In vivo screen reveals Plasmodium falciparum targets for mosquito-based malaria intervention

Interventions that directly target malaria parasites within the mosquito represent a promising tool to disrupt parasite transmission and reduce disease burden. In order to identify additional inhibitors of mosquito stage P. falciparum development, we conducted an in vivo topical application screen with a diverse library of 81 small molecule compounds with known asexual blood stage antimalarial activity. In this topical system, compounds are solubilized in acetone and 2% DMSO and directly pipetted onto the dorsal thorax of An. gambiae females prior to P. falciparum infection, which is then analyzed at 7 days post infection (dpi) by counting surviving oocysts. This screen identified 22 compounds that significantly reduced parasite infection, including several endochin-like quinolones (ELQs) targeting P. falciparum cytochrome bc1 complex (CytB) at either its oxidation (Qo) or reduction (Qi) site. To assess the propensity for cross resistance between Qo and Qi site inhibitors and the potential transmissibility of resistance, we performed asexual blood stage drug selections in transmissible NF54 parasites with the Qo site inhibitors atovaquone, ELQ-121 (the active form of our most potent drug ELQ-453), and ELQ-400 (the active form of ELQ-458) as well as the Qi site inhibitors ELQ-432 (the active form of ELQ-613) and ELQ-300 (the active form of ELQ-331). Resistant parasites from the selections were subject to whole genome sequencing, identifying nonsynonymous point mutations in CytB (Qo site: M133I, V259L, V284L; Qi site: H12Q). Multiple attempts at NF54 drug selection with ELQ-432 failed to produce any resistant parasites, consistent with previous attempts showing low propensity for in vitro resistance of Qi site. In asexual blood stages, we found that some Qo mutations were highly compound specific, while other mutations such as V259L conferred some level of resistance (~5-20 fold EC50 shift) to all of the tested Qo site inhibitors. Importantly, none of the parasite lines with mutations in their Qo site were resistant to any of the tested Qi site inhibitors, and vice versa. We also performed infection experiments with the M133I, V259L, and V284L mutant parasites and found that all three lines had impaired development in the mosquito, suggesting that such resistance would not be transmissible through the mosquito.