Pseudokinases: From Allosteric Regulation of Catalytic Domains and the Formation of Macromolecular Assemblies to Emerging Drug Targets

Round 1

Reviewer 1 Report

The manuscript describes the existence of Pseudokinases, structural comparison of pseudokinases with bona fide kinases and the protein-protein interactions between pseudokinases with bona fide kinases. Authors describe different types of protein-protein interactions and the analysis of their interactions between different family classes for identification of pseudokinases as suitable drug targets. The topic is interesting but several review articles have been published in the past couple of years. The authors have failed to explain what makes the manuscript sufficiently novel which could not be found in the other reviews. The interesting part of the manuscript is "Pseudokinases protein-protein interaction analysis", however, this is not sufficient enough to suggest any novel approaches or methods for selection of Pseudokinases as potential drug targets. I encourage the authors to resubmit the review with more focus on this topic demonstrating this as a new application for drug target identification.

The manuscript is not suitable for publication in present form due to the lack of the novelty.         

Author Response

Our response: we thank the reviewer for the feedback. We acknowledge that recent reviews published by others have discussed pseudokinases. We are pleased the reviewer found our discussion about "Pseudokinases protein-protein interaction analysis" interesting. We agreed that such analysis per se is not sufficient to suggest any novel approaches or methods for selection of Pseudokinases as potential drug targets. However, our point was to bring to the attention of the reader that interfering with specific protein-protein interactions has been demonstrated to be an effective strategy to target diverse signalling systems with small size drugs. The fact that pseudokinases function often involves their physical interaction with other proteins, in principle represents an opportunity for the design of drugs that target specific pseudokinase protein-protein interfaces. We have rephrased sentences in the sections protein-protein interaction and extended our discussion about current trends in drug discovery. In an attempt to make our point clearer, we have included recent reports by others confirming the druggability of pseudokinases and the promise pseudokinases represent as suitable drug targets for the treatment of human malignancies of poor prognosis

Reviewer 2 Report

In this review authors have presented an overview of the Pseudokinases and its role in cellular signaling. Further, the dysregulation of Pseudokinases and its associated role in various human diseases including cancer is also addressed. This review is well organized, well written and the selected case studies are discussed in detail. This current review may enhance the understanding of role of Pseudokinases as target for treatment of various diseases. This may be helpful in the field of drug development. Each section of this review is clearly described and discussed in detail, including types of pseudokinases and mechanism of action. Line 27: period is missing

Author Response

we thank the reviewer for bringing this error to our attention and for the revision of our manuscript.

Reviewer 3 Report

This relatively short review by Tomoni, Bastida and associates is a primer from several groups not very well know currently for their work on pseudokinases, and they cover most of the key points found in other reviews in this area. It enters a very crowded field of studies and recent reviews, and suffers due to poor citation of the recent field and some repetition (e.g. mechanisms of action of pseudokinases well studied and reviewed), mixed with a lack of focus. What is positive, is that there are some newish sections towards the end on potential drugs and binding partners, which is certainly worthy of review. Prior to publication, the major movements in the field that need to be included to bring the review to the relevant level for dissemination is: 

Major points:

i) the recognition that pseudokinases are part of a much bigger field of pseudoenzymes, and I recommend that the authors include a small section on this at the beginning, stating that pseudokinases are not unique amongst enzyme families but rather part of the bigger pseudoenzyme superfamily, and several reviews are available to highlight this (e.g. PMID: 31409758) ii) pseudokinases are not restricted to multicellular organisms (the recent resource from Kannan and colleagues demonstrates the presence of pseudokinases across life, and this reference is missing PMID: 31015289) and the earlier work of Manning in Giardia (PMID 21787419) and new studies of Eph receptors (PMID: 31406248) need to be included and briefly discussed. 

The drug targetting section should include a brief discussion of some of the new work with Tribbles pseudokinases, building on the cited Kung review and explaining how unique conformations are targetable with drugs, mention that TRIB1 R107L mutation is linked to Down Syndrome (PMID:22294728) and a mention of the new generation of TYK2 pseudokinase ligands (eg. PMID: 31341059 and 31314518). The section on subcellular localisation should also mention TRIB1 and the new work from Jura and colleagues looking at COP1 nuclear export (PMID: 30692133)

Minor points:

Drosophila BubR1 has recently been described to possess catalytic activity as a CENP-E kinase, and this might also be mentioned in the text as an example of the challenges in the field, and to compare with human BubR1 PMID: 31201382).

WNKs are also linked to the regulation of blood pressure through regulation of SPAK, but this was not mentioned, and WNK inhibitors such as WNK463 might be antihypertensives, which should be cited (e.g. 27595330) 

 

Spelling:

 

Figure 1 is the general kinase fold, rather than a pseudokinase in particular. 

 

Figure 2. Is this human or murine MLKL?

 

Line 143: POMK is a sugar kinase, and not a protein pseudokinase

 

Line 265: their, not there

 

Figure 6: Anchor is mis-spelled as Ancor in the diagram and also in the legend.

 

Author Response

Our response: we thank the reviewer for the thorough revision of our manuscript and for the constructive feedback. We address the reviewer comments as follows:

“This relatively short review by Tomoni, Bastida and associates is a primer from several groups not very well know currently for their work on pseudokinases, and they cover most of the key points found in other reviews in this area”.

Our response: we respectfully disagree with the reviewer about our lack of experience in the study of pseudokinases. For example, one of us has a track record in the study of BubR1, which in humans function as a pseudokinase (please see the following publications for details: PMID: 25482201; PMID: 25964054; PMID: 22000412 and PMID: 20220147) and another on the development of computational approaches to predict protein function and for protein classsification into families and superfamilies, which includes pseudokinases (please see the following contributions for details: PMID: 26139634; PMID: 25964299;  PMID: 24943848;

“It enters a very crowded field of studies and recent reviews, and suffers due to poor citation of the recent field and some repetition (e.g. mechanisms of action of pseudokinases well studied and reviewed), mixed with a lack of focus”.

Our response: we agree the wording throughout the manuscript could be improved and that important, recent reports and reviews by others were not included, for which we apologise. We have amended this as follows:

 “What is positive, is that there are some newish sections towards the end on potential drugs and binding partners, which is certainly worthy of review”.

Our response: we agree and have extended the discussion on this important aspect of pseudokinases and the promise they represent for the treatment of human diseases.

Prior to publication, the major movements in the field that need to be included to bring the review to the relevant level for dissemination is: 

Major points:

the recognition that pseudokinases are part of a much bigger field of pseudoenzymes, and I recommend that the authors include a small section on this at the beginning, stating that pseudokinases are not unique amongst enzyme families but rather part of the bigger pseudoenzyme superfamily, and several reviews are available to highlight this (e.g. PMID: 31409758) ii) pseudokinases are not restricted to multicellular organisms (the recent resource from Kannan and colleagues demonstrates the presence of pseudokinases across life, and this reference is missing PMID: 31015289) and the earlier work of Manning in Giardia (PMID 21787419) and new studies of Eph receptors (PMID: 31406248) need to be included and briefly discussed.

Our response: we agree. A small section that aims to provide a broader perspective of pseudokinases as part of the larger class of pseudoenzymes has been included at the beginning.

The drug targeting section should include a brief discussion of some of the new work with Tribbles pseudokinases, building on the cited Kung review and explaining how unique conformations are targetable with drugs, mention that TRIB1 R107L mutation is linked to Down Syndrome (PMID:22294728) and a mention of the new generation of TYK2 pseudokinase ligands (eg. PMID: 31341059 and 31314518). The section on subcellular localisation should also mention TRIB1 and the new work from Jura and colleagues looking at COP1 nuclear export (PMID: 30692133)

Our response: we agree and we thank the reviewer for bringing this to our attention. A discussion of the advances reported in these publications and others has now been included.

 

Minor points:

Drosophila BubR1 has recently been described to possess catalytic activity as a CENP-E kinase, and this might also be mentioned in the text as an example of the challenges in the field, and to compare with human BubR1 PMID: 31201382).

Our response: we agree. An extended commentary about this interesting pseudokinase has been included in the revised manuscript.

WNKs are also linked to the regulation of blood pressure through regulation of SPAK, but this was not mentioned, and WNK inhibitors such as WNK463 might be antihypertensives, which should be cited (e.g. 27595330) 

Our response: we agree. This work is now cited.

 

Spelling:

 Figure 1 is the general kinase fold, rather than a pseudokinase in particular. 

 Figure 2. Is this human or murine MLKL?

 Line 143: POMK is a sugar kinase, and not a protein pseudokinase

 Line 265: their, not there

 Figure 6: Anchor is mis-spelled as Ancor in the diagram and also in the legend.

Our response: we agree with all the spelling comments and the commentary about the kinase PMOK. Any mention to this sugar kinase has been removed from the manuscript. All the typos, grammar and clarification issues listed above have now been amended.

Round 2

Reviewer 1 Report

The authors have given due consideration to the suggestions and made significant changes in the manuscript accordingly. The changes are satisfactory. The manuscript is suitable for the publication in the present form.