Review 1: "A Fast-Acting Inhibitor of Blood-Stage P. falciparum with Mechanism Distinct from Artemisinin and Chloroquine"

RR\ID Evidence Scale rating by reviewer:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review: The emergence of resistance to artemisinin has spurred an urgent need for the development of alternative fast-acting antimalarial therapies. In this context, the preprint by Kabeche, Meister and Yeh titled ‘A fast-acting inhibitor of blood-stage Plasmodium falciparum with mechanism distinct from artemisinin and chloroquine’, highlights a promising discovery. The study focuses on MMV1580853, one of the open-access Pandemic Response Box compounds released by Medicines for Malaria Venture. The compound demonstrates potent, fast-acting in vitro antiplasmodial activity against P. falciparum blood stages, suggesting a low probability of resistance development, which operates through a mechanism of action likely different from those of artemisinin and chloroquine.

Strengths of the study:

• The authors provide a wide array of experimental settings to demonstrate the fast-acting activity of MMV1580853, including in all cases different known antimalarial compounds as controls. Moreover, the selected approaches are nicely conducted to draw the main conclusions of the manuscript, based on previous findings available in the cited literature. For instance, the inclusion of conditional knockdown P. falciparum strains of the 3 prenyltransferases annotated in the genome of the parasite, to try to unveil the mode of action behind MMV1580853.

• The Materials and Methods section is sufficiently detailed, with some minor exception (see below).

Limitations of the study:

Major:

• In addition to Giemsa staining, it would be advisable to observe the ultrastructural changes induced by MMV1580853 treatment compared to the effects of chloroquine and artemisinin, for instance using transmission electron microscopy. This approach could help identify differences that may not be visible using light microscopy. Testing the compound in chloroquine-resistant lines would also contribute to strengthen the suggestion that the sought-after mode of action is different from that of quinoline antimalarials, a statement that so far is mainly substantiated by a non-quantitative microscopic study and a Western blot showing hemoglobin levels.

• Although the authors did not succeed in the identification of the molecular target of MMV1580853 through the selection of resistant parasites, other approaches are available to achieve this goal. These alternative strategies might include expanding drug exposure beyond 60 days or using a P. falciparum strain with an intrinsically high mutation rate, to avoid the risk of artifacts resulting from potential toxic effects of EMS used at high concentrations like 1.5 mM. Furthermore, as the authors mention at the end of the manuscript, it would be highly valuable to explore other fast-acting antimalarial phenotypes or, even better, to use approaches based on barcoded parasite lines with known mutations that confer resistance to a variety of antimalarial drugs. Including at least a literature review of these techniques in the discussion section would significantly enhance the value of the manuscript.

Minor:

• The data illustrating the generation of conditional knockdown strains is missing despite being cited in the text as present in Figure S1. The current Figure S1 does not include this information.