Myeloperoxidase: Regulation of Neutrophil Function and Target for Therapy

Round 1

Reviewer 1 Report

The current manuscript represents an overall well written, interesting and up to date review article on the role of myeloperoxidase in neutrophil function.  This review is topical and will be of considerable interest to those interested in the fields of inflammation and oxidative stress.  There are, however, some key issues that require the authors attention.

1. Section 3 on MPO enzymatic reactions contains some errors that require correction.  Thus the authors contend that compound II can react with halides to generate hypohalous acids. This is not correct.  Only compound I can mediate the formation of the hypohalous acids and diversion of compound I via a one electron reaction to form compound II inhibits the halogenation cycle of MPO. Therefore, the authors are requested to correct this error. Moreover, it would be helpful for the authors to provide a clear description of the halogenation and peroxidase cycles to help readers who are not familiar with the complex catalytic cycles and reactions of MPO. 

The authors also refer to the reaction of proteins containing glutathione.  Do they mean reaction of HOCl with glutathione, which is a small molecular weight tripeptide antioxidant molecule?  The authors also state that HOCl oxidizes GSH into glutathione sulfonamide (GSSG). While HOCl can form glutathione sulfonamide when oxidizing GSH, the GSSG abbreviation refers to glutathione disulfide that is also a product of the reaction of HOCl with GSH. Therefore this also needs to be corrected by the authors.

2. In section 5 when discussing the signaling properties of oxidants it is important for the Authors to also include details on HOSCN, which is an important MPO-derived oxidant that targets protein cysteine residues similar to H2O2 and hence capable of mediating redox cell signaling.

3. Section 10 on MPO inhibitors claims that 4-ABAH is the most potent inhibitor of peroxidation reported to date.  This is incorrect. Various new classes of more potent and selective irreversible and reversible MPO inhibitors have been discovered since 4-ABAH, most notably the 2-thioxanthine irreversible class of MPO inhibitors that are superior to 4-ABAH in inhibiting various MPO-catalyzed oxidative reactions (J Biol Chem, 2011;286:37578-89; Biochem Pharmacol2017;135:90-115) and are in fact the AZD compounds discussed by the authors that are in clinical trials.  The authors are therefore requested to improve this section by providing an up to date appraisal of the latest MPO inhibitors available, including a brief description of their mechanism of action (e.g., irreversible, reversible), selectivity and potency.   

 

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Author Response

1. Section 3 on MPO enzymatic reactions contains some errors that require correction.  Thus the authors contend that compound II can react with halides to generate hypohalous acids. This is not correct.  Only compound I can mediate the formation of the hypohalous acids and diversion of compound I via a one electron reaction to form compound II inhibits the halogenation cycle of MPO. Therefore, the authors are requested to correct this error. Moreover, it would be helpful for the authors to provide a clear description of the halogenation and peroxidase cycles to help readers who are not familiar with the complex catalytic cycles and reactions of MPO. 

The authors also refer to the reaction of proteins containing glutathione.  Do they mean reaction of HOCl with glutathione, which is a small molecular weight tripeptide antioxidant molecule?  The authors also state that HOCl oxidizes GSH into glutathione sulfonamide (GSSG). While HOCl can form glutathione sulfonamide when oxidizing GSH, the GSSG abbreviation refers to glutathione disulfide that is also a product of the reaction of HOCl with GSH. Therefore this also needs to be corrected by the authors.

Re: We thank the reviewer for the careful reading of our manuscript as well as pointing out factual errors. We apologize for these errors and rewrote Section 3.

2. In section 5 when discussing the signaling properties of oxidants it is important for the Authors to also include details on HOSCN, which is an important MPO-derived oxidant that targets protein cysteine residues similar to H2O2 and hence capable of mediating redox cell signaling.

Re: Thank you for raising this important issue. We have expanded Section 5 to discuss redox signaling by MPO-derived HOSCN.

3. Section 10 on MPO inhibitors claims that 4-ABAH is the most potent inhibitor of peroxidation reported to date.  This is incorrect. Various new classes of more potent and selective irreversible and reversible MPO inhibitors have been discovered since 4-ABAH, most notably the 2-thioxanthine irreversible class of MPO inhibitors that are superior to 4-ABAH in inhibiting various MPO-catalyzed oxidative reactions (J Biol Chem,2011;286:37578-89; Biochem Pharmacol2017;135:90-115) and are in fact the AZD compounds discussed by the authors that are in clinical trials.  The authors are therefore requested to improve this section by providing an up to date appraisal of the latest MPO inhibitors available, including a brief description of their mechanism of action (e.g., irreversible, reversible), selectivity and potency.   

Re: We accept your criticism. We expanded Section 10 to introduce the mechanisms of irreversible and reversible inhibitors, and detailed the effects and mechanism of action of the 2-thioxanthine class inhibitors. We also included a brief discussion of melatonin as a promising irreversible MPO inhibitor. However, we feel that extensive description of selectivity and potency of the MPO inhibitors was beyond the scope of our review as these have been discussed in more depth in previous excellent reviews.  

 

Reviewer 2 Report

This review covers an interesting topic, and it is clear and well written.

It does, however, contain disturbing factual errors with regards the enzymatic properties of MPO. This is obviously outside the area of expertise of the authors, but it is important to get correct, and additional literature needs to be read, understood and cited. Examples of errors in section 3 include: primary amines are not halides/pseudohalides and are not oxidised to hypohalous acids by MPO; the reaction of HOCl and HOBr with amines generates chloramines and bromamines, but they are not reactive nitrogen species (line 119/120) ; Compound I is part of both the halogenation and the peroxidation cycles; Compound I gains one electron to become compound II (not loses two); only compound I reacts with halides, not compound II; glutathione is not a protein; and glutathione sulfonamide is abbreviated to GSA not GSSG. In the abstract and introduction the authors also wrote “formation/production of chlorinating oxidants, such as HOCl”, when HOCl is the only chlorinating oxidant that MPO produces, but it also produces oxidants such as HOSCN and HOBr that are not chlorinating oxidants. In section 10 they write that ABAH is a potent inhibitor of peroxidation, but it also inhibits chlorination.

The feature of the review that I found the most interesting was description of the “non-enzymatic functions of MPO”. This is obviously an important consideration when developing inhibitors that only focus on targeting enzyme activity. It would be valuable for the authors to describe how others have determined enzyme activity is not important when assessing function. For example in section 5 and 6 (refs 61, 65, 99, 100) was inactivated MPO used to prove this? MPO binding may be important for its function, but it may also have to be catalytically active.

 

Author Response

It does, however, contain disturbing factual errors with regards the enzymatic properties of MPO. This is obviously outside the area of expertise of the authors, but it is important to get correct, and additional literature needs to be read, understood and cited. Examples of errors in section 3 include: primary amines are not halides/pseudohalides and are not oxidised to hypohalous acids by MPO; the reaction of HOCl and HOBr with amines generates chloramines and bromamines, but they are not reactive nitrogen species (line 119/120) ; Compound I is part of both the halogenation and the peroxidation cycles; Compound I gains one electron to become compound II (not loses two); only compound I reacts with halides, not compound II; glutathione is not a protein; and glutathione sulfonamide is abbreviated to GSA not GSSG.

Re: Thank you for your appreciation of our manuscript and constructive criticism. We regret our oversight over these factual errors and clumsy sentences, indeed these are embarrassing. We have thoroughly revised Section 3.

In the abstract and introduction the authors also wrote “formation/production of chlorinating oxidants, such as HOCl”, when HOCl is the only chlorinating oxidant that MPO produces, but it also produces oxidants such as HOSCN and HOBr that are not chlorinating oxidants.

Re: You are right. The Abstract has been corrected accordingly.

In section 10 they write that ABAH is a potent inhibitor of peroxidation, but it also inhibits chlorination.

Re: We agree, and corrected the statement on ABAH.

The feature of the review that I found the most interesting was description of the “non-enzymatic functions of MPO”. This is obviously an important consideration when developing inhibitors that only focus on targeting enzyme activity. It would be valuable for the authors to describe how others have determined enzyme activity is not important when assessing function. For example in section 5 and 6 (refs 61, 65, 99, 100) was inactivated MPO used to prove this? MPO binding may be important for its function, but it may also have to be catalytically active.

Re: We appreciate raising this critical issue. Indeed, previous studies utilized MPO inactivated with 4-ABAH or 3-amino-1,2,4-triazol to show that MPO-evoked neutrophil responses do not require catalytic activity (refs. 61, 65). By contrast, the pro-apoptotic action of MPO on HL-60 cells were partially attenuated by blocking enzymatic activities (101, 102). These differences likely reflect differences in response of primary and leukemic cells to MPO, and are discussed in the revised Section 6.   

Reviewer 3 Report

Multifaceted pro-inflammatory and protective functions of the heme protein myeloperoxidase, the most abundant protein in neutrophils, have extensively been reviewed. The focus is directed on non-enzymatic functions of this protein. Myeloperoxidase inhibitors are also reviewed. Except section 3, the review is well written and informative.

A schematic overview about multiple functions of myeloperoxidase is given in Figure 1. In this figure, pro-inflammatory activities of MPO are given in red, and protective functions in green. In the legend, no link is given to this color selection. It remains unknown, why the authors selected NO, Cl^-, and O^- (this species does not exist!) as precursors for reactive species. Moreover, in this figure several abbreviations are contained that are not specified in the legend.

Section 3 (enzymatic properties) needs to be revised or better newly written as several wrong statements are given. For example, Compound II of MPO is unknown to oxidize any (pseudo)halides. NO₂^- is not a (pseudo)halide. The formation of Compound I from resting MPO is known as the first reaction in both the halogenation and peroxidase cycles of MPO. GSSG is not glutathione sulfonamide.

Author Response

Except section 3, the review is well written and informative.

Re: Thank you for your evaluation and criticism.

A schematic overview about multiple functions of myeloperoxidase is given in Figure 1. In this figure, pro-inflammatory activities of MPO are given in red, and protective functions in green. In the legend, no link is given to this color selection. It remains unknown, why the authors selected NO, Cl^-, and O^- (this species does not exist!) as precursors for reactive species. Moreover, in this figure several abbreviations are contained that are not specified in the legend.

Re: We have revised Figure 1 and replaced NO, Cl^- or superoxide (that was erroneously labeled as O^-) with ROS. The legend has also been modified to indicate the functions of red and green colors on the figure as well as to complete the list abbreviations.

Section 3 (enzymatic properties) needs to be revised or better newly written as several wrong statements are given. For example, Compound II of MPO is unknown to oxidize any (pseudo)halides. NO₂^- is not a (pseudo)halide. The formation of Compound I from resting MPO is known as the first reaction in both the halogenation and peroxidase cycles of MPO. GSSG is not glutathione sulfonamide.

Re: We accept your criticism and apologizes for the clumsy description and incorrect statements. We hope that the rewritten section correctly present both the halogenation and peroxidation cycles of MPO and provide a better support for the discussion of MPO inhibitors.

Round 2

Reviewer 1 Report

I thank the authors for their responses and amendments.  I have no further comments.

Reviewer 2 Report

The revised version addresses my original concerns.

Reviewer 3 Report

Section 3 is now much better written. I have no further amendments.