Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents an innovative application of the modified bioluminescence-based rate-of-kill assay to screen a diverse microbial natural product library for anti-plasmodial activity. The approach is well-structured and demonstrates clear utility for early-stage triage. However, several aspects require improvement to strengthen the translational relevance of the findings. First, the study does not address compound purity, drug-likeness beyond basic molecular weight and lipophilicity, or bioavailability considerations. Including these parameters would enhance confidence in the chemical suitability of the leads. Second, the pharmacodynamic evaluation is limited to trophozoite stages; incorporating stage-specific assays, particularly ring-stage and gametocyte activity, would provide a more comprehensive profile. Similarly, testing against additional drug-resistant strains, such as artemisinin-resistant parasites, is strongly recommended to assess clinical potential.

Cytotoxicity assessment using HepG2 cells is appropriate, but the discussion should acknowledge that these assays also reflect metabolite toxicity, which is relevant for predicting off-target liabilities. Media composition differences between parasite and cytotoxicity assays, especially the presence of serum proteins, should be clarified, as protein binding can influence compound activity and selectivity indices. The manuscript specifies the use of Dd2 luc and NF54 luc strains and describes culture conditions but does not provide the original source or accession details for these lines; including this information would improve reproducibility and transparency. 

Statistical rigor could be improved by reporting confidence intervals for all EC₅₀ values and including P-values for key comparisons.

 

Optional recommendations include expanding cytotoxicity profiling to additional mammalian cell lines, integrating in silico target prediction or preliminary mechanistic studies, and providing chemical annotation for all prioritized compounds to enable structure–activity relationship analysis. Finally, benchmarking against a broader panel of reference antimalarials would strengthen pharmacodynamic interpretation.

Overall, the study offers a promising screening framework, but addressing these points will significantly enhance its robustness and impact.

 

Comments on the Quality of English Language

Needs improvement. A lot of rare synonyms were chosen, impacting understanding by non-native English-speaking peers.  And for writing purposes, the lab number can be refered by a,b c d  , defining symbol used at first instance.  

Author Response

Responses to Reviewer 1:

We sincerely thank the reviewer for the time, expertise, and careful consideration devoted to evaluating our manuscript. We are greatly appreciative of the careful evaluation of our manuscript and for the constructive and insightful comments, which have been instrumental in refining the clarity, rigor, and translational framing of our study. 

Below, we provide a point-by-point response to the reviewers' invaluable comments.

Comment 1: Clarification regarding compound purity, drug-likeness descriptors, and bioavailability considerations. 

Response: We appreciate this important observation. The PhytoQuest library comprises purified natural products isolated and chemically characterised by the provider. Although full analytical datasets remain proprietary, the chemical information supplied by Phytoquest has now been clarified in the manuscript (Lines 46-52; Lines 387-401) in the revised manuscript. We have expanded the Discussion to explicitly acknowledge that, in addition to molecular weight and lipophilicity, further drug-likeness parameters (e.g., solubility, permeability, metabolic stability, and broader ADME profiling) will be essential during downstream optimization (Lines 452-458). 

Comment 2: Consideration of additional parasite stages and strains to strengthen translational relevance.

Response: We are grateful for this thoughtful suggestion. We agree that broader stage-specific profiling would strengthen translational interpretation. The present study focused on trophozoite-stage parasites because luciferase expression in the Dd2^luc and NF54^luc reporter lines is optimal at this stage, ensuring robust signal detection within the mBRRoK platform. This rationale has now been clarified in the Methods (Lines 109-115) section. We additionally acknowledge in the revised Discussion that evaluation against additional P. falciparum, including artemisinin-resistant lines, as well as evaluating compound activity across other parasite stages such as ring-stage and gametocytes would provide a more comprehensive pharmacodynamic assessment and have identified this as an important direction for future work (Lines 442-446).

Comment 3: Clarification regarding metabolite-associated toxicity and assay conditions. 

Response: We thank the reviewer for highlighting this critical point. The Discussion has been revised to explicitly state that HepG2 cytotoxicity assays may reflect both parent compound effects and metabolite-associated liabilities. We have also clarified differences in media composition, including serum content, between parasite and mammalian assays, noting that protein binding may influence apparent selectivity indices (Lines 429-438).

Comment 4: Additional detail regarding parasite strains to enhance reproducibility. 

Response: We sincerely appreciate the reviewer's suggestion to provide additional details on parasite strains to enhance reproducibility. In response, we have expanded the Methods section 2.2 (Lines 109-115) to include detailed information on the origin of the Dd2^luc and NF54^luc lines, with appropriate references, to improve methodological transparency and reproducibility.  

Comment 5: Inclusion of confidence intervals and clarification of statistical comparisons.

Response: We thank the reviewer for highlighting the importance of comprehensive statistical reporting to strengthen the robustness of our results. We have now included 95% confidence intervals for all EC₅₀ values and clarified statistical comparisons throughout the Methods, Results, and Supplementary materials sections where applicable.

Optional recommendations (expanded cytotoxicity panels, in silico target prediction, broader benchmarking)

Response: We are grateful for the reviewer's insightful optional recommendations. While these were beyond the scope of this initial triage-focused study, we have addressed them in the Discussion section (Lines 438-441). Specifically, we note that future studies will expand cytotoxicity profiling across multiple cell lines, incorporate in silico target prediction for prioritized hits, and include broader benchmarking against reference compounds. These enhancements will further strengthen translational relevance and guide downstream optimization.

We are grateful for your detailed and constructive feedback, which has significantly strengthened the manuscript. Thank you again for your time and thoughtful recommendations.

Language and Formatting Comments

Response: We sincerely appreciate your careful assessment of the manuscript and thank you for highlighting concerns regarding language clarity and formatting. We fully acknowledge the importance of clear and accessible scientific communication, particularly for an international readership. In response to your comment regarding the use of rare synonyms and wording that may impact understanding for non-native English-speaking readers, we have carefully revised the manuscript to improve clarity, simplify terminology where appropriate, and enhance overall readability. Unnecessarily complex vocabulary has been replaced with more standard scientific phrasing to ensure precision while maintaining accessibility.

Regarding the notation of laboratory compound numbers, we agree that simplified labeling improves readability. Compound identifiers have now been standardised using alphabetical notation (e.g., a, b, c, d) where appropriate, with the symbol definitions clearly provided at first mention in the manuscript. This adjustment has improved consistency and reduced potential confusion.

Reviewer 2 Report

Comments and Suggestions for Authors

The presented work seeks to characterize the antiparasitic properties of several natural compounds in an effort to curb malaria drug resistance. The authors validate their findings using a Modified Bioluminescence Relative Rate of Kill assay. The presented study has the potential for a significant impact.

 

The authors do a spectacular job at summarizing the growing health concern of artemisinin combination therapy resistance, and screen a very impressive library of natural compounds provided by PhytoQuest, which is an appropriate source for obtaining natural compounds. The authors also introduce the modified assay and cite appropriate literature to support its use. The modified assay was tested against trophozoites at 6 and 48 hours. I am curious as to why the authors chose to measure antimalarial activity against trophozoites instead of schizonts, where the most replication takes place.

 

The materials and methods section is well-written and easy to understand. The parasite was revived and grown in appropriate conditions. Appropriate statistics were used for data analysis. I have no concerns regarding the materials and methods.

 

The results are well-presented and organized in an orderly fashion. The data is presented with common antimalarial compounds as an internal control. I have no concerns with the results section.

 

The authors summarize their findings in the discussion section. I was pleased to see them consider the cytotoxic effects of high-performing compounds. The authors adequately address the study's shortcomings and open a discussion for future studies and validation. I have no concerns with the discussion. Overall, I do recommend this work for publication in your journal, with my only question being the reason for choosing the troph stage.

Comments on the Quality of English Language

The English is clean and free from errors.

Author Response

Response to reviewer 2:

We sincerely thank the reviewer for the thorough evaluation of our manuscript and for the generous and encouraging comments. We greatly appreciate the positive assessment of the study design, data presentation, and discussion, as well as the recommendation for publication.

In response to the question regarding the choice of the trophozoite stage for assessing antimalarial activity, this decision was guided by the characteristics of the luciferase-expressing parasite lines used in the study. The Dd2^luc strain, described by Wong et al. (2011), expresses luciferase predominantly at the trophozoite stage under proliferating cell nuclear antigen regulatory sequences and uses blasticidin S deaminase for selection. The genetically distinct NF54^luc line was subsequently developed to express luciferase using the same reporter and regulatory elements, with human dihydrofolate reductase as the selectable marker for WR99210 selection. As luciferase expression is driven during the trophozoite stage in these reporter lines, measuring activity at this stage provides optimal signal detection and assay sensitivity for the mBRRoK platform. To clarify this point, we have now added this explanation to the Materials and Methods section (Lines 109-115) in the revised manuscript.

Thank you again for your time and constructive feedback. We are pleased that you found the work to be of potential impact.