Defining the complex genetic basis of Plasmodium falciparum resistance to artemisinin and quinine and identifying resistance-refractory therapeutics

PROJECT SUMMARY Malaria morbidity and mortality rates have rebounded in recent years and threaten to further increase, as a predicted consequence of the recent emergence and spread in east Africa of Plasmodium falciparum (Pf) strains resistant to first-line artemisinin (ART) derivatives. ART resistance is likely to increase selective pressure on the partner drugs used in ART-based combination therapy, as evidenced in Uganda where parasites are also displaying decreased susceptibility to the first-line partner drug, lumefantrine. Recent data suggest that the dissemination of ART-resistant strains across sub-Saharan Africa will require mutations in the primary determinant k13 as well as additional determinants that can augment resistance and overcome fitness costs associated with mutant k13. To identify these determinants, we genetically crossed a drug-sensitive African parasite with an ART-resistant parasite from Southeast Asia, where resistance arose a decade earlier. By comparing the genome sequences and ART susceptibility profiles of recombinant progeny, we associated ART resistance with mutant k13 and two additional, physically unlinked loci. In Aim 1, we propose to test the role of the prioritized candidates mrp1 and arps10, by CRISPR/Cas9 editing their mutations into the ART-resistant parent and several African culture-adapted strains. We will then assess their ability to mitigate mutant k13’s fitness costs, thus enabling ART-resistant parasites to successfully compete with drug-sensitive strains and disseminate throughout African parasite populations. We will also assess the contribution of other genes implicated from in vitro or small animal model studies. In Aim 2, we will define the multigenic basis of Pf resistance to quinine, a former first-line drug that represents a viable alternative to intravenous artesunate for the management of severe malaria in regions where ART resistance becomes prevalent. Candidate physically unlinked genes, including dmt1, a putative transporter related to pfcrt, as well as ftsh1 and samc, have been identified from a second genetic cross involving an Asian quinine-resistant parasite and a sensitive African strain. In Aim 3, we will leverage preliminary data to explore Pf resistance to ZY-19489 and MMV688533, two promising antimalarial candidates in clinical trials. We will also assess the resistance risks of the registered drugs lumefantrine and pyronaridine where the knowledge gap is acute, as well as ferroquine and INE963 that are in clinical trials. These experiments, which take advantage of our unique panel of hyper-mutable African and Asian strains, will identify which of these candidate therapeutics are resistance-refractory. Achieving the goals of our project will yield important insights into the multigenic basis of ART and quinine resistance, deliver novel molecular markers to track the dissemination of multidrug-resistant Pf malaria, and inform the development of future therapeutics.