Peer-reviewed by 2 reviewers with median rating of 13.5/20. Review process was triple-blinded.
Round 1 (13.5/20)
Conceptual advance and Impact7
Comment: While realising that it is contentious to use the level of unbound compound in plasma as a proxy for efficacy without knowing the plasma-protein off-rate or target affinity (as the equilibrium can settle on high levels of target binding even with low free compound levels) I think it is justified in this case, as the IC50 for each compound in the near-absence of plasma protein is known.
As a suggestion, perhaps in vitro IC50 values could be titrated with % of FBS in the culture media to weed out compounds that are inactive in blood due to high plasma protein binding or other reasons.
By "activity in vivo" the context suggests that they mean activity as promoters of procyclic form differentiation. However they actually tested for parasite load reduction, so they need to specify this.
Again, unclear whether by "efficacy" they mean differentiation-promoting activity of the compounds, or the trypanocidal activity.
Results & Discussion
I have a mix of major changes and minor changes.
1 - They need to present actual tail blood parasite load data for experiments 1, 2 and 3, instead of just saying "no significant effect". Or at least clarify what criteria they use to qualify a blood parasite sample as "no significant effect".
2 - They need to state in main body of text that blood samples between 2 mice were pooled before measuring. Was pooling only done for the PK study or for all of the experiments including the preceding parasite load assays? Were equal volumes of blood from each mouse added?
1 - To clarify, after the sentence "Mice were infected with T. b. rhodesiense one day before the start of treatment" they should insert something like "and the effect of the treatments on blood parasite load was subsequently assessed."
2 - In general they don't refer much to their figures when discussing the experiments. Its difficult for the reader to match up each experiment to the dosing regime. They need to at least refer to which of the 3 regimes in figure 1A they are referring to while discussing each experiment. Better still, present the dosing regime and a plot of the tail blood parasite concentration (this data warrants inclusion as per Major Changes, point 1) together. ie. make 3 different panels, one per experiment, each showing dosing regime and blood parasite load results together, with x-axes aligned.
3 - Likewise, Fig 1b could be merged with 1c to make a new figure, as the x-axes should align.
4 - Mention somewhere in the main body of the manuscript that UPLC MS/MS was how they quantified their compounds.
5 - Consider including all PPB and t1/2 values for the 3 tested compounds in main manuscript body, maybe as a small table, rather than relegating them to the supplementary material.
for Snapshot PK experiment section, add something like: "Blood compound concentrations were measured by UPLC MS/MS, see additional supplementary methods."
Conceptual advance and Impact6
The study is well done and the limitations well explained. I'd argue differently if it were a full sized article, but given it is a short communication, the lower sample sizes and less consumption of material and methods seem necessary. Cannot comment on the impact of this study on progression of the science in this field.