Defining the resistome in P. falciparum: evolution and mechanism

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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.