Mechanistic insights into dual-active liver and blood-stage antiplasmodials.

The identification of novel antimalarials with activity against both the liver and blood stages of the parasite lifecycle would have the dual benefit of prophylactic and curative potential. However, one challenge of leveraging chemical hits from phenotypic screens is subsequent target identification. Here, we use in vitro evolution of resistance to investigate nine compounds from the Tres Cantos Antimalarial Set (TCAMS) with dual liver and asexual blood stage activity. We succeeded in eliciting resistance to four compounds, yielding mutations in acetyl CoA synthetase (AcAS), cytoplasmic isoleucine tRNA synthetase (cIRS), and protein kinase G (PKG), respectively. Using a combination of CRISPR editing and in vitro activity assays with recombinant proteins, we validate these as targets for TCMDC-125075 (AcAS), TCMDC-124602 (cIRS), TCMDC-141334, and TCDMC-140674 (PKG). Notably, for the latter two compounds, we obtained a T618I mutation in the gatekeeper residue of PKG, consistent with direct interaction with the active site, which we modeled with molecular docking. Finally, we performed cross-resistance evaluation of the remaining five resistance-refractory compounds using the Antimalarial Resistome Barcode sequencing assay (AReBar), which examined a pool of 52 barcoded lines with mutations covering >30 common modes of action. None of the five compounds where in vitro evolution of resistance was not successful yielded validated hits using AReBar, indicating they likely act via novel mechanisms and may be candidates for further exploration.IMPORTANCEDespite recent progress in the development of the next generation of antimalarial drug candidates, the risk of resistance remains. Thus, the identification of new targets that are essential at multiple stages of the malaria parasite lifecycle is an important priority. Validation of targets essential for the liver-stage not only would be relevant for prophylaxis but may also limit the potential generation of resistance due to the lower parasite numbers encountered at this stage. Here, we have leveraged compounds with dual liver- and blood-stage activity, combined with evolution of resistance, to chemically validate targets with multi-stage essentiality. We successfully evolved resistance to four, which we further characterize by genome sequencing, CRISPR editing and biochemical assays. The remaining five resistance-refractory compounds showed no interactions when profiled against a barcoded parasite library representing >30 known modes of action, suggesting these may have novel targets and represent interesting starting points for further exploration.