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    Reply to the reviewers

    Response to reviewers


    __Reviewer #1 (Evidence, reproducibility and clarity (Required)): __ The authors develop a previously identified lead compound for the blocking of malaria transmission from humans to mosquitoes further and identified a protein target of the chemical. The protein target, Pfs16 is long known to be upregulated in gametocytes and has been speculated to be a target for small molecules. The work is well (if at time maybe too well/too detailed) described and potential shortfalls are highlighted.

    My major comment is that without a deletion mutation of Pfs16, the paper will remain somewhat preliminary. I would strongly encourage the authors to generate such a mutant and compare it to the parasites treated with their drug candidate. I feel the text can be much shortened and a lot of information moved to the materials and methods. The conclusions should be toned down on several occasions (abstract, introduction, discussion). Avoid adjectives, e.g. what is a 'powerful starting point' (abstract) or 'compelling interdisciplinary evidence' but hot air?

    We thank the reviewer for this comment. However, we would like to reiterate (as stated in the manuscript) that knockout of Pfs16 in *P. falciparum *is transmission lethal, i.e. you do not get progression of male gametogenesis. Thus, whilst re-generation of a Pfs16 KO would be interesting in terms of comparing phenotypically with the drug treated parasites, we are not convinced it would add any further evidence of support for or against our conclusion in terms of the ability of the N-4HCS scaffold to target this protein. E.g. we could drug treat a Pfs16 KO but this would not be expected to show gametogenesis irrespective of treatment. Therefore, whilst of academic interest, we believe it is satisfactory to judge our phenotypic work based on published accounts of the Pfs16 KO without having to engage in the costly experiments to regenerate the parasite and work on it side-by-side, especially given the limited resolution it would give towards the overall goal of the work in terms of defining the effect and likely target of this drug class on parasites.

    Addressing the second comment, we are happy to alter areas of the paper that may have over-stated the conclusions of the work including the abstract/introduction and discussion.

    CROSS-CONSULTATION COMMENTS I think these three reviews are pretty much in line with their overall assessment. I am happy if send as is to authors as it will help them shape a much better paper

    Reviewer #1 (Significance (Required)):

    The paper shows that very likely a new chemical with some potential for transmission inhibition of malaria parasites for mosquitoes binds to a Plasmodium protein that is specifically expressed in the sexual stages of the parasite.

    The paper compares to good papers published in journals like ACS Infectious Diseases or Antimicrobial Agents and Chemotherapy, but I am not sure which of the Review Commons sister journals it would fit to. I am a molecular parasitologist.

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __ Transmission blocking drugs are of high interest as a strategy to combat malaria but they are difficult to study. For instance it is problematic to raise resistant parasites to find mode of action of transmission blocking drugs and to identify their targets in the cell. In this manuscript Yahiya et al. build on previous work which identified the N-4HCS scaffold, of which DDD01035881 is the lead compound, as an inhibitor P. falciparum male gametocytes. Using PAL to enrich for target proteins Pfs16 was identified and validated as a possible target of DDD01035881. Binding was validated through CESTA. Determination of the phenotype following DDD01035881 treatment was found to partially match the previously published Pfs16 KO phenotype. However curiously no impact was seen in gametocytogenesis despite published evidence of Pfs16 being involved in sexual conversion. The authors speculate as to reasons but a direct experimental comparison with Pfs16 mutant parasites (which likely would have been revealing) is not provided. On the positive side, this analysis of the stage-specific effect of the drug pinpoint the stage inhibited during microgamete development which is a very interesting part of the manuscript.

    We thank the reviewer for this positive assessment of our work. Mirroring comments above, our challenge with Pfs16 knockout or mutation is that if we ablate Pfs16 function we cannot assess the effect of drug action. Definition of a mutant that would demonstrate precisely the drug mode of action would require structural resolution of drug bound to target (i.e. to identify which residues to target) – this is a major goal for our research group moving forwards, but likely many years’ work. In general, our core approach here has been one of chemo-proteomic based methods and phenotypic investigation of the novel antimalarial. Further evidence might be forthcoming from molecular genetics/structural biology, but we believe these are beyond the scope of the current work (and our available resources at present). We state future directions in the discussion and can add more to this in any revised manuscript.

    This work deepens the understanding of a novel class of transmission blocking drugs with reasonable potency (foremost (-)-DDD01028076, which has low nanomolar activity, the modified versions considerably less). Question on how to achieve serum concentrations for sufficient potency aside, these compounds will in the very least provide experimental tools to study their mode of action and might reveal interesting biology. This work is therefore of interest to the malaria field.

    The experimental methodology seems excellent but some of the results raise questions that make definite conclusions difficult and this should be addressed. Overall, this is very solid work but leaves some doubts whether Pfs16 is indeed the (only) target of this class of compounds.

    Major comments:

    1. The reasons for excluding Etramp10.3 are not convincing. In fact it could be argued it is nearly as good a candidate as Pfs16. Contrary to the author's statements in the results section, etramp10.3 transcription is highly upregulated in gametocytes (see e.g. PMID: 22129310) with a generally very low transcription in asexual stages. It is argued that Etramp10.3 is essential in blood stages because MacKellar et al failed to disrupt the gene and because the PiggyBAC screen predicted it to be essential. However, if this is an argument for exclusion then this would also apply to Pfs16 which is also predicted by the PiggyBAC screen to be essential (likely both are non-essential in blood stages as they are barely expressed but Pfs16 and Etramp10.3 might by chance have not received an insertion in the PiggyBAC screen due to their very small size which may also explain failure of disrupting integration in MacKellar). Given the finding that the drug binds Pfs16 only in late gams it might also be argued that an essential function in asexuals might not be affected if they behave similarly to young gams and hence this criterion is not valid anyway.

    Further following this line of thought that ETRAMP10.3 could be a hit equivalent to Pfs16, Figure 2D shows a band below the band considered to be Pfs16. It would not be all surprising if this were ETRAMP10.3 (the size would fit).

    We don’t disagree with reviewer 2’s comments that ETRAMP10.3 *could *be an additional target. Although not traditionally related there is some similarity between these proteins and it may be that at the macroscopic level there is a structural homology between them. As stated elsewhere we are happy to tone down the assertion that Pfs16 is the *only *drug target candidate, leaving open the possibility of future follow up work that may yet reveal additional targets. This cannot be explored much further without extensive experimentation, which is beyond our current capacity. Given the strong phenotypic effect on gametocytes, whilst ETRAMP may be upregulated, this paper naturally focused its core attention on Pfs16 as a candidate target. We certainly subscribe to the view that absence of evidence is not evidence of absence.

    Both, Pfs16 and ETRAMP10.3 can be expected to be very abundant proteins in the parasite periphery in gams. Can the authors exclude that these simply are the first to encounter the N-4HCS photoaffinity probe and that this may have led to their enrichment in the target identification experiments. The biochemical data argues for a specific interaction with Pfs16, but by itself is not that strong. Given the discrepancies of the phenotype with the Pfs16 disruption and the peculiar finding that the drug binds Pfs16 only in later stage gametocytes, it might be a good idea to further caution the conclusion of Pfs16 as the inhibited target.

    We don’t necessarily agree that the evidence is not strong (three methods pointing to the same target is by many accounts solid evidence). Additionally, whilst it is true that the N-4HCS photoaffinity probes likely interact with PVM proteins in first instance, it is also worth noting that this doesn’t necessarily deduct from their likelihood to be true targets, but instead fits with the N-4HCS phenotype. We observe the compounds to inhibit microgametogenesis without any prior incubation and to retain this activity even beyond activation of microgametogenesis, specifically during the window in which the PVM remains associated with the parasite. Our phenotypic observations therefore fit with the notion that the molecules target proteins that lie within the PVM and interact with the molecules at first instance. Whilst we understand the concern that PVM proteins may be likely to be enriched given their abundance and localisation, we believe this to support our phenotypic findings.

    The phenocopy evidence of the NH compounds with the Pfs16 disruption is based on comparison with published evidence. It would have been much preferred to have a side-by-side comparison with the (or an) actual Pfs16 disruption parasite line. Although the authors stress that the phenotype with DD01035881 fits the phenotype of the targeted gene disruption in the results, this only partially matches the cited publication (PMID: 14698439) which concludes there is an effect on the number of gametocytes produced. The exflagellation phenotype in that publication was classified as preliminary. Although this is discussed, the main results text should be adapted to reflect this and the conclusion that Pfs16 may be the target should be further cautioned.

    As stated, we are happy to tone down conclusions in this direction. We also note comments above about Pfs16 disruption.

    Minor comments:

    1. From the modifications of the compounds it seems the chemical space for further modification to achieve higher potency is limited with this scaffold. Maybe the authors can comment whether they envisage this to be a potential obstacle.

    The modification space of the compounds is explored extensively in previous work from our group, which we feel more than adequately addresses this question. See Rueda-Zubiaurre et al (2020) J Med Chem.

    Line 67: references are superscript.

    We can change this

    Line 77: I would recommend replacing 'quiescence' here, a cell that matures is not quiescent.

    We can change this

    Line 116: consider removing 'interdisciplinary'.

    We can change this

    Line 120: I would caution here (see major comments) and recommend a less definite proclamation of Pfs16 as a promising new drug target

    We can change this along with the general “tone” of the manuscript.

    Page 7: compounds 9 is still considered active ("retained micromolar activity"), but in Table 1 this is given as >1000nM. Please add the actual IC50.

    We can add this to the final version. The actual IC50 for this compound was 1.7uM. For the SAR study we grouped compounds with IC50 >1uM into discrete groups based on rough IC50 (>1uM, >10uM etc.) hence this fell in the intermediate group.

    Line 138- 173: The order in which this is discussed makes it unclear that the work described was done prior to, and guided, the synthesis of compound 1 and probe 2

    This can be addressed in a revised manuscript.

    Line 194: was the data deposited in a database?

    The proteomics data has not been deposited in a database but is accessible in the extended SI.

    Line 202: introduction as to the benefits of using a competition + probe condition here could aid reader understanding. The interpretation of this data is complicated by the covalent and reversible binding of the two compounds and the weight of this control is therefore difficult to gage.

    We can embellish the description here.

    Table 2 and Extended Data Table 1 show different p values and enrichments for the same hits. This is confusing. It would also be useful to label the hits in the scatter plots in Figure 2 for easy identification and comparison to the tables.

    We can amend this and label each hit within the scatter plot.

    Line 215-218, please correct the data on Etramp10.3 (see major points) and put in perspective to Pfs16 (Etramp10.3 is similarly upregulated in gams where it is highly expressed; PiggyBAC predicts essentiality for Pfs16 and Etramp10.3 in blood stages).

    We can discuss this to a limited extent for future exploration of Etramp10.3.

    Line 221: the results from the PiggyBAC screen are stated as fact, but what the screen provides is a prediction of the probability of importance for parasite growth. I would replace 'is' with 'is predicted' (even though in the case of Rab1b it seems likely the prediction is correct).

    We can change this

    Line 233 and elsewhere: define 'reversibility' (binding? activity?).

    We can change this

    Line 240: clarify what is in the cited paper (see major points).

    We can clarify this

    Line 297: We utilised in-lysate...... clunky sentence, please rephrase.

    We can change this

    Line 325: reference is missing the year.

    We can change this

    Line 343: It is utterly puzzling that binding is specific to Pfs16 in mature gametocytes and I do not find the explanation in the discussion convincing (see point 28 below). Do the authors have another explanation? Could Pfs16 be modified in later gams (or vice versa)?

    We believe that Pfs16 is functionally different at different stages of gametocyte development, this is either in terms of its presentation (e.g. perhaps due to complex formation, though this remains elusive) or the functionality of different domains, as per the effect of different truncation mutants. We can address some of these concerns in a revised manuscript.

    Line 388: Justification seems odd as a PV protein would be unlikely to directly impact DNA replication. Please rephrase the sentence.

    We can change this

    Line 405: remove the 'to'

    We can change this

    Line 411: it would be useful to the reader to state at what IC-value the drug was used in these experiments.

    We can state this

    Line 431: While the alpha-tubulin staining indicates exflagellation and is similar to the DMSO only control, the staining for the RBC membrane (Glycophorin A) and DNA (DAPI) appear different, yet this is ignored. One interpretation of this could be that while late treatment doesn't block exflagellation, it still impacts other aspects of microgamete development.

    We can make mention of this

    Line 436: IFA work was done with drug treatment post activation while EM was done post activation but drug treatment prior to activation. Is there a reason for this?

    The reviewer is astute to point this out. Limitations with access to the EM facility meant that whilst IFAs were completed for pre-activation treated samples, the post-activation EM became impossible as the EM facility closed during the COVID lockdown. Thus, we do not have a complete set here. However, we do not feel this takes away from the EM observations presented. We can clarify this incompleteness in the revised manuscript.

    Line 450: is this really CytB, or was it CytD?

    We did indeed used Cytochalasin B here, which whilst less potent than D does still target microfilament formation.

    Line 465: Pfs16 localised to vesicles: there is no data showing the dots in the micrograph are vesicles, please rephrase.

    We can change this

    Page 19 and 20, discussion on stage-specific differences of Pfs16 during gametocytogenesis to explain the difference in binding: without experimental data using H-4HCS in the parasites of the publication cited to explain this (PMID: 21498641), this is very speculative. The cited work used episomal expression of Pfs16 tagged with fluorescent proteins. This would be the first integral PVM protein that is actually inserted into the PV membrane when tagged in that way (usually this results in a PV location), casting some doubt on the findings in that paper. All in all the provided explanation is not very convincing.

    We can attempt to clarify this in a revised discussion.

    Line 519: if with the conserved part the N-terminus is meant, then this has for other PVM proteins already been shown to be PVM internal, not facing the erythrocyte (show in very early work; PMID: 1852170 but also multiple times after that).

    We can clarify this

    Line 534: consider replacing 'highly plausible' with something more cautious.

    We can change this

    Line 550: Given this discussion how stable are N- 4HCS compounds?

    We can clarify this.

    Table 1: Having all chemical structures in same orientation would be nicer visually. I assume blue indicates modification but this is not stated.

    We can change this

    Figure 1: Please use different colours or symbols. The dark green crosses and the blue Pfs16 cross are hard to distinguish.

    We can change this

    Figure 3d: Unclear as to why a difference temperature range is displayed here.

    We can clarify this

    Figure 3e: Unclear % Inhibition compared to what.

    We can clarify this

    Figure 5G: What is the white arrow pointing to?

    We can clarify this

    Figure 5j: Given how the explanation is written this would make more sense between current image 5G and 5J.

    We are not sure what the comment relates to here but we can endeavour to clarify this

    Figure 6: Erythrocyte membrane colour not stated in legend.

    We can change this

    Figure 6A: were the exposure times similar? How can so little be left after ~4-5.5 minutes but at later time points there seems to be much more Pfs16 signal left? Maybe amount of signal should be taken into consideration to establish the fate of Pfs16 in the process.

    We can endeavour to clarify this

    Figure 6B: is the second phenotype (successful but aberrant egress) shown? The only image where WGA is not circular around the parasite is an exact match of Pfs16 which is in dots (image at 7.5-8.5 minutes). The imaging data for this phenotype should be presented more clearly.

    We can attempt to clarify this

    Reviewer #2 (Significance (Required)):

    Nature and significance: a lot of weight has been placed on transmission blocking drugs although there are also a number of problems associated with them (ethics for testing and use etc; drugs acting on asexuals and transmission stages alike might be even more useful). Transmission blocking drugs are difficult to study and this work is therefore important. The experiments are well done, but the conclusions are not fully convincing, leaving some doubts in regard to Pfs16 being the actual target of the class of drugs studied.

    Compare to existing published evidence: it is a logic continuation of previous work and this is appropriately highlighted in the manuscript.

    Audience: medium interest for malaria researchers; high interest for researchers working on transmission blocking drugs and those studying microgametes.

    Your expertise: malaria, P. falciparum, biology of apicomplexans

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)): __ The manuscript by Yahiya et al describes an extensive investigation of the mode of action of DDD01028076, which specifically inhibits microgametogenesis in Plasmodium falciparum. The phenotypic characterisation of the MOA uses some very nice imaging to demonstrate the point at which this compound inhibits microgametogenesis. The authors have also attempted to identify the molecular target using chemoproteomics and label-free CETSA techniques. The photoaffinity labelling and pull-down approach suggested the Pfs16 may be preferentially enriched by a PAL probe that is representative of this series. However, the data supporting the validation of this target is not very conclusive, and in some cases argues against Pfs16 being a specific target of DDD01028076. Whilst the presented data makes a significant contribution to the literature regarding a novel drug candidate that targets microgametogenesis, it does not support the author's claims that Pfs16 is the target.

    Major Concerns: The strongest evidence for Pfs16 being the target comes from the chemoproteomics pull-down study that found Pfs16 to be the most significantly enriched protein by compound 2 vs DMSO. However, this should be interpreted with caution as it is based on only 3 replicates and omics studies are prone to false-positives. That only 125 proteins were detected also raises questions about the coverage of the proteomics, it is quite possible that the actual target is not detectable using this method, and the Pfs16 appears because it is one of the more abundant proteins during this stage of the lifecycle.

    As discussed, we are happy to tone down the conclusions about Pfs16 being an exclusive target for the N-4HCS drug class, however, we feel the reviewer is being unnecessarily negative. There are myriad papers in the literature based on singular proteomics experiments (given their cost, complexity and time -consuming nature) that then facilitate downstream experiments that support findings. We have endeavoured to be as thorough as we could in the work and believe, like others, three replicates of a massive experimental pipeline should be sufficient to make a defined conclusion – whether the additional downstream evidence we have then leaned on is supportive of this (as we judge it to be) is another matter. We agree, proteomics often suffers with low protein abundance. The complexity of growing large quantities of gametocytes is familiar to anyone who has struggled to grow these finicky parasites at a larger scale than 10-25mL dishes. Given the scales we have reached, we believe these might in fact be some of the most comprehensive proteomics studies to date!

    Somewhat concerningly, the control with 1 as the competitor did not show significant enrichment of Pfs16, although a trend was observed. More concerning, was the lack of enrichment when using DDD01028076 as the competitor. This result essentially proves that Pfs16 is not the specific target (and the argument about reversibility is unlikely since most drugs are reversible binders, but many have worked with this type of approach). It is surprising that DDD01028076 (ideally the (-) form) wasn't used as the competitor for the proteomics study. This compound has ~100-fold better potency than the probe 2, which should provide much better competition that 1. It would also be more specific than 1, which is an important control considering that (-)-DDD01028076 has activity in the low nanomolar range, whereas 2 acts in the micromolar range. Non-specific interactions are an important consideration to exclude, and whilst 1 is structurally similar, it is not very potent and therefore not the best control to find the target associated with activity.

    Whilst we understand the concerns with insignificant enrichment in the competition labelling, we believe the enrichment in the presence of photoaffinity probe 2 over background (i.e. DMSO vs. probe experiments) to be of more value given the design of the experiment. The competition experiments were performed by co-treating gametocytes with photoaffinity probe 2 and parent molecule 1 prior to UV irradiation, to enable irreversible conjugation to protein target(s). However, given that both compounds, probe and parent, theoretically bind to Pfs16 at the PVM in a reversible manner (i.e. losing interaction with even gentle washing), UV irradiation is likely to favour probe-binding irrespective of competition with a marginally more potent parent molecule (in this case, parent molecule 1). This is especially true as treated parasites were very thoroughly washed after irradiation, so should the parent molecule have bound the target protein(s), these drug-target interactions were likely lost during stringent washing. The drug-target interactions with parent molecule 1 wouldn’t have been aided by UV irradiation, as the molecule lacks the functional group required for bioconjugation. So, even if parent molecule-target interactions were more abundant than probe-target interactions, interactions between parent molecule 1 were most likely lost and proteins bound by probe were enriched.

    This would have been true with more potent N-4HCS derivatives such and DDD01028076 and (-)-DDD01028076 (where potency is tested in the DGFA, independent of bioconjugation), and here we opted for a structurally similar compound of similar potency to not skew competition solely based on potency.

    We can embellish on this in the revised manuscript to make our conclusions from this part clear.

    A closer look at the gels in the supplementary data raises many questions that undermine the authors conclusions:

    • Fig S1a - The lane without probe (2) still identifies Pfs16 (or a protein at that MW) as the most abundant protein. Also, as the Pfs16 band increases, you can see that most other proteins also increase in abundance, so either the loading is inconsistent, or the probe actually causes non-specific enrichment of many proteins. This figure also indicates that the washing protocol is not sufficient to remove non-specific binders. Given the covalent nature of the PAL approach I would think a very thorough washing protocol could be employed.

    It is certainly the case that Pfs16 is abundant in gametocytes, a reason behind its early discovery. Thus it is challenging to remove it from background. We still believe the enrichment to be specific, highlighting the comparative work with Pfg377 in Figure 2. Further repetitions with more stringent washing might resolve the background, however, this is beyond our current resources to repeat.

    -- Running another negative control in the proteomics using one of the inactive controls from table 1 might help to disambiguate specificity.

    We don’t disagree with this though this would involve an entire re-running of the experimental workflow which is not possible.

    • Fig S2a - The anti-Pfs16 Western blots show that this protein is actually enriched more in the flow-through than the eluates. This shows that this protein is not specifically enriched by the PAL-CuAAC pull-down, it is just more abundant in the treated samples.

    Again, the presence of Pfs16 in the flow-through is unsurprising, given its abundance in stage V gametocytes. The relative abundance in the eluate is not an indication that the binding and subsequent enrichment is not specific, rather this shows the compound does not necessarily bind each and every protein – which is not unexpected. The crucial conclusion to be drawn here is the concentration-dependent enrichment of Pfs16 in the eluate in the presence of probe.

    • Fig S2b - The darkest Pfs16 spot is actually the sample with no UV treatment. This is a negative control, so should not enrich the target protein. This sample also has significant signal in replicates A and C.

    As we have noted above, it is not unsurprising that modification of the N-4HCS scaffold to yield this probe may introduce a level of irradiation-independent binding, which explains the presence of signal in the UV-independent sample.

    • Fig S2c - This blot is very messy and difficult to read, but in general the Pfs16 spots in the IGF don't correlate with the intensities in the anti-Pfs16 western.

    These experiments are extremely challenging (something that is perhaps beyond the expertise of the reviewer) and what is presented is the result of substantial optimisation. Loss of AzTB fluorescence in the gel which is subsequently analysed by western blot explains this.

    • Fig S2 - This data, and the main figures based on this data, generally don't support the hypothesis that Pfs16 is the specific target. The controls are not as would be expected, and there are no loading controls. Looking at the flow-throughs suggests that there was just more Pfs16 (and possibly total protein) in the treated samples before the enrichment step. The Pfg377 also appears quite variable in the different samples, with replicates B and C not consistent with A.

    We do not concur with the reviewer here and their dismissal of what was extremely thorough and well-executed experimtns. These are not like traditional western blots and require substantial optimisation. We refer them to our previous point in reference to the UV controls. With regards to the Pfg377 variability, the experiment itself is inherently variable with such large volumes of parasites. In many cases, for example, the male:female ratio within a mature gametocyte culture can vary and this can contribute to the variability in 377 abundance between replicates.

    The other major concern is with the CETSA analysis, which appears to show very minor stabilisation of Pfs16, but the specificity of this target is questionable, and the data has the following inconsistencies.

    • The supplementary data only shows n=1, yet there are error bars in the main figures. Where did these come from?

    The individual western blot replicates can be provided in a revised manuscript if judged important.

    • The samples with apparent destabilisation are all near the edge of large western blots, which often doesn't run straight and has no loading controls. We need to see the loading controls.

    Given all proteins within a lysate will aggregate with thermal treatment, antibody loading controls are not feasible with these experiments. Each sample is normalised prior to thermal stabilisation (ensuring the same protein quantity is treated in both DMSO and drug, at each temperature) and any protein that is not aggregated is loaded – the nature of CETSA itself is to compare the stabilisation between DMSO and drug.

    • The melting temperature of Pfs16 is extremely high at around 85 degrees C. Most plasmodium proteins melt at around 50-60 degrees (Dzekian et al, 2019). Even the cited work on membrane proteins didn't go to those temperatures (Kawatkar et al, 2019) Can this high temperature be explained, and has the CETSA approach been validated at such high temperatures where additional physical and chemical processes may be occurring in the sample?

    We agree that this temperature of stabilisation is unusually high and may require further biochemical validation. Without further investigation we cannot say definitively why the melting temperature of Pfs16 is so high, but suspect its size and membrane localisation may play a role.

    • The lack of difference between + and - isomers suggests that the very small stabilisation observed here is not specific to drug activity, but is more likely a non-specific binding effect. Additional negative control compounds might help here, but the + isomer is probably the best negative control (albeit the concentrations were not ideal in the presented data).

    Please we have already addressed this in the text – refer to line 312 and beyond.

    • The very high concentration (100uM) increases the chances of non-specific effects being observed here (especially since the authors claim to see stabilisation at about 10nM). The study should be repeated at lower concentrations (with negative controls) in order to confirm a specific binding effect.

    Whilst further replicates with different conditions might be preferable, as discussed extensively here, this would be beyond the scope of what we are able to achieve for a revision.

    • The concentration-ranging study was performed at 78.4 degrees, at which temperature very little denaturation of Pfs16 occurs fig S4a (and Fig 3b-c). Therefore, you would not expect to see any drug-induced stabilisation, and it is not plausible that significant stabilisation could occur at this temperature. Therefore, the apparent destabilisation at sub-10nM drug concentrations is highly questionable.

    We would have to agree to disagree on this point.

    • Stabilisation of Pfs16 did not occur in lysates from younger gametocytes (fig s4g-h), but this is a biophysical assay, so regardless of the function of this protein at different stages, the biophysical interaction between the drug and the protein should be the same regardless of the source of the protein. This data argues against Pfs16 being a specific binding target of Pfs16.

    We don’t agree with this statement, since the drug is binding the protein in native lysate – this may be a multi-meric complex (homo or hetero) which only exists at certain stages. As such we disagree with the reviewer that this argues against Pfs16 being the target.

    In addition to the above concerns, the fact that this compound doesn't inhibit the earlier functions of Pfs16 in gametocytogenesis, and that it doesn't inhibit P. berghei, also argue against this being the specific target of this drug. Whilst the authors have a valid argument that these findings don't exclude the possibility of stage-specific targeting of Pfs16, we could also argue that all the phenotypic data in figures 4-6 is merely correlative of a drug that acts at the same point in the lifecycle as Pfs16.

    We have discussed this in the manuscript and strongly feel the reviewer is being unnecessarily dismissive of a body of work that is coherent. We are happy to tone down the narrative of the paper with Pfs16 being the exclusive target. Structural homology of P. berghei Pfs16 orthologues has never been done but it would not be unprecedented if another target was functionally homologous (an idea we are currently pursuing). Stage specificity is also possible given the nature of Pfs16 (e.g. if it is in a complex). The reviewer appears fixated on a singular entity and unable to imagine a complex scenario where structure or protein-protein interactions might affect drug binding (as it does with other proteins present in complexes, e.g. proteasomal targeting drugs).

    Overall, I believe that significant additional studies would be required to identify the target of this compound. Either by repeating the included studies with additional controls and conditions, or by follow-up studies such as genetic manipulation (knock-down or overexpression) or heterologous expression and biophysical binding studies.

    Alternatively, the manuscript could be restructured as primarily a report on the phenotypic effect of this compound on microgametogenesis, with the target identification work reported as a hypothesis-generating chemoproteomics study that provides some ideas about possible targets, but requires substantial follow-up to confirm the target (which may be beyond the scope of this report?).

    We strongly disagree with this reviewer’s entire dismissal of an extensive body of work. In line with other reviewers comments we accept a need to tone down our conclusions, but do not consent to dropping the majority of the paper in favour of a phenotypic descriptive work.

    MINOR COMMENTS The manuscript is very well-written and presented.

    Several of the conclusions are overstated (as detailed above) and several statements should be tempered based on this data (e.g. statements linking DDD01028076 effects to Pfs16 function).

    We can address the overstatement of conclusions in a revised manuscript.

    I find the term 'crosslinking' confusing for the photo-affinity labelling, as crosslinking in proteomics often refers to crosslinking between proteins (not between protein and drug).

    This is simple to address – to minimise confusion for readers, we can simply state where photoaffinity labelling and bioconjugation were performed (and not refer to the latter as crosslinking).

    The data and terminology around activity (IC50) for compounds in table 1 is a little confusing. Some IC50 values are reported as >1000, while others have precise mean values reported over 1000, and others are >10,000 or >25,000. This is especially confusing where 9 is claimed to have retained activity, but is >1000. If consistent thresholds are not appropriate then perhaps including dose response curves in the supp data might be necessary to explain these?

    We can simply provide the provide IC50s for compounds of greater potency. We are also happy to provide the curves but with such a large body of work already, this might be unnecessary.

    Reviewer #3 (Significance (Required)):

    The work is potentially interesting to Plasmodium biology and drug discovery researchers. The concept of a transmission-blocking drug is quite attractive to this community, so the topic is highly relevant. Keeping in mind that this compound was reported previously, the main novelty is in defining it's window of activity during the microgametogenesis process, and differentiating this from other drugs/compounds that inhibit this process. There is clearly an advance in knowledge presented here.

    If Pfs16 were to be confirmed as the target of this series then I think that this study would have much greater impact and attract interest from a broad audience. However, at this stage I don't see strong evidence for this hypothesis, and some of this data casts significant doubt on the likelihood that Pfs16 is the direct target.

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Yahiya et al describes an extensive investigation of the mode of action of DDD01028076, which specifically inhibits microgametogenesis in Plasmodium falciparum. The phenotypic characterisation of the MOA uses some very nice imaging to demonstrate the point at which this compound inhibits microgametogenesis. The authors have also attempted to identify the molecular target using chemoproteomics and label-free CETSA techniques. The photoaffinity labelling and pull-down approach suggested the Pfs16 may be preferentially enriched by a PAL probe that is representative of this series. However, the data supporting the validation of this target is not very conclusive, and in some cases argues against Pfs16 being a specific target of DDD01028076. Whilst the presented data makes a significant contribution to the literature regarding a novel drug candidate that targets microgametogenesis, it does not support the author's claims that Pfs16 is the target.

    Major Concerns:

    The strongest evidence for Pfs16 being the target comes from the chemoproteomics pull-down study that found Pfs16 to be the most significantly enriched protein by compound 2 vs DMSO. However, this should be interpreted with caution as it is based on only 3 replicates and omics studies are prone to false-positives. That only 125 proteins were detected also raises questions about the coverage of the proteomics, it is quite possible that the actual target is not detectable using this method, and the Pfs16 appears because it is one of the more abundant proteins during this stage of the lifecycle.

    Somewhat concerningly, the control with 1 as the competitor did not show significant enrichment of Pfs16, although a trend was observed. More concerning, was the lack of enrichment when using DDD01028076 as the competitor. This result essentially proves that Pfs16 is not the specific target (and the argument about reversibility is unlikely since most drugs are reversible binders, but many have worked with this type of approach). It is surprising that DDD01028076 (ideally the (-) form) wasn't used as the competitor for the proteomics study. This compound has ~100-fold better potency than the probe 2, which should provide much better competition that 1. It would also be more specific than 1, which is an important control considering that (-)-DDD01028076 has activity in the low nanomolar range, whereas 2 acts in the micromolar range. Non-specific interactions are an important consideration to exclude, and whilst 1 is structurally similar, it is not very potent and therefore not the best control to find the target associated with activity.

    A closer look at the gels in the supplementary data raises many questions that undermine the authors conclusions:

    • Fig S1a - The lane without probe (2) still identifies Pfs16 (or a protein at that MW) as the most abundant protein. Also, as the Pfs16 band increases, you can see that most other proteins also increase in abundance, so either the loading is inconsistent, or the probe actually causes non-specific enrichment of many proteins. This figure also indicates that the washing protocol is not sufficient to remove non-specific binders. Given the covalent nature of the PAL approach I would think a very thorough washing protocol could be employed. -- Running another negative control in the proteomics using one of the inactive controls from table 1 might help to disambiguate specificity.
    • Fig S2a - The anti-Pfs16 Western blots show that this protein is actually enriched more in the flow-through than the eluates. This shows that this protein is not specifically enriched by the PAL-CuAAC pull-down, it is just more abundant in the treated samples.
    • Fig S2b - The darkest Pfs16 spot is actually the sample with no UV treatment. This is a negative control, so should not enrich the target protein. This sample also has significant signal in replicates A and C.
    • Fig S2c - This blot is very messy and difficult to read, but in general the Pfs16 spots in the IGF don't correlate with the intensities in the anti-Pfs16 western.
    • Fig S2 - This data, and the main figures based on this data, generally don't support the hypothesis that Pfs16 is the specific target. The controls are not as would be expected, and there are no loading controls. Looking at the flow-throughs suggests that there was just more Pfs16 (and possibly total protein) in the treated samples before the enrichment step. The Pfg377 also appears quite variable in the different samples, with replicates B and C not consistent with A.

    The other major concern is with the CETSA analysis, which appears to show very minor stabilisation of Pfs16, but the specificity of this target is questionable, and the data has the following inconsistencies.

    • The supplementary data only shows n=1, yet there are error bars in the main figures. Where did these come from?
    • The samples with apparent destabilisation are all near the edge of large western blots, which often doesn't run straight and has no loading controls. We need to see the loading controls.
    • The melting temperature of Pfs16 is extremely high at around 85 degrees C. Most plasmodium proteins melt at around 50-60 degrees (Dzekian et al, 2019). Even the cited work on membrane proteins didn't go to those temperatures (Kawatkar et al, 2019) Can this high temperature be explained, and has the CETSA approach been validated at such high temperatures where additional physical and chemical processes may be occurring in the sample?
    • The lack of difference between + and - isomers suggests that the very small stabilisation observed here is not specific to drug activity, but is more likely a non-specific binding effect. Additional negative control compounds might help here, but the + isomer is probably the best negative control (albeit the concentrations were not ideal in the presented data).
    • The very high concentration (100uM) increases the chances of non-specific effects being observed here (especially since the authors claim to see stabilisation at about 10nM). The study should be repeated at lower concentrations (with negative controls) in order to confirm a specific binding effect.
    • The concentration-ranging study was performed at 78.4 degrees, at which temperature very little denaturation of Pfs16 occurs fig S4a (and Fig 3b-c). Therefore, you would not expect to see any drug-induced stabilisation, and it is not plausible that significant stabilisation could occur at this temperature. Therefore, the apparent destabilisation at sub-10nM drug concentrations is highly questionable.
    • Stabilisation of Pfs16 did not occur in lysates from younger gametocytes (fig s4g-h), but this is a biophysical assay, so regardless of the function of this protein at different stages, the biophysical interaction between the drug and the protein should be the same regardless of the source of the protein. This data argues against Pfs16 being a specific binding target of Pfs16.

    In addition to the above concerns, the fact that this compound doesn't inhibit the earlier functions of Pfs16 in gametocytogenesis, and that it doesn't inhibit P. berghei, also argue against this being the specific target of this drug. Whilst the authors have a valid argument that these findings don't exclude the possibility of stage-specific targeting of Pfs16, we could also argue that all the phenotypic data in figures 4-6 is merely correlative of a drug that acts at the same point in the lifecycle as Pfs16.

    Overall, I believe that significant additional studies would be required to identify the target of this compound. Either by repeating the included studies with additional controls and conditions, or by follow-up studies such as genetic manipulation (knock-down or overexpression) or heterologous expression and biophysical binding studies. Alternatively, the manuscript could be restructured as primarily a report on the phenotypic effect of this compound on microgametogenesis, with the target identification work reported as a hypothesis-generating chemoproteomics study that provides some ideas about possible targets, but requires substantial follow-up to confirm the target (which may be beyond the scope of this report?).

    Minor comments

    The manuscript is very well-written and presented.

    Several of the conclusions are overstated (as detailed above) and several statements should be tempered based on this data (e.g. statements linking DDD01028076 effects to Pfs16 function).

    I find the term 'crosslinking' confusing for the photo-affinity labelling, as crosslinking in proteomics often refers to crosslinking between proteins (not between protein and drug).

    The data and terminology around activity (IC50) for compounds in table 1 is a little confusing. Some IC50 values are reported as >1000, while others have precise mean values reported over 1000, and others are >10,000 or >25,000. This is especially confusing where 9 is claimed to have retained activity, but is >1000. If consistent thresholds are not appropriate then perhaps including dose response curves in the supp data might be necessary to explain these?

    Significance

    The work is potentially interesting to Plasmodium biology and drug discovery researchers. The concept of a transmission-blocking drug is quite attractive to this community, so the topic is highly relevant. Keeping in mind that this compound was reported previously, the main novelty is in defining it's window of activity during the microgametogenesis process, and differentiating this from other drugs/compounds that inhibit this process. There is clearly an advance in knowledge presented here.

    If Pfs16 were to be confirmed as the target of this series then I think that this study would have much greater impact and attract interest from a broad audience. However, at this stage I don't see strong evidence for this hypothesis, and some of this data casts significant doubt on the likelihood that Pfs16 is the direct target.

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    Referee #2

    Evidence, reproducibility and clarity

    Transmission blocking drugs are of high interest as a strategy to combat malaria but they are difficult to study. For instance it is problematic to raise resistant parasites to find mode of action of transmission blocking drugs and to identify their targets in the cell. In this manuscript Yahiya et al. build on previous work which identified the N-4HCS scaffold, of which DDD01035881 is the lead compound, as an inhibitor P. falciparum male gametocytes. Using PAL to enrich for target proteins Pfs16 was identified and validated as a possible target of DDD01035881. Binding was validated through CESTA. Determination of the phenotype following DDD01035881 treatment was found to partially match the previously published Pfs16 KO phenotype. However curiously no impact was seen in gametocytogenesis despite published evidence of Pfs16 being involved in sexual conversion. The authors speculate as to reasons but a direct experimental comparison with Pfs16 mutant parasites (which likely would have been revealing) is not provided. On the positive side, this analysis of the stage-specific effect of the drug pinpoint the stage inhibited during microgamete development which is a very interesting part of the manuscript.

    This work deepens the understanding of a novel class of transmission blocking drugs with reasonable potency (foremost (-)-DDD01028076, which has low nanomolar activity, the modified versions considerably less). Question on how to achieve serum concentrations for sufficient potency aside, these compounds will in the very least provide experimental tools to study their mode of action and might reveal interesting biology. This work is therefore of interest to the malaria field.

    The experimental methodology seems excellent but some of the results raise questions that make definite conclusions difficult and this should be addressed. Overall, this is very solid work but leaves some doubts whether Pfs16 is indeed the (only) target of this class of compounds.

    Major comments:

    1. The reasons for excluding Etramp10.3 are not convincing. In fact it could be argued it is nearly as good a candidate as Pfs16. Contrary to the author's statements in the results section, etramp10.3 transcription is highly upregulated in gametocytes (see e.g. PMID: 22129310) with a generally very low transcription in asexual stages. It is argued that Etramp10.3 is essential in blood stages because MacKellar et al failed to disrupt the gene and because the PiggyBAC screen predicted it to be essential. However, if this is an argument for exclusion then this would also apply to Pfs16 which is also predicted by the PiggyBAC screen to be essential (likely both are non-essential in blood stages as they are barely expressed but Pfs16 and Etramp10.3 might by chance have not received an insertion in the PiggyBAC screen due to their very small size which may also explain failure of disrupting integration in MacKellar). Given the finding that the drug binds Pfs16 only in late gams it might also be argued that an essential function in asexuals might not be affected if they behave similarly to young gams and hence this criterion is not valid anyway. Further following this line of thought that ETRAMP10.3 could be a hit equivalent to Pfs16, Figure 2D shows a band below the band considered to be Pfs16. It would not be all surprising if this were ETRAMP10.3 (the size would fit).
    2. Both, Pfs16 and ETRAMP10.3 can be expected to be very abundant proteins in the parasite periphery in gams. Can the authors exclude that these simply are the first to encounter the N-4HCS photoaffinity probe and that this may have led to their enrichment in the target identification experiments. The biochemical data argues for a specific interaction with Pfs16, but by itself is not that strong. Given the discrepancies of the phenotype with the Pfs16 disruption and the peculiar finding that the drug binds Pfs16 only in later stage gametocytes, it might be a good idea to further caution the conclusion of Pfs16 as the inhibited target.
    3. The phenocopy evidence of the NH compounds with the Pfs16 disruption is based on comparison with published evidence. It would have been much preferred to have a side-by-side comparison with the (or an) actual Pfs16 disruption parasite line. Although the authors stress that the phenotype with DD01035881 fits the phenotype of the targeted gene disruption in the results, this only partially matches the cited publication (PMID: 14698439) which concludes there is an effect on the number of gametocytes produced. The exflagellation phenotype in that publication was classified as preliminary. Although this is discussed, the main results text should be adapted to reflect this and the conclusion that Pfs16 may be the target should be further cautioned.

    Minor comments:

    1. From the modifications of the compounds it seems the chemical space for further modification to achieve higher potency is limited with this scaffold. Maybe the authors can comment whether they envisage this to be a potential obstacle.
    2. Line 67: references are superscript.
    3. Line 77: I would recommend replacing 'quiescence' here, a cell that matures is not quiescent.
    4. Line 116: consider removing 'interdisciplinary'.
    5. Line 120: I would caution here (see major comments) and recommend a less definite proclamation of Pfs16 as a promising new drug target
    6. Page 7: compounds 9 is still considered active ("retained micromolar activity"), but in Table 1 this is given as >1000nM. Please add the actual IC50.
    7. Line 138- 173: The order in which this is discussed makes it unclear that the work described was done prior to, and guided, the synthesis of compound 1 and probe 2
    8. Line 194: was the data deposited in a database?
    9. Line 202: introduction as to the benefits of using a competition + probe condition here could aid reader understanding. The interpretation of this data is complicated by the covalent and reversible binding of the two compounds and the weight of this control is therefore difficult to gage.
    10. Table 2 and Extended Data Table 1 show different p values and enrichments for the same hits. This is confusing. It would also be useful to label the hits in the scatter plots in Figure 2 for easy identification and comparison to the tables.
    11. Line 215-218, please correct the data on Etramp10.3 (see major points) and put in perspective to Pfs16 (Etramp10.3 is similarly upregulated in gams where it is highly expressed; PiggyBAC predicts essentiality for Pfs16 and Etramp10.3 in blood stages).
    12. Line 221: the results from the PiggyBAC screen are stated as fact, but what the screen provides is a prediction of the probability of importance for parasite growth. I would replace 'is' with 'is predicted' (even though in the case of Rab1b it seems likely the prediction is correct).
    13. Line 233 and elsewhere: define 'reversibility' (binding? activity?).
    14. Line 240: clarify what is in the cited paper (see major points).
    15. Line 297: We utilised in-lysate...... clunky sentence, please rephrase.
    16. Line 325: reference is missing the year.
    17. Line 343: It is utterly puzzling that binding is specific to Pfs16 in mature gametocytes and I do not find the explanation in the discussion convincing (see point 28 below). Do the authors have another explanation? Could Pfs16 be modified in later gams (or vice versa)?
    18. Line 388: Justification seems odd as a PV protein would be unlikely to directly impact DNA replication. Please rephrase the sentence.
    19. Line 405: remove the 'to'
    20. Line 411: it would be useful to the reader to state at what IC-value the drug was used in these experiments.
    21. Line 431: While the alpha-tubulin staining indicates exflagellation and is similar to the DMSO only control, the staining for the RBC membrane (Glycophorin A) and DNA (DAPI) appear different, yet this is ignored. One interpretation of this could be that while late treatment doesn't block exflagellation, it still impacts other aspects of microgamete development.
    22. Line 436: IFA work was done with drug treatment post activation while EM was done post activation but drug treatment prior to activation. Is there a reason for this?
    23. Line 450: is this really CytB, or was it CytD?
    24. Line 465: Pfs16 localised to vesicles: there is no data showing the dots in the micrograph are vesicles, please rephrase.
    25. Page 19 and 20, discussion on stage-specific differences of Pfs16 during gametocytogenesis to explain the difference in binding: without experimental data using H-4HCS in the parasites of the publication cited to explain this (PMID: 21498641), this is very speculative. The cited work used episomal expression of Pfs16 tagged with fluorescent proteins. This would be the first integral PVM protein that is actually inserted into the PV membrane when tagged in that way (usually this results in a PV location), casting some doubt on the findings in that paper. All in all the provided explanation is not very convincing.
    26. Line 519: if with the conserved part the N-terminus is meant, then this has for other PVM proteins already been shown to be PVM internal, not facing the erythrocyte (show in very early work; PMID: 1852170 but also multiple times after that).
    27. Line 534: consider replacing 'highly plausible' with something more cautious.
    28. Line 550: Given this discussion how stable are N- 4HCS compounds?
    29. Table 1: Having all chemical structures in same orientation would be nicer visually. I assume blue indicates modification but this is not stated.
    30. Figure 1: Please use different colours or symbols. The dark green crosses and the blue Pfs16 cross are hard to distinguish.
    31. Figure 3d: Unclear as to why a difference temperature range is displayed here.
    32. Figure 3e: Unclear % Inhibition compared to what.
    33. Figure 5G: What is the white arrow pointing to?
    34. Figure 5j: Given how the explanation is written this would make more sense between current image 5G and 5J.
    35. Figure 6: Erythrocyte membrane colour not stated in legend.
    36. Figure 6A: were the exposure times similar? How can so little be left after ~4-5.5 minutes but at later time points there seems to be much more Pfs16 signal left? Maybe amount of signal should be taken into consideration to establish the fate of Pfs16 in the process.
    37. Figure 6B: is the second phenotype (successful but aberrant egress) shown? The only image where WGA is not circular around the parasite is an exact match of Pfs16 which is in dots (image at 7.5-8.5 minutes). The imaging data for this phenotype should be presented more clearly.

    Significance

    Nature and significance: a lot of weight has been placed on transmission blocking drugs although there are also a number of problems associated with them (ethics for testing and use etc; drugs acting on asexuals and transmission stages alike might be even more useful). Transmission blocking drugs are difficult to study and this work is therefore important. The experiments are well done, but the conclusions are not fully convincing, leaving some doubts in regard to Pfs16 being the actual target of the class of drugs studied.

    Compare to existing published evidence: it is a logic continuation of previous work and this is appropriately highlighted in the manuscript.

    Audience: medium interest for malaria researchers; high interest for researchers working on transmission blocking drugs and those studying microgametes.

    Your expertise: malaria, P. falciparum, biology of apicomplexans

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    Referee #1

    Evidence, reproducibility and clarity

    The authors develop a previously identified lead compound for the blocking of malaria transmission from humans to mosquitoes further and identified a protein target of the chemical. The protein target, Pfs16 is long known to be upregulated in gametocytes and has been speculated to be a target for small molecules. The work is well (if at time maybe too well/too detailed) described and potential shortfalls are highlighted.

    My major comment is that without a deletion mutation of Pfs16, the paper will remain somewhat preliminary. I would strongly encourage the authors to generate such a mutant and compare it to the parasites treated with their drug candidate. I feel the text can be much shortened and a lot of information moved to the materials and methods. The conclusions should be toned down on several occasions (abstract, introduction, discussion). Avoid adjectives, e.g. what is a 'powerful starting point' (abstract) or 'compelling interdisciplinary evidence' but hot air?

    Referees cross-commenting

    I think these three reviews are pretty much in line with their overall assessment. I am happy if send as is to authors as it will help them shape a much better paper

    Significance

    The paper shows that very likely a new chemical with some potential for transmission inhibition of malaria parasites for mosquitoes binds to a Plasmodium protein that is specifically expressed in the sexual stages of the parasite.

    The paper compares to good papers published in journals like ACS Infectious Diseases or Antimicrobial Agents and Chemotherapy, but I am not sure which of the Review Commons sister journals it would fit to. I am a molecular parasitologist.