters may differ substantially between current field isolates exposed to modern first-line drugs and other selective pressures, as compared with reference laboratory strains isolated decades ago. Our hypotheses will be tested by pursuing three specific aims. In Aim 1, we will use adaptive laboratory evolution and deep whole-genome sequencing to obtain a high-resolution view of drug resistance acquisition. To accomplish this, we will define the extent to which a parasite’s genetic background plays a role by comparing results from recent African, Asian and South American clinical isolates to those obtained with laboratory strains dating back >40 years. We will also test whether these strains differ fundamentally in their mutational paths, levels of and time to resistance, the minimum inoculum of resistance and the impact of resistance on parasite fitness. We will also answer the critical question of whether resistance liabilities are more a function of the target or of the chemotype, parameters that contribute to resistance emergence such as number of genome replication events and the number of different alleles and whether different chemical chemotypes interacting with a given drug target give different results. In Aim 2, we will seek to understand mechanisms of resistance in a panel of poorly understood mediators. These studies will combine conditionally regulated genetic, proteomic, cellular and structural approaches to studying the impact of genetic changes conferring resistance on parasite biology. In Aim 3, we will explore the role that P. falciparum genes play in mediating drug tolerance as a means to survive antiplasmodial pressure. Innovation includes characterizing the evolution of resistance in geographically distinct modern field isolates instead of relying entirely on historical laboratory strains. Novelty includes assessing whether the resistance risk is driven by the target or the chemotype,and defining the role for tolerance in surviving antimalarial exposure. This research is significant because it will alter the way in which drug candidates are selected prior to extensive clinical and preclinical studies, ideally at the early lead stage.