Chemogenetic characterization through in vitro evolution combined with whole genome analysis is a powerful tool to discover novel antimalarial drug targets and identify drug resistance genes. Our comprehensive genome analysis of 262 Plasmodium falciparum parasites treated with 37 diverse compounds reveals how the parasite evolves to evade the action of small molecule growth inhibitors. This detailed data set revealed 159 gene amplifications and 148 nonsynonymous changes in 83 genes which developed during resistance acquisition. Using a new algorithm, we show that gene amplifications contribute to 1/3 of drug resistance acquisition events. In addition to confirming known multidrug resistance mechanisms, we discovered novel multidrug resistance genes. Furthermore, we identified promising new drug target-inhibitor pairs to advance the malaria elimination campaign, including: thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This deep exploration of the P. falciparum resistome and drug-able genome will guide future drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms of the deadliest malaria parasite.One Sentence Summary: Whole genome sequencing reveals how Plasmodium falciparum evolves resistance to diverse compounds and identifies new antimalarial drug targets.peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/139386 doi: bioRxiv preprint first posted online 3
Main Text:Malaria has a disproportionately negative impact on human health because its causal protozoan parasites are adept at changing their genomes to evade antimalarial drugs and the human immune system. A human infection may contain upwards of to 10 12 asexual blood stage parasites.Thus, even with a relatively slow random parasite mutation rate (~10 -9 ), within a few cycles of replication each base in the P. falciparum genome can acquire a random genetic change that may render at least one parasite resistant to the activity of a small molecule or a human-encoded antibody. The recent evolution of artemisinin-resistant parasites in Southeast Asia now threatens a life-saving treatment in addition to malaria control efforts(1).Although this rapid evolution impedes our ability to control the disease, it can also be used to our advantage since in vitro evolution in the presence of known antimalarials followed by whole-genome sequencing of resistant clones can be used to discover important mediators of drug resistance. For example, the kelch13 allele that is associated with artemisinin resistance was first identified in the genome sequence of an in vitro cultured P. falciparum parasite line exposed to sublethal concentrations of dihydroartemisinin in the laboratory(2). This method of resistance evolution can also reveal new antimalarial drug targets(3). Unlike drug targets that are val...