Optimization and Characterization of N-Acetamide Indoles as Antimalarials that Target PfATP4

Emerging resistance to frontline antimalarials is resulting in them becoming increasingly ineffective, highlighting the need for new antimalarials. To discover new antimalarials a screen of the Janssen Jumpstarter library against the asexual stage parasite uncovered an N-acetamide indole hit class. The structure-activity relationship of this chemotype was defined by generating optimized frontrunner analogs including WJM664 with potent asexual stage activity and high metabolic stability. To determine the mechanism of action, resistance was selected and whole genome sequencing revealed mutations in PfATP4. PfATP4 was validated as the target of the N-acetamide indole class showing cross-resistance to PfATP4 drug-resistant strains, and a metabolomic signature consistent with the PfATP4 inhibitor KAE609. The indole acetamide class exhibited a fast-to-moderate rate of kill and a vacuolized asexual blood stage phenotype, phenocopying known PfATP4 inhibitors. Moreover, N-acetamide indole analogs increased cytosolic Na+ demonstrating on-target inhibition of PfATP4. N-Acetamide indole derivatives inhibited male and female gamete development and blocked transmission from infected blood to the mosquito. WJM664 exhibited low oral efficacy in an asexual stage P. berghei mouse model which may be attributed to the low potency against PbATP4, and the low aqueous solubility and Caco-2 permeability. Further optimization of these attributes is required for the N-acetamide indole class to be considered for development in a curative or transmission blocking combination therapy.