Review 2: "Anti-tubercular Potential and pH-driven Mode of Action of Salicylic Acid Derivatives"

RR\ID Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review: Laudouze et al test a series of phenolic acids for growth inhibitory activity against four bacteria, including Mycobacterium tuberculosis (Mtb). They find that some of these phenolic acids block growth of Mtb in a pH-dependent manner by acidifying the cytoplasm. The results of the presented experiments are broadly consistent with published literature and provide further support for cytoplasmic acidification as a relevant mechanism of action of potential antituberculars.

Items for consideration:

1. This reviewer would appreciate an expanded discussion on the discrepancy between the observed pH dependence of the growth inhibitory properties of the compounds and the compound pKa- what do the authors propose is happening?

2. The authors observe some degree of cytoplasmic acidification for salicylic acid even at pH 6.8. At this concentration of SA, is there sufficient protonated SA species to mediate cytoplasmic acidification?

3. The implications of this sentence were unclear to me: “We demonstrate that iodination of SA does not alter the antibacterial activity of the resulting 5ISA and 3,5diISA derivatives, and could constitute an interesting way to synthetize new antibacterial molecules.”

4. Framing the results of this paper in light of (one of) the proposed mechanism(s) of action of PZA would be welcomed in the concluding paragraph.