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  1. Evaluation Summary:

    This submission reports an approach to identify bacterial specific carboxyesterases that can be exploited to activate prodrugs of antibiotics in Staphylococcus aureus. The main premise is that charged functional groups found in some antibiotics prevent entry of these into bacteria, an effect that can be circumvented through esterification of the antibiotic to allow entry. Thereafter, activation of the antibiotic will occur in the bacterial cytoplasm through hydrolysis by bacterial esterases. This could lead to new antibiotic delivery strategies.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    This manuscript describes an effort to identify and study bacterial carboxyesterases that can be exploited for the activation of antibiotic prodrugs. The overall premise is that highly charged functionality like phosphonates render molecules impermeable to the bacterial membrane. Masking these groups can allow permeation but will require some form of activation within the bacterial to liberate the active drug. Equally important will be the stability of the prodrugs towards human hydrolases such that the drugs are not prematurely activated. This work recounts an effort to identify and characterize appropriate carboxyesterases from S. aureus. The overall approach is very elegant and logical. Utilizing a known antibiotic prodrug (Hex) in combination with a library of strains with specific esterases knocked out, the authors identified two enzymes needed to activation of HEX. The importance of these was confirmed by generating lab raised mutants to HEX which showed several SNPs in the enzymes (GloB and FrmB). Characterization studies included examination of substrate specificity and determination of high-resolution crystal structures of the enzymes. Several pro-moieties that were good substrates for the bacterial enzymes were identified and counter-screened against serum esterases (human and mouse) to demonstrate that selective activation would be possible. Overall, the study is very well executed and the methods and analysis appear to have a very high level of rigor and represents a nice mixture of genomic, structural and chemical analysis. The overall impact of this work will be determined by the utility of such prodrugs in the treatment of infections. Some additional considerations that will need to be addressed are the overall metabolic stability of the prodrug, the possibility for facile resistance, spectrum of activity and suitability of the application.

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  3. Reviewer #2 (Public Review):

    In this article, the authors describe how new prodrugs can be used in antibiotics against S. aureus bacteria. Antimicrobial resistance is a serious problem and S. aureus infections are still a serious public health threat. With prodrugs we can improve the properties of molecules and in this case improve the oral absorption of cellular uptake. The authors discovered suitable carboxy-ester-based prodrugs that can be specifically hydrolyzed in bacteria by the two enzymes GloB and FrmB, but not in human plasma. They determined the substrate specificities for FrmB and GloB and demonstrated the structural basis of these preferences. The authors identified several promoters that are likely to be serum esterase resistant and microbially labile.

    The advantage of this study is that it allows structure-based design of new molecules that target staphylococcal pathogens. It would be beneficial to have a proof-of-concept of this approach on real antibiotic molecules. The approach, which is interesting, may have limitations in the discovery of new antibacterial agents. The introduction of lipophilic ester groups increases the logP and consequently the plasma protein binding could be high, limiting the in vivo efficacy of such prodrugs.

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