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

TY - JOUR

T1 - Mechanistic Insights into Dual-Active Liver and Blood-Stage Antiplasmodials

AU - Rawat, Mukul

AU - Boonyalai, Top

AU - Smidt, Cindy

AU - Luth, Madeline R.

AU - Chen, Daisy

AU - Plater, Andrew

AU - Post, John

AU - Lin, De

AU - McMillan, Joel

AU - Eadsforth, Thomas

AU - Moliner Cubel, Sonia

AU - Billker, Oliver

AU - Rayner, Julian C.

AU - Gamo, Fransisco-Javier

AU - Baragana, Beatriz

AU - Winzeler, Elizabeth A.

AU - Lee, Marcus C.S.

N1 - Copyright: © 2025 Rawat et al.

PY - 2026/1/14

Y1 - 2026/1/14

N2 - 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.

AB - 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.

KW - malaria

KW - antimalarial agents

KW - drug resistance mechanisms

UR - https://www.scopus.com/pages/publications/105027477294

U2 - 10.1128/mbio.02423-25

DO - 10.1128/mbio.02423-25

M3 - Article

C2 - 41277867

SN - 2150-7511

VL - 17

SP - e0242325

JO - MBio

JF - MBio

IS - 1

M1 - e02423-25

ER -