Efficacy and mechanism of action of cipargamin as an antibabesial drug candidate

Reviewer #2 (Public review):

Summary:

In this manuscript, authors have tried to repurpose cipargamin (CIP), a known drug against Plasmodium and Toxoplasma against Babesia. They proved the efficacy of CIP on Babesia in nanomolar range. In silico analyses revealed the drug resistance mechanism through a single amino acid mutation at amino acid position 921 on the ATP4 gene of Babesia. Overall, the conclusions drawn by the authors are well justified by their data. I believe this study opens up a novel therapeutic strategy against babesiosis.

Strengths:

Authors have carried out a comprehensive study. All the experiments performed were carried out methodically and logically.

Reviewer #3 (Public review):

Summary:

The authors aim to establish that cipargamin can be used for the treatment of infection caused by Babesia organisms.

Strengths:

The study provides strong evidence that cipargamin is effective against various Babesia species. In vitro growth assays were used to establish that cipargamin is effective against Babesia bovis and Babesia gibsoni. Infection of mice with Babesia microti demonstrated that cipargamin is as effective as the combination of atovaquone plus azithromycin. Cipargamin protected mice from lethal infection with Babesia rodhaini. Mutations that confer resistance to cipargamin were identified in the gene encoding ATP4, a P-type Na ATPase that is found in other apicomplexan parasites, thereby validating ATP4 as the target of cipargamin. A 7-day treatment of cipagarmin, when combined with a single dose of tafenoquine, was sufficient to eradicate Babesia microti in a mouse model of severe babesiosis caused by lack of adaptive immunity.

Weaknesses:

Cipargamin was tested in vivo at a single dose administered daily for 7 days. Despite the prospect of using cipargamin for the treatment of human babesiosis, there was no attempt to identify the lowest dose of cipagarmin that protects mice from Babesia microti infection. In the SCID mouse model, cipargamin was tested in combination with tafenoquine but not with atovaquone and/or azithromycin, although the latter combination is often used as first-line therapy for human babesiosis caused by Babesia microti.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors address an important issue in Babesia research by repurposing cipargamin (CIP) as a potential therapeutic against selective Babesia spp. In this study, CIP demonstrated potent in vitro inhibition of B. bovis and B. gibsoni with IC₅₀ values of 20.2 ± 1.4 nM and 69.4 ± 2.2 nM, respectively, and the in vivo efficacy against Babesia spp. using mouse model. The authors identified two key resistance mutations in the BgATP4 gene (BgATP4^L921I and BgATP4^L921V) and explored their implications through phenotypic characterization of the parasite using cell biological experiments, complemented by in silico analysis. Overall, the findings are promising and could significantly advance Babesia treatment strategies.

Strengths:

In this manuscript, the authors effectively repurpose cipargamin (CIP) as a potential treatment for Babesia spp. They provide compelling in vitro and in vivo data showing strong efficacy. Key resistance mutations in the BgATP4 gene are identified and analyzed through both phenotypic and in silico methods, offering valuable insights for advancing treatment strategies.

Thank you for your insightful comments and for taking the time to review our manuscript.

Weaknesses:

The manuscript explores important aspects of drug repurposing and rational drug design using cipargamin (CIP) against Babesia. However, several weaknesses should be addressed. The study lacks novelty as similar research on cipargamin has been conducted, and the experimental design could be improved. The rationale for choosing CIP over other ATP4-targeting compounds is not well-explained. Validation of mutations relies heavily on in silico predictions without sufficient experimental support. The Ion Transport Assay has limitations and would benefit from additional assays like Radiolabeled Ion Flux and Electrophysiological Assays. Also, the study lacks appropriate control drugs and detailed functional characterization. Further clarity on mutation percentages, additional safety testing, and exploration of cross-resistance would strengthen the findings.

We appreciate your feedback and for giving us the chance to improve our paper. We have specified how we revised the below comments one by one. I hope these address your concerns.

Comment 1: It is commendable to explore drug repurposing, drug deprescribing, drug repositioning, and rational drug design, especially using established ATP4 inhibitors that are well-studied in Plasmodium and other protozoan parasites. While the study provides some interesting findings, it appears to lack novelty, as similar investigations of cipargamin on other protozoan parasites have been conducted. The study does not introduce new concepts, and the experimental design could benefit from refinement to strengthen the results. Additionally, the rationale for choosing CIP over other MMV compounds targeting ATP4 is not clearly articulated. Clarifying the specific advantages CIP may offer against Babesia would be beneficial. Finally, the validation of the identified mutations might be strengthened by additional experimental support, as reliance on in silico predictions alone may not fully address the functional impact, particularly given the potential ambiguity of the mutations (BgATP4 L to V and I).

Thank you for your thoughtful feedback. We have addressed the concerns as follows: (1) Introduction of new concepts and experimental design: While our study primarily builds on existing frameworks, it provides novel insights into the interaction of CIP with Babesia parasites, which we believe contribute to the field. Regarding the experimental design, we acknowledge its limitations and have revised the manuscript to include additional experiments to strengthen the robustness of our findings. Specifically, we have added experiments on the detection of BgATP4-associated ATPase activity (Figure 3H), the evaluation of cross-resistance to antibabesial agents (Figures 5A and 5B), and the efficacy of CIP plus TQ combination in eliminating B. microti infection with no recrudescence in SCID mice (Figure 5C).

(2) Rationale for choosing CIP over other MMV compounds targeting ATP4: We appreciate this point and have expanded the introduction section to articulate our rationale for selecting CIP (Lines 94-97). Specifically, CIP was chosen due to its previously demonstrated efficacy against Plasmodium and other protozoan parasites.

(3) Validation of identified mutations: We agree that additional experimental data would strengthen the validation of the identified mutations. In response, we have indicated the ratio of wild-type to mutant parasites by Illumina NovaSeq6000 to validate the impact of the BgATP4 C-to-G and A mutations (Figure 2D).

Comment 2: Conducting an Ion Transport Assay is useful but has limitations. Non-specific binding or transport by other cellular components can lead to inaccurate results, causing false positives or negatives and making data interpretation difficult. Indirect measurements, like changes in fluorescence or electrical potential, can introduce artifacts. To improve accuracy, consider additional assays such as

a. Radiolabeled Ion Flux Assay: tracks the movement of Na⁺ using radiolabeled ions, providing direct evidence of ion transport.

b. Electrophysiological Assay: measures ionic currents in real-time with patch-clamp techniques, offering detailed information about ATP4 activity.

Thank you for highlighting the limitations of the ion transport assay and suggesting alternative approaches to improve accuracy. However, they require specialized equipment and expertise not currently available in our laboratory. We have acknowledged these limitations and included these alternative methods as part of the study's future directions. Thank you for your suggestions which will undoubtedly enhance the rigor and depth of our research.

Comment 3: In-silico predictions can provide plausible outcomes, but it is essential to evaluate how the recombinant purified protein and ligand interact and function at physiological levels. This aspect is currently missing and should be included. For example, incorporating immunoprecipitation and ATPase activity assays with both wild-type and mutant proteins, as well as detailed kinetic studies with Cipargamin, would be recommended to validate the findings of the study.

Thank you for your insightful suggestions regarding the validation of in-silico predictions. We recognize the importance of evaluating the interaction and function of recombinant purified proteins and ligands at physiological levels to strengthen the study's findings. (1) Incorporating experimental validation:

a. Immunoprecipitation assays: We agree that immunoprecipitation could provide valuable evidence of protein-ligand interactions. While this was not included in the current study due to limitations in sample availability, we plan to incorporate this assay in follow-up experiments.

b. ATPase activity assays: Assessing ATPase activity in both wild-type and mutant proteins is a crucial step in validating the functional impact of the identified mutations. We included the results in the revised manuscript (Figure 3H).

(2) Detailed kinetic studies with cipargamin: We appreciate the recommendation to conduct detailed kinetic analyses. These studies would provide deeper insights into the binding affinity and inhibition dynamics of cipargamin. We have included the results of these experiments in the current study (Figure 3I).

Comment 4: The study lacks specific suitable control drugs tested both in vitro and in vivo. For accurate drug assessment, especially when evaluating drugs based on a specific phenotype, such as enlarged parasites, it is important to use ATP4 gene-specific inhibitors. Including similar classes of drugs, such as Aminopyrazoles, Dihydroisoquinolines, Pyrazoleamides, Pantothenamides, Imidazolopiperazines (e.g., GNF179), and Bicyclic Azetidine Compounds, would provide more comprehensive validation.

Thank you for emphasizing the importance of including suitable control drugs. We acknowledge the absence of specific control drugs in the previous version of the manuscript. To date, no drug targeting ATP4 proteins in Babesia has been definitively identified. The suggested drugs could potentially disrupt the parasite's ability to regulate sodium levels by inhibiting PfATP4, a protein essential for its survival. This highlights PfATP4 as an attractive target for antimalarial drug development. However, further studies are required to evaluate whether these drugs exhibit similar activity against ATP4 homologs in Babesia.

Comment 5: Functional characterization of CIP through microscopic examination and quantification for assessing parasite size enlargement is not entirely reliable. A Flow Cytometry-Based Assay is recommended instead 9 along with suitable control antiparasitic drugs). To effectively monitor Cipargamin's action, conducting time-course experiments with 6-hour intervals is advisable rather than relying solely on endpoint measurements. Additionally, for accurate assessment of parasite morphology, obtaining representative qualitative images using Scanning Electron Microscopy (SEM) or Transmission Electron Microscopy (TEM) for treated versus untreated samples is recommended for precise measurements.

Thank you for your constructive feedback regarding the methods for functional characterization of CIP and the evaluation of parasite morphology.

(1) Flow Cytometry-Based Assay: We agree that a flow cytometry-based assay would enhance the accuracy of detecting changes in parasite size and morphology. We will implement this method in future studies as our laboratory currently does not have the capability to conduct such experiments.

(2) Microscopy for Morphology Assessment: We acknowledge the importance of obtaining high-resolution, representative images of treated and untreated samples. Utilizing Scanning Electron Microscopy (SEM) or Transmission Electron Microscopy (TEM) for qualitative analysis will significantly improve the precision of our morphological assessments. However, both methods have limitations.

a. SEM: This technique can only scan the erythrocytes' surface; it cannot scan the parasite itself because it is inside the erythrocytes.

b. TEM: Since the parasite is fixed, observations from various angles may reveal longitudinal or cross-sectional portions, making it impossible to precisely view the parasite's dimensions. As a result, we employed TEM to precisely observe the parasite's internal structure alterations both before and after treatment, as seen in Figure 3C.

Comment 6: A notable contradiction observed is that mutant cells displayed reduced efficacy and affinity but more pronounced phenotypic effects. The BgATP4^L921I mutation shows a 2x lower susceptibility (IC₅₀ of 887.9 ± 61.97 nM) and a predicted binding affinity of -6.26 kcal/mol with CIP. However, the phenotype exhibits significantly lower Na⁺ concentration in BgATP4^L921I (P = 0.0087) (Figure 3E).

The seemingly contradicting observation of reduced CIP binding and efficacy in the BgATP4^L921I mutant with a significant decrease in intracellular Na⁺ concentration may be explained by factors other than the direct CIP interaction. Logically, we consider that CIP binds less effectively to its target in the BgATP4^L921I mutant, but the observed phenotype may be attributed to the functional consequences of the mutation. The BgATP4^L921I mutation probably directly impacts the function of BgATP4's ion transport mechanism, which likely disrupts Na⁺ homeostasis independently. Thus, we hypothesize that the dysregulated Na⁺ homeostasis is driven by the mutation itself rather than the already weakened inhibitory effect of CIP.

Comment 7: The manuscript does not clarify the percentage of mutations, and the number of sequence iterations performed on the ATP4 gene. It is also unclear whether clonal selection was carried out on the resistant population. If mutations are not present in 100% of the resistant parasites, please indicate the ratio of wild-type to mutant parasites and represent this information in the figure, along with the chromatograms.

Thank you for your valuable comments. We appreciate your detailed observations and giving us the opportunity to clarify these points. During the long-term culture process, subculturing was performed every three days. Although clonal selection was not conducted, mutant strains were effectively selected during this process. Using the Illumina NovaSeq6000 sequencing platform, high-throughput next-generation sequencing was performed to detect ratio of wild-type to mutant parasites. Results showed that for BgATP4^L921V, 99.97% of 7,960 reads were G, and for BgATP4^L921I, 99.92% of 7,862 reads were A. To enhance clarity, we have included a new figure (Figure 2D) illustrating the sequencing results. We believe this addition will help provide a clearer understanding for the readers.

Comment 8: While the compound's toxicity data is well-established, it is advisable to include additional testing in epithelial cells and liver-specific cell lines (e.g., HeLa, HCT, HepG2) if feasible for the authors. This would provide a more comprehensive assessment of the compound's safety profile.

Thank you for your thoughtful suggestion. We included toxicity testing in human foreskin fibroblasts (HFF) as supplemental toxicity data to provide a more comprehensive evaluation of the compound's safety profile (Figure supplement 1B).

Comment 9: In the in vivo efficacy study, recrudescent parasites emerged after 8 days of treatment. Did these parasites harbor the same mutation in the ATP4 gene? The authors did not investigate this aspect, which is crucial for understanding the basis of recrudescence.

Thank you for raising this important point. We acknowledge that understanding the genetic basis of recrudescence is critical for elucidating mechanisms of resistance and treatment failure. Although our current study did not include an analysis of the BrATP4 gene in relapse parasites due to limitations in sample availability, we evaluated CIP efficacy in SCID mice and performed sequencing analysis of the BmATP4 gene in recrudescent samples. However, no mutation points were identified (Lines 211-212). We believe that if a relapse occurs after the 7-day treatment, it is unlikely that the parasites would easily acquire mutations.

Comment 10: The authors should explain their choice of BABL/c mice for evaluating CIP efficacy, as these mice clear the infection and may not fully represent the compound's effectiveness. Investigating CIP efficacy in SCID mice would be valuable, as they provide a more reliable model and eliminate the influence of the immune system. The rationale for not using SCID mice should be clarified.

We appreciate the reviewer's suggestion regarding the use of SCID mice to evaluate the efficacy of CIP. In response to your suggestion, we have now included an experiment using SCID mice to evaluate the efficacy of CIP and to eliminate the confounding influence of the immune system. We further investigated the potential of combined administration of CIP plus TQ to eliminate parasites, as we are concerned that the long-term use of CIP as a monotherapy may be limited due to its potential for developing resistance. The results are shown in Figure 5C.

Comment 11: Do the in vitro-resistant parasites show any potential for cross-resistance with commonly used antiparasitic drugs? Have the authors considered this possibility, and what are their expectations regarding cross-resistance?

Thank you for your insightful question regarding the potential for cross-resistance between in vitro-resistant parasites and commonly used antiparasitic drugs. In response to your suggestion, we have now included experiments to assess whether B. gibsoni parasites that are resistant to CIP exhibit any cross-resistance to other commonly used antiparasitic drugs, such as atovaquone (ATO) and tafenoquine (TQ). The IC₅₀ values for both ATO and TQ in the resistant strains showed only slight changes compared to the wild-type strain, with less than a onefold difference (Figure 5A, 5B). This minimal variation suggests that the resistant strain has a mild alteration in susceptibility to ATO and TQ, but not enough to indicate strong resistance or significant cross-resistance. This suggests that CIP could be used in combination with TQ to treat babesiosis.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors have tried to repurpose cipargamin (CIP), a known drug against plasmodium and toxoplasma against babesia. They proved the efficacy of CIP on babesia in the nanomolar range. In silico analyses revealed the drug resistance mechanism through a single amino acid mutation at amino acid position 921 on the ATP4 gene of Babesia. Overall, the conclusions drawn by the authors are well justified by their data. I believe this study opens up a novel therapeutic strategy against babesiosis.

Strengths:

The authors have carried out a comprehensive study. All the experiments performed were carried out methodically and logically.

Thank you for the comments and your time to review our manuscript.

Weaknesses:

The introduction section needs to be more informative. The authors are investigating the binding of CIP to the ATP4 gene, but they did not give any information about the gene or how the ATP4 inhibitors work in general. The resolution of the figures is not good and the font size is too small to read properly. I also have several minor concerns which have been addressed in the "Recommendations for the authors" section.

We thank the reviewer for their valuable comments. In response, we have revised the introduction to include a more detailed explanation of the ATP4 gene, its biological significance, and the mechanism of ATP4 inhibitors to provide a better context of the study (Lines 86-93). Additionally, we have reformatted the figures to enhance resolution and increased the font size to ensure improved readability. We also appreciate the reviewer's careful assessment of the manuscript and have addressed all minor concerns outlined in the "Recommendations for the Authors" section. A detailed, point-by-point response to each concern is provided in the response letter, and the corresponding revisions have been incorporated into the manuscript.

Reviewer #3 (Public review):

Summary:

The authors aim to establish that cipargamin can be used for the treatment of infection caused by Babesia organisms.

Strengths:

The study provides strong evidence that cipargamin is effective against various Babesia species. In vitro, growth assays were used to establish that cipargamin is effective against Babesia bovis and Babesia gibsoni. Infection of mice with Babesia microti demonstrated that cipargamin is as effective as the combination of atovaquone plus azithromycin. Cipargamin protected mice from lethal infection with Babesia rodhaini. Mutations that confer resistance to cipargamin were identified in the gene encoding ATP4, a P-type Na⁺ ATPase that was found in other apicomplexan parasites, thereby validating ATP4 as the target of cipargamin.

We appreciate the reviewer for taking the time to review our manuscript.

Weaknesses:

Cipargamin was tested in vivo at a single dose administered daily for 7 days. Despite the prospect of using cipargamin for the treatment of human babesiosis, there was no attempt to identify the lowest dose of cipagarmin that protects mice from Babesia microti infection. Exposure to cipargamin can induce resistance, indicating that cipargamin should not be used alone but in combination with other drugs. There was no attempt at testing cipargamin in combination with other drugs, particularly atovaquone, in the mouse model of Babesia microti infection. Given the difficulty in treating immunocompromised patients infected with Babesia microti, it would have been informative to test cipargamin in a mouse model of severe immunosuppression (SCID or rag-deficient mice).

We thank the reviewer for raising these important comments. We address each concern as follows:

(1) Identifying the lowest protective dose of CIP:

Although our current study was designed to assess the efficacy of CIP at a single therapeutic dose over a 7-day period, we acknowledge that identifying the lowest effective dose would provide valuable information for optimizing treatment regimens. We plan to address this in future studies by conducting a dose-response experiment to identify the minimal protective dose of CIP.

(2) Testing CIP in combination with other drugs:

In the current study, we have tested the efficacy of tafenoquine (TQ) combined with CIP, as well as CIP or TQ administered individually, in a mouse model of B. microti infection. Our results demonstrated that, compared with monotherapy, the combination of CIP and TQ completely eliminated the parasites within 90 days of observation (Figure 5C).

(3) Testing in an immunocompromised mouse model:

We agree with the reviewer that evaluating CIP in immunocompromised models is critical for understanding its potential in treating immunocompromised patients. To address this, we have conducted experiments using SCID mice infected with B. microti. Our results indicated that the combination therapy of CIP plus TQ was effective in eliminating parasites in the severely immunocompromised model (Figure 5D).

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Comment 1: Table: Include the in-silico binding energies for each mutation and ligand.

We have added binding energies for each mutation and ligand in Table supplement 3.

Comment 2: Did the authors investigate the potential of combination therapies involving CIP?

We have tested the efficacy of TQ combined with CIP in a mouse model of B. microti infection.

Comment 3: Does this mutation affect the transmission of the parasite?

Based on our observations, the growth and generation rates of the mutant strain are comparable to those of the wild-type strain. These findings suggest that the mutation does not significantly affect the spread or transmission of the parasite. We have included this observation in the revised manuscript (Lines 243-244).

Comment 4: 60: Use abbreviations CLN for clindamycin and QUI for quinine.

We have revised them accordingly (Lines 59-60).

Comment 5: 86: The hypothesis is not strong or convincing; it needs to be modified to be more specific and convincing.

We have revised the hypothesis to reflect the rationale behind the study better and to support our claim more strongly (Lines 94-97).

Comment 6: 93: Change to: "In vitro efficacy of CIP against B. bovis and B. gibsoni.".

We have changed the suggested content in the manuscript (Line 104).

Comment 7: 96: Define CC₅₀.

We have added the definition of CC₅₀ (Line 106).

Comment 8: 102: Change to: "...Balb/c mice increased dramatically in the...".

We have changed the word following your recommendation (Line 114).

Comment 9: 108: "...significant decrease at 12 DPI...".

We have revised it according to your suggestion (Line 120).

Comment 10: 110: "This indicates that the administration...".

We have revised it according to your suggestion (Line 122).

Comment 11: Figure 1:

(1) Panels A and B should clearly indicate parasite species within the graph for better self-explanation.

We have indicated parasite species within the graph.

(2) For panels C, D, and E, if mice were eliminated or euthanized in the study, include a symbol in the graph to indicate this.

For panels C and D, no mice were eliminated during the study; therefore, no symbol was added to these graphs. Panel F already provides information about the number of eliminated mice, which corresponds to the data in Panel E.

(3) In panels C, D, and E, use a continuation arrow for drug treatment rather than a straight line, to cover the duration of the treatment.

We have updated the figures to use continuation arrows instead of straight lines to represent the duration of drug treatment.

Comment 12: Figure 2: The color combination for the WT and mutant curves is hard to read; consider using regular, less fluorescent, and more distinguishable colors.

We have adjusted the color scheme to use more distinguishable and less fluorescent colors, ensuring better readability and clarity. The revised figure with the updated color scheme has been included in the updated manuscript, and we hope this resolves the readability concern.

Comment 13: Figure 3:

(1) Panel A: Represent a single infected iRBC rather than a field for better visualization.

We have updated Panel A to display a single infected iRBC instead of a field.

(2) Panels E and F: Change the color patterns, as the current colors, especially the green variants (WT and mutant L921V), are difficult to read.

To improve readability, we have updated the color patterns for these panels by selecting more distinguishable colors with higher contrast (Figure 3F, 3G).

Comment 14: Figure 4: Panels B, C, and D: The text is too small to read; increase the font size or change the resolution.

We have increased the font size and replaced the panels with high-resolution versions (Figure 4B, 4C, 4D).

Reviewer #2 (Recommendations for the authors):

Comment 1: In the last paragraph of the introduction, the authors mentioned determining the activity of CIP in vitro in B. bovis and B. gibsoni while in vivo in B. microti and B. rodhaini. It is not explained why they are testing the in vitro and in vivo effects on different Babesia species. Could you please add some logic there? Also, why did they mention measuring the inhibitory activity of CIP by monitoring the Na⁺ and H⁺ balance? This part needs to be rewritten with more information. The ATP4 gene is not properly introduced in the manuscript.

We thank the reviewer for raising these important points. Below, we address each aspect of the comment in detail:

(1) Rationale for testing different Babesia spp. in vitro and in vivo:

B. bovis and B. gibsoni are well-established Babesia models for in vitro culture systems, allowing evaluation of CIP's inhibitory activity under controlled laboratory conditions. B. microti and B. rodhaini, on the other hand, are commonly used rodent models for the in vivo studies of babesiosis, enabling the assessment of drug efficacy in a mammalian host system. This multi-species approach provides a comprehensive evaluation of CIP's efficacy across Babesia spp. with different biological characteristics.

(2) Measuring CIP's inhibitory activity via Na⁺ and H⁺ balance:

We acknowledge that this section of the introduction requires more context. The revised manuscript now includes additional information explaining that the ATP4 gene, which encodes a Na⁺/H⁺ transporter, is the proposed target of CIP (Lines 86-93). CIP disrupts the ion homeostasis maintained by ATP4, leading to an imbalance in Na⁺ and H⁺ concentrations. Monitoring these ionic changes provides a mechanistic understanding of CIP's mode of action and its impact on parasite viability. This rationale has been expanded in the introduction to clarify its significance.

Comment 2: The figure fonts are too small. The resolution for the images is also poor.

We have increased the font size in all figures to improve readability. Additionally, we have replaced the figures with high-resolution versions to ensure clarity and visual quality.

Comment 3: Figures 1A and 1B: one of the error bars merged to the X-axis legend. Please modify these panels. Which curve was used to determine the IC₅₀ values (although it's mentioned in the methods section, would it be better to have the information in the figure legends as well)?

We thank the reviewer for their comments regarding Figures 1A and 1B.

(1) Error bars overlapping the X-axis legend:

The error bars in the figures were automatically generated using GraphPad Prism9 based on the data and are determined by the values themselves. Unfortunately, this overlap cannot be avoided without altering the data representation.

(2) IC₅₀ curve information:

To clarify the determination of IC₅₀ values, we have already included gray dashed lines in the graphs to indicate where the IC₅₀ values were derived from the curves. This visual representation provides clear information about the IC₅₀ points.

Comment 4: Supplementary Figure 1: what are MDCK cells? What is CC₅₀? Please mention their full forms in the text and figure legends (they should be described here because the methods section comes later). What is meant by a predicted selectivity index? There should be an explanation of why and how they did it. Which curve was used to determine the IC₅₀ values?

We thank the reviewer for pointing out the need to clarify terms and provide additional context in the supplementary figure and text. We have updated the figure legend and text to include the full forms of MDCK (Madin-Darby canine kidney) cells and CC₅₀ (50% cytotoxic concentration), ensuring clarity for readers encountering these terms for the first time. In text, now we have included a brief explanation of the selectivity index as a measure of a drug's safety and specificity (Lines 108-110). The selectivity index is calculated as the ratio between the half maximal inhibitory concentration (IC₅₀) and the 50% cytotoxic concentration (CC₅₀) values (Lines 333-335). We also have already included gray dashed lines in the graphs to indicate where the IC₅₀ values were derived from the curves (Figure supplement 1).

Comment 5: Figures 1C-F: It feels unnecessary to write down n=6 for each panel and each group. Since "n" is equal for all, it would be nice to just mention it in the figure legend only.

We appreciate the reviewer's suggestion regarding the notation of "n=6" in Figures 1C-F. To improve clarity and reduce redundancy, we have removed the "n=6" notation from the individual panels and included it in the figure legend instead.

Comment 6: Figure 2A: was never mentioned in the text.

We have described the sequencing results for the wild-type B. gibsoni ATP4 gene with a reference to Figure 2A in the revised manuscript (Lines 134-135).

Comment 7: Figure 2D: some of the error bars merged to the X-axis legend. Please modify. Again, which curve was used to determine the IC₅₀ values? Can the authors explain why the pH declined after 4 minutes?

We thank the reviewer for this insightful question.

(1) Error bars overlapping the X-axis legend:

The error bars in Figure 2E were automatically generated using GraphPad Prism9 and are determined by the underlying data values. Unfortunately, this overlap cannot be avoided without altering the data representation.

(2) IC₅₀ curve information:

Since Figure 2E contains three separate curves, adding dashed lines to indicate the IC₅₀ for each curve would make the figure overly cluttered and reduce readability. To address this, we have clearly indicated the IC₅₀ values in Figures 1A and 1B and described the methodology for determining IC₅₀ values in the Methods section. We believe this approach provides sufficient clarity without compromising the visual experience of Figure 2E.

(3) The pH decline observed after 4 minutes (Figure 3E) may be attributed to the following factors:

a. Ion transport dynamics:

The initial rise in pH likely reflects the rapid inhibition of Na⁺/H⁺ exchange mediated by CIP, which temporarily alkalinizes the intracellular environment. However, after this initial phase, compensatory mechanisms, such as proton influx or metabolic acid production, may lead to a subsequent decline in pH.

b. Drug kinetics and target interaction:

The decline could also result from the time-dependent effects of CIP on ATP4-mediated ion transport. As the drug action stabilizes, the parasite may partially restore ionic balance, leading to a decrease in intracellular pH.

Comment 8: Supplementary Figure 2: It's difficult to distinguish between red and pink colors, so it would be wise to use two contrasting colors to distinguish between Pf and Tg CIP resistant cites.

We have updated the figure to enhance clarity. Purple squares and arrows now represent sites linked to P. falciparum CIP resistance, replacing the previous red squares. Similarly, gray squares and arrows have replaced the green squares to denote sites associated with T. gondii (Figure supplement 2).

Comment 9: Line 65: Is it possible to add a reference here?

We have added a reference in line 65.

Comment 10: Line 69: Please spell the full form of G6PD as it was never mentioned before.

We have added the full form of G6PD in lines 69-70.

Comment 11: Line 103: mention what DPI is (irrespective of the methods section which comes later).

We have spelled out DPI (days postinfection) in line 115.

Comment 12: Line 120: It's not explained why B. gibsoni ATP4 gene was investigated? There should be more explanation and references to previous work.

We thank the reviewer for pointing out the need to provide more context for investigating the B. gibsoni ATP4 gene. To address this, we have added more information to the introduction, explaining that the ATP4 gene, which encodes a Na⁺/H⁺ transporter, is the proposed target of CIP (Lines 86-93).

Comment 13: Line 203-219: line spacing seems different from the rest of the manuscript.

We have corrected the incorrect format (Lines 262-278).

Reviewer #3 (Recommendations for the authors):

Comment 1: Lines 66-68: The report by Marcos et al. 2022 did not demonstrate that tafenoquine was effective in curing relapsing babesiosis. In the discussion of that article, the authors state that "it is impossible to conclude that the drug tafenoquine provided any clinical benefit." The first demonstration of tafenoquine efficacy against relapsing babesiosis was reported by Rogers et al. 2023 and confirmed by Krause et al. 2024. Please rephrase the statement and use relevant citations.

We thank the reviewer for pointing out this issue and we have rephrased the statement and used relevant citations (Lines 66-68).

Comment 2: Line 103: mean parasitemia at 10 DPI is reported to be 35.88% but Figure 1C appears to indicate otherwise.

We are sorry for the carelessness, the correct mean parasitemia at 10 DPI is 38.55%, and this has been updated in line 115 of the revised manuscript to reflect the data shown in Figure 1C.

Comment 3: Line 116: parasitemia is said to recur on day 14 post-infection but Figure 1E indicates that recurrence was already noted on day 12 post-infection.

We thank the reviewer for pointing out this inconsistency. We have corrected the relapse day to reflect that recurrence was noted on day 12 post-infection, as shown in Figure 1E. This correction has been made in the revised manuscript (Line 128).

Comment 4: Line 120: Replace "wells" with "strains". Also, start the paragraph with one brief sentence to state how resistant parasites were generated.

We have replaced "wells" with "strains" and added one brief sentence to explain how resistant parasites were generated (Lines 132-134).

Comment 5: Line 169: is Ji et al, 2022b truly the appropriate reference to support a statement on tafenoquine?

We thank the reviewer for highlighting this point. We have added one other reference to support a statement on tafenoquine. The IC₅₀ value of TQ was 20.0 ± 2.4 μM against B. gibsoni (Ji et al., 2022b), and 31 μM against B. bovis (Carvalho et al., 2020) (Lines 223-225).

Comment 6: Lines 184-185: given that exposure to CIP induces mutations in the ATP4 gene and therefore resistance to CIP, what is the prospect of using CIP for the treatment of babesiosis? Can the authors speculate on whether CIP should not be used alone but rather in combination with other drugs currently used for the treatment of human babesiosis?

We thank the reviewer for raising this important question. Given that exposure to CIP induces mutations in the ATP4 gene, leading to resistance, we acknowledge that the long-term use of CIP as a monotherapy may be limited due to the potential for resistance development. To address this concern, we investigated the combination therapy of TQ and CIP to achieve the complete elimination of B. microti in infected mice (a model for human babesiosis). The results of this study are presented in Figure 5C.

Comment 7: Lines 258-259: it is stated that drug treatment was initiated on day 4 post-infection when mean parasitemia was 1% and that drug treatment was continued for 7 days. This is not the case for B. rodhaini infection. As reported in Figure 1E, treatment was initiated on day 2 post-infection.

We apologize for the oversight and any confusion caused. We have corrected the statement to reflect that drug treatment for B. rodhaini-infected mice was initiated at 2 DPI, as reported in Figure 1E (Lines 347-349).

Comment 8: Lines 282-285: RBCs are said to be exposed to CIP for 3 days but parasite size is said to be measured on day 4. Which is correct?

We thank the reviewer for pointing out this discrepancy. To clarify, the infected erythrocytes were exposed to CIP for three consecutive days (72 hours). Blood smears were then prepared at the 73^rd hour, corresponding to the fourth day.

Comment 9: Lines 35-37: this sentence can be omitted from the abstract as it does not summarize additional insight or additional data.

We have omitted this sentence from the abstract.

Comment 10: Line 55: replace Drews et al. 2023 with Gray and Ogden 2021 (doi: 10.3390/pathogens10111430). This excellent article directly supports the statement made by the authors.

We appreciate the reviewer's suggestion and have replaced the reference with Gray and Ogden, 2021 (doi: 10.3390/pathogens10111430) (Line 54).

Comment 11: Line 55: modify the start of sentence to read "The disease is known as babesiosis ...".

We have modified the sentence (Line 54).

Comment 12: Line 56: rephrase to read ".... but chronic infections can be asymptomatic".

We have modified the sentence (Line 55).

Comment 13: Line 57: rephrase to read "The fatality rate ranges from 1% among all cases to 3% among hospitalized cases but has been as high as 20% in immunocompromised patients."

We have rephrased the sentence (Lines 55-57).

Comment 14: Line 61: replace Holbrook et al. 2023 with Krause et al. 2021 (doi: 10.1093/cid/ciaa1216).

We have replaced Holbrook et al. 2023 with Krause et al. 2021 (doi: 10.1093/cid/ciaa1216) (Line 60).

Comment 15: Line 62: rephrase to read "... cytochrome b, which is targeted by atovaquone, were identified in patients with relapsing babesiosis." Here, also cite Lemieux et al., 2016; Simon et al., 2017; Rosenblatt et al, 2021, Marcos et al., 2022; Rogers et al., 2023; Krause et al., 2024.

We have rephrased the sentence and cited the suggested references (Lines 61-64).

Comment 16: Line 65: rephrase "Despite its efficacy, this combination can elicit adverse drug reactions (Vannier and Krause, 2012)."

We have rephrased the sentence (Lines 65-66).

Comment 17: Lines 75-77: rephrase to read "... of the drug indicated that CIP taken orally had good absorption, a long half-life, and ...".

We have rephrased the sentence (Lines 76-77).

Comment 18: Line 79: remove "the".

We have removed "the" (Lines 79-80).

Comment 19: Lines 83-85: rephrase to read "Mice infected with T. gondii that were treated with CIP on the day of infection and the following day had 90% fewer parasites 5 days post-infection (Zhou et al., 2014).".

We have rephrased the sentence (Lines 83-85).

Comment 20: Line 90: shorten the sentence to end as follows "... of CIP on Babesia parasites.".

We have shortened the sentence in line 100 with your suggestion.

Comment 21: Line 96: spell out CC₅₀.

We have spelled out the full form of CC₅₀ (Line 106).

Comment 22: Line 104: remove "of body weight".

We have removed "of body weight" (Line 116).

Comment 23: Line 108: delete "from 8 DPI to 24 DPI, with statistically significant decreases".

We have deleted "from 8 DPI to 24 DPI, with statistically significant decreases" (Line 120).

Comment 24: Line 111: start a new paragraph with the sentence "BALB/c mice infected ...".

We have started a new paragraph with the sentence "BALB/c mice infected ..." (Line 124).

Comment 25: Line 123: replace "showed" with "occurred".

We have replaced "showed" with "occurred" (Line 138).

Comment 26: Line 127: rephrase to read "... sensitivity of the resistant parasite lines ...".

We have rephrased the sentence (Line 144).

Comment 27: Lines 137-140: rephrase to read ".... lines were lower when compared with ..." .

We have rephrased the sentence (Line 158).

Comment 28: Line 149: replace "BgATP4" with "B. gibsoni ATP4".

We have replaced "BgATP4" with "B. gibsoni ATP4" (Line 183).

Comment 29: Line 154: spell out "pLDDT" prior to pLDDT.

We have provided the full form of pLDDT in the revised manuscript (Line 188).

Comment 30: Lines 165-166: rephrase to read "CIP is a novel compound that inhibits Plasmodium development by targeting ATP4 and has been ...".

We have rephrased the sentence (Lines 219-220).

Comment 31: Lines 171-172: rephrase to read "...AZI, the combination recommended by the CDC in the United States.

We have rephrased the sentence (Lines 226-227).

Comment 32: Line 173: rephrase to read "... B. rodhaini infection, with survival up to 67%.".

We have rephrased the sentence (Line 228).

Comment 33: Lines 175-178: rephrase to read "In a previous study, a P. falciparum Dd2 strain that acquired resistance to CIP carried the G358S mutation in the ...".

We have rephrased the sentence (Lines 230-231).

Comment 34: Lines 179-180: rephrase to read "ATP4 is found in the parasite plasma membrane and is specific to the subclass of apicomplexan parasites.".

We have rephrased the sentence (Lines 232-233).

Comment 35: Lines 182-184: rephrase to read "In another study of Toxoplasma gondii, a cell line that carried the mutation G419S in the TgATP4 gene was 34 times ...".

We have rephrased the sentence (Lines 235-237).

Comment 36: Lines 201-202: deleted the last sentence of this paragraph.

We have deleted the last sentence of the paragraph (Line 261).

Comment 37: Line 228: rephrase to read "... that CIP had a weaker binding to BgATP4^L921I than to BgATP4^L921V.".

We have rephrased the sentence (Lines 294-295).

Comment 38: Lines 261-262: please state that drugs were prepared in sesame oil. Add "20 mg/kg" in front of AZI.

We have stated that drugs were prepared in sesame oil and added "20 mg/kg" in front of AZI (Lines 350-352).

Comment 39: Line 265: replace "care" with "treatments".

We have replaced "care" with "treatments" (Line 355).

Comment 40: Line 267: replace "observe" with "assess".

We have replaced "observe" with "assess" (Line 357).

Comment 41: Lines 269-271: please provide the absolute numbers of B. gibsoni infected RBCs and the absolute numbers of uninfected RBCs that were added to the culture medium.

We thank the reviewer for this suggestion. In the revised manuscript, we have included the absolute numbers of B. gibsoni-infected RBCs and uninfected RBCs added to the culture medium. Specifically, the culture medium contained 10 μL (5×10 ⁶) B. gibsoni iRBCs mixed with 40 μL (4×10 ⁸) uninfected RBCs (Lines 360-361).

Comment 42: Line 279: replace "confirmed" with "identified".

We have replaced "confirmed" with "identified" (Line 370).

Comment 43: Figure Supplement 2: the squares are not readily visible. Could the entire column corresponding to the mutation position be highlighted?

We thank the reviewer for this suggestion. To improve visibility, we have changed the color of the squares and added arrows to make the mutation sites as prominent as possible. Unfortunately, due to software limitations, we were unable to highlight the entire column corresponding to the mutation position.

Comment 44: Figure Supplement 4: for the parasite that carries a mutation in BgATP4, please delete the arrows that are next to BgATP4. These arrows send the message that the mutation ATP4 has an active role in pumping back Na⁺ and H⁺ back in their compartment, which is not the case.

We thank the reviewer for their observation. The dotted arrows next to BgATP4 are intended to indicate the recovery of H⁺ and Na⁺ balance facilitated by the mutated ATP4, which reduces susceptibility to ATP4 inhibitors. To avoid potential confusion, we have revised the figure legend to clearly explain the role of the arrows, ensuring the intended message is accurately conveyed.