Mechanistic Insights into Dual-Active Liver and Blood-Stage Antiplasmodials

Abstract

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), and 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 modelled 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.

Figure 1.. Antiplasmodial activity of selected compounds.

(A) Chemical structures of nine compounds from the Tres Cantos Antimalarial Compound Set (TCAMS) with dual-stage activity against both blood and liver stages of Plasmodium. Liver-stage activity was obtained from (9). (B) In vitro efficacy of the nine compounds against P. falciparum laboratory strains 3D7 and Dd2. (C) Antiplasmodial activity of the compounds across a panel of geographically diverse P. falciparum strains including Dd2 (Laos), GB4 (Ghana), UG654 (Uganda), Cam3.II and Cam/PH0212C (Cambodia). Activity data for all compounds are detailed in Supplementary Tables 1 and 2. In (B) and (C), each dot represents a biological replicate (n = 3); error bars indicate mean ± SD.

Figure 2.. TCMDC-125075 targets P. falciparum acetyl-CoA synthetase (PfAcAS).

(A) Single-step resistance selection against TCMDC-125075 was performed in triplicate using 5 × 10⁷ DNA polymerase-mutator parasites (Dd2-Polδ). All three selections yielded mutations in PfAcAS. (B) Clonal lines derived from two independent flasks (Flask 2 and Flask 3) were tested for resistance to TCMDC-125075. The parental Dd2-Polδ line served as a control. (C) The same clones (Flask2-D6 and Flask3-F7) were also tested against a structurally related PfAcAS inhibitor, MMV019721. (D) CRISPR-edited lines carrying PfAcAS mutations T648M and A597V displayed reduced sensitivity to TCMDC-125075. Dd2 parental line was used as a control. (E) In vitro enzymatic inhibition assays with recombinant PfAcAS, human AcAS (HsAcAS), and mutant PfAcAS proteins (T648M and A597V) demonstrated selective activity of TCMDC-125075 against PfAcAS. (B- E), each dot represents a biological replicate (n = 4); bars indicate mean ± SD and statistical significance determined by Mann-Whitney U tests (*p<0.05, **p<0.01).

Figure 3.. TCMDC-124602 targets P. falciparum cytoplasmic isoleucyl-tRNA synthetase (PfcIRS).

(A) Single-step resistance selection with TCMDC-124602 was performed in triplicate using 5 × 10⁷ Dd2-Polδ mutator parasites. Mutations in PfcIRS were identified in all three selections. (B) Clonal lines from two independent flasks (Flask 2-C3 and Flask 3-G1) were tested for resistance to TCMDC-124602. The parental Dd2-Polδ line was included as a control. (C) Schematic representation of PfcIRS architecture, coloured by domain, indicating the locations of resistance-associated mutations identified during compound selection. (D–E) Clones (Flask2-C3 and Flask3-G1) were further evaluated for cross-resistance to structurally related PfcIRS inhibitors MMV019869 and MMV1091186. (F) CRISPR-edited parasite lines bearing the V500A and E180D mutations in PfcIRS exhibited reduced sensitivity to TCMDC-124602, whereas the L810F mutant showed hypersensitivity. Dd2 parental line served as a control. (G) In vitro enzymatic assays confirmed inhibition of recombinant PfcIRS by TCMDC-124602. In (B,D- G), each dot represents a biological replicate (n = 4); bars indicate mean ± SD and statistical significance determined by Mann-Whitney U tests (*p<0.05).

Figure 4.. TCMDC-141334 and TCMDC-140674 target P. falciparum cGMP-dependent protein kinase (PfPKG).

(A) Single-step resistance selection with TCMDC-141334 was conducted in triplicate using 5 × 10⁷ Dd2-Polδ mutator parasites. Mutations in PfPKG were identified in clones from two flasks, while a mutation in PfTKL3 was observed in one flask. (B) Clonal lines from two independent flasks (Flask 2-G6 and Flask 3-C2) were evaluated for resistance to TCMDC-141334. The Dd2-Polδ parental line served as a control. (C) Resistance selection with TCMDC-140674, also performed in triplicate using 5 × 10⁷ Dd2-Polδ parasites, yielded PfPKG mutations in clones from two flasks. One clone from Flask 2 additionally carried a mutation in PfCDPK3. (D) Clones from Flask 2-D7 and Flask 3-D4 were tested for resistance to TCMDC-140674, with Dd2-Polδ used as a control. (E–F) Cross-resistance profiles of clones selected with TCMDC-140674 and TCMDC-141334 were tested against the reciprocal compounds. (G–H) CRISPR-edited parasite lines carrying the PfPKG T618I mutation, as well as a double mutant line with PfPKG T618I and PfTKL3 I1342N, exhibited reduced sensitivity to both TCMDC-141334 and TCMDC-140674. The Dd2 parental line was included as a reference. In (B,D-H), each dot represents a biological replicate (n = 4); bars indicate mean ± SD and statistical significance determined by Mann-Whitney U tests (*p<0.05, **p<0.01).

Figure 5.. Cross-resistance profiling of non-resistant compounds using the AReBar (Antimalarial Barcode Sequencing) assay.

(A) Growth curves for five compounds that previously failed to generate resistant parasites, alongside DSM265 (positive control with a known target) and a no-drug control. (B) Stacked bar plots showing the relative abundance of different barcoded parasite lines before and after 14 days of AReBar assay treatment with each compound. (C) Log₂ fold change (LFC) values for each barcode population following treatment with the five test compounds and DSM265, indicating potential compound-specific selection. Plots show LFC (y-axis) for individual lines in the pool, indicated by circles (Dd2 background) or triangles (3D7 background); dotted lines indicate 2.5 LFC. (D) Bar graph showing the drug response of MDR2 and ACS10 mutant cell lines (two candidate targets identified from AReBar analysis) following treatment with TCMDC-134122.