Project Summary With widespread resistance of malaria parasites to older agents, artemisinin-based combination therapies are the mainstay for antimalarial treatment, but efficacy is threatened by resistance to artemisinins and partner drugs. New antimalarial drugs are needed. Spearheaded by the Medicines for Malaria Venture (MMV), a robust pipeline of new lead antimalarial compounds is under development. However, resistance to new agents can be anticipated. In a number of cases drug targets and resistance mechanisms have been identified, but studies have focused on small numbers of P. falciparum laboratory strains. It is critical also to consider sensitivity to lead antimalarials of fresh P. falciparum field isolates, especially isolates from Africa. With these data, genotypic and additional phenotypic analysis can allow identification of mechanisms underlying varied susceptibility, as we have described for multiple compounds to date, informing optimal development of next- generation combination antimalarials. This application seeks continued funding for a project characterizing susceptibilities of malaria parasites isolated in Uganda and Burkina Faso to lead antimalarials under development. We offer state-of-the-art assessment of ex vivo P. falciparum susceptibilities linked to high throughput genotypic characterization to improve our understanding of mechanisms of drug action and resistance. As supported by data generated to date, we hypothesize that African P. falciparum isolates will demonstrate varied sensitivity to lead antimalarial compounds, and that characterization of genotypes and phenotypes of field isolates will identify shared resistance mechanisms and guide selection of optimal combination therapies. These results will be of great value as we develop next-generation combination antimalarials and continue efforts toward discovery of additional novel compounds. Our specific aims will be: (1) to characterize ex vivo susceptibilities to lead antimalarial compounds of P. falciparum field isolates, (2) to characterize genotypes to identify mediators of decreased susceptibility in field isolates to lead antimalarial compounds, and (3) to characterize phenotypes of drug sensitivity outliers to elucidate mechanisms of resistance of lead antimalarial compounds. Our studies will define resistance mechanisms for the most important new compounds under development as antimalarials and inform choices of optimal antimalarial drug combinations and the direction of continued drug discovery efforts.