From the journal RSC Chemical Biology Peer review history

Berlin, March 23, 2020

Dear Dr Tate:

Manuscript ID: CB-ART-02-2020-000020
TITLE: Wheat pathogen Zymoseptoria tritici N-myristoyltransferase inhibitors: on-target antifungal activity and an unusual metabolic defense mechanism

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Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

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Reviewer 1

In the last years, the group of Ted Ate has extensively studied the development of N-myristoytransferase (NMT) inhibitors as well as of chemical proteomics methodologies to determine NMT modifications in living systems. In the present study, they have now extended this research line to the important crop fungal pathogen Zymoseptoria tritici that causes huge crop losses and whose agrochemical treatment is hampered by rising resistances. The authors therefore propose that NMT inhibition might represent a novel strategy to develop urgently required, alternatively acting antifungals.

The authors started their investigations by identifying a set of Z. tritici NMT inhibitors via biochemical screening of a compound library of known NMT inhibitors (of other organisms). This resulted in the identification of two structurally different compound classes inhibiting in the nanomolar range. In the present study, they focused on a thorough characterization of one of these two compound classes. Subsequent assays however revealed that the identified inhibitors displayed an only rather low in vivo bioactivity. To study the molecular mechanisms underlying this unexpected low bioactivity, they first showed via chemical proteomics (more precisely metabolic labeling) that the inhibitors hit the NMT target, however with much lower activity than in the biochemical assays. They therefore then used proteomics to determine enhanced expression of detoxification proteins as a potential mechanism underlying the different biochemical and in vivo bioactivity.

Overall, this study is well written, logical in order and all experiments have been performed with the necessary care. In my view, the study also addresses a timely and scientific relevant topic that warrants publication in RSC ChemBiol. The highlights in my view are 1) the demonstration that previous results from biomedical research (i.e. development of NMT inhibitors) can be translated to new applications, e.g. in the field of the development of agrochemicals, 2) the power of chemical proteomics to study the impact of inhibitors of living organisms with limited alternative research approaches (e.g. classical molecular biology) and 3) the demonstration that in case of missing genome information, genome data from a related organism (here yeast) can be used.

I therefore recommend publication after a very minor correction:

1) In Fig. 3, the authors show the result of the full proteome analysis in the form of a heat map. However, I realized that the observed differences in abundance span only a region of -0.25 to 0.25. These seem to be rather low alterations. Could the authors comment on this?

Reviewer 2

The manuscript describes the isolation of the Z. tritici NMT enzyme, development of enzyme inhibitors and proteome profiling. The results are relevant and the experiments reported well described. There are however some points to be addressed to make the work is suitable for publication in RCS Chemical Biology.
In the content it is missing enzyme kinetics and docking to support the enzyme activity and the inhibition mode. Secondly the authors do not give any data related to the inhibitors selectivity to N-myristoyltransferases from humans and other organisms, an important aspect in the development of pesticides.
For the format it is more a report of all the experiments performed than a paper for a scientific journal. The manuscript would greatly improved if the authors provide more concise and clear tables and figures in the paper.

Referee: 1

Comments to the Author

1) In Fig. 3, the authors show the result of the full proteome analysis in the form of a heat map. However, I realized that the observed differences in abundance span only a region of -0.25 to 0.25. These seem to be rather low alterations. Could the authors comment on this?

Whilst the differences observed in this experiment appear modest, they are both reproducible (measured in triplicate) and relatively substantial considering that the changes are measured at only 4 hours of inhibitor treatment, which is the earliest timepoint one would expect to see measurable changes in the proteome as a result of still earlier changes in transcription. These changes therefore reflect the acute response mounted by the fungus in response to compound. A sentence has been added to MS to reflect these points.


Referee: 2

Comments to the Author
1) The manuscript describes the isolation of the Z. tritici NMT enzyme, development of enzyme inhibitors and proteome profiling. The results are relevant and the experiments reported well described. There are however some points to be addressed to make the work is suitable for publication in RCS Chemical Biology.
In the content it is missing enzyme kinetics and docking to support the enzyme activity and the inhibition mode.
This comment is incorrect; we have provided both kinetics data (SI Fig S1) to characterise enzyme activity and docking (SI Fig S2) to characterise inhibition mode; the inhibition mode is peptide-competitive, as for all other examples in this inhibitor class (e.g. Mousnier Nature Chem 2018, 599).

2) Secondly the authors do not give any data related to the inhibitors selectivity to N-myristoyltransferases from humans and other organisms, an important aspect in the development of pesticides.
In this manuscript we used a set of NMT inhibitors with known activity against human NMT. To satisfy the reviewer’s concern, we added all previously reported HsNMT1 IC50 values as a resource to the supplementary table 1, which lists all the inhibitors used. We have also added a comment to the conclusion highlighting that selectivity of an antifungal over other NMTs (particularly human) is a significant challenge for future studies to address. We will be collaborating with our colleagues at Syngenta with the hope of solving this issue in the future, and will be reporting the results in separate publications.

3) For the format it is more a report of all the experiments performed than a paper for a scientific journal. The manuscript would greatly improved if the authors provide more concise and clear tables and figures in the paper.
The intent of this comment is unclear. We believe we have reported the key data in a format consistent with high standards of presentation and clarity, as appreciated by referee #1. This is a data-rich study with multiple complex datasets, and only the key conclusions have been included in the main text figures, the remainder is presented in the supporting information.
We updated visual presentation of the tables on Figure 3, without altering any content.

Berlin, 27. May 2020

Dear Dr Tate, dear Ed:

Manuscript ID: CB-ART-02-2020-000020.R1
TITLE: Wheat pathogen Zymoseptoria tritici N-myristoyltransferase inhibitors: on-target antifungal activity and an unusual metabolic defense mechanism

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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With best wishes,

Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

Reviewer 1

The authors have adequately addressed all concerns. This is an important and timely study. I am therefore happy to recommend its publication in its present form.

Reviewer 2

The authors make additions to the manuscript to clarify the compounds activity in different enzymes. I recommend publication in RSC Chemical Biology.