Review 3: "Anti-tubercular Potential and pH-driven Mode of Action of Salicylic Acid Derivatives"

RR\ID Evidence Scale rating by reviewer:

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

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Review: The manuscript demonstrates that salicylic acid (SA) and two iodinated derivatives as well as related aspirin (ASP) have growth inhibitory activity against an avirulent strain of Mtb, but little activity against other mycobacterial species. The activity was pH dependent for SA and ASP; but, unexpectedly, the activity of the iodinated derivates was not pH dependent. The minimal pH dependence of the iodinated derivatives suggests their activity may not be via intrabacterial acidification as lower external pH should promote internal acidification.

It is clearly demonstrated that the intrabacterial pH acidifies at concentrations which inhibit or kill Mtb. This is nicely demonstrated using a pH-sensitive GFP.  If the compounds are inhibitory but did not kill, this acidification would likely be a direct result of the drug. However, if the drugs kill then it is possible the internal pH measurements are a result of membrane depolarization and the internal pH equilibrating with the external pH which is much lower than mycobacterial internal pH which is over 7. As only MIC data were provided, it is not possible to conclude the pH measurements were not a result of death. The fact that the MES buffer was only used in the pH 5.5 medium and not also in the pH 6.8 medium leaves the possibility, although unlikely, that the MES itself was increasing activity in the low pH conditions.

In the materials and methods section, it is claimed that the protonophore CCCP and the ionophore Monensin were used as internal controls, but no data was presented from use of these molecules. CCCP could be used at different external pHs to generate a standard curve to convert the fluorescent ratio to internal pH which would be more useful in interpreting the pH GFP results.

Importantly, they demonstrate the molecules used in the study have low cytotoxicity making them potential drug candidates. It is however premature to suggest these molecules are very attractive drug candidates as they could be antagonistic to other TB drugs and this was not tested.

Interestingly, they show a related TB drug, para-aminosalicylic acid (PAS), was not pH-dependent and did not acidify the intrabacterial pH. Demonstration that SA and ASP activity is not blocked by methionine is a strong finding that likely rules out that they work via the same mechanism as PAS and supports the overall conclusion of activity via internal acidification.

In line 69 the following statement is incorrect. “PAS was then introduced in anti-TB therapies and has been proven to be effective to cure TB when administered alone or co-administered with streptomycin” No single drug alone cures TB and PAS is known to be one of the weakest of drugs used for TB.