Advancements in Serine Protease Inhibitors: From Mechanistic Insights to Clinical Applications
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsSerine proteases are validated drug targets in several pathologies and physiological processes. Using molecules that inhibit these protases is a valuable strategy to regulate the serine protease activity. The manuscript titled Advancements in Serine Protease Inhibitors: From Mechanistic 2 Insights to Clinical Applications summarizes the serine protease inhibitors in clinical trials and describes their protease targets and clinical applications. The authors make a detailed description of the molecules, and the challenges encountered during their development as drugs. They also propose some general strategies to overcome the difficulties in the development of drugs based on these inhibitors. I consider this a valuable contribution to the efforts to develop new protease inhibitors to be used in clinical treatment and diagnosis.
However, I would like to suggest some changes to improve the quality of this manuscript.
I suggest improving the quality of Figures 3 and 4.
In section 2 “Mechanisms and Classification of Serine Protease Inhibitors”, the authors describe three mechanisms of inhibition of serine proteases. Two of these mechanisms classify serine protease inhibitors as classical and non-canonical while the third is based on targeting the autolysis loop with allosteric inhibitors. However, classical (canonical) or non-canonical classification is used mainly with peptide, proteinaceous, or peptidomimetic inhibitors, not small molecule inhibitors. Some small molecules could use these mechanisms, but I suspect that some others do not.
What is the meaning of peptide inhibitors in the document? Sometimes they use the term for peptide molecules (less than 50 amino acids, eg: Leupeptin) and sometimes for polypeptides (Kunitz inhibitors).
Concerning Kunitz inhibitors, there is an error with their classification. Kunitz serine protease inhibitors are not a family of proteins secreted by various plants and animals. There are two families of Kunitz inhibitors: one from animals (one described in section 3.2) and another from plants (mentioned in lines 560-566). Both families have completely different folds, sizes, and binding modes, although some inhibitors of these families use the same canonical inhibition mechanism to inhibit serine proteases. I suggest modifying this section.
The authors mentioned (lines 276-278) that aprotinin can inhibit proteolytic activation of SARS-CoV-2 but both proteases present in this virus are cysteine proteases. Is there any evidence that aprotinin can inhibit these or other cysteine proteases?
In section 3.3 a different kind of protease inhibitors is introduced because they are not enzymatic inhibitors at least not in the classical or most common definition. The nucleotide drugs described act by blocking or inhibiting the expression of the proteases and not inhibiting the enzyme activity. This is quite interesting, but I was surprised by their inclusion as protease inhibitors because their mechanism of action was not included in Section 2 (Mechanisms and Classification of Serine Protease Inhibitors).
The title of section 4 referred to biomacromolecular inhibitors as endogenous protein inhibitors and antibodies but some of the peptide inhibitors mentioned in previous sections are also biomacromolecular inhibitors. Besides, it is written in a way that suggests SPINK1 is a serpin inhibitor when it belongs to the Kazal family, previously described in another section of the manuscript. I recommend, checking and correcting these issues.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsSerine protease inhibitors represent a significant class of proteins that, through the regulation of serine proteases, play an important role in maintaining diverse physiological processes. This has facilitated their implementation in a multitude of fields, including biochemistry, medicine, and pharmacology. These inhibitors are indispensable for both natural physiological regulation and therapeutic applications. Serine proteases and their inhibitors play a key role in numerous immune and inflammatory processes, including T- and B-cell differentiation, cytokine and complement activation, and the repair of inflammatory tissues. In recent years, there has been a notable increase in the number of studies examining protease dysregulation in gastrointestinal diseases, which has led to protease inhibition becoming one of the most promising new therapeutic strategies. As research progresses, the potential of these inhibitors in the treatment of various diseases continues to expand, underscoring their importance for modern science and medicine. In light of the aforementioned considerations, a review of serine protease inhibitors, their medical applications, and the challenges encountered along this path, along with strategies for overcoming them, is both interesting and pertinent. The following comments and suggestions for the authors are offered for their consideration:
Lines 50-51, 295 – It would be beneficial to explain what is meant by "the chymotrypsin symbol for thrombin"? Does this imply that it has chymotrypsin activity?
Line 263 – It would be beneficial to include serine protease inhibitors of the Bowman–Birk family, which are more resistant than Kunitz inhibitors and are also used in the treatment of malignant neoplasms
Lines 453-471 – It would be more appropriate to relocate this paragraph from the section "4.2. Antibody", to which it has nothing to do, to the sections corresponding to the inhibitors in question
Furthermore, the authors should consider in the discussion the possibility that some inhibitors may act on proteases that perform multiple functions. By modulating the activity of these proteases, you can improve the situation (physiological state of the body) in one area, but at the same time lose something important for the body in another.
It would be very clear if the authors included a table that lists the serine protease inhibitors used, the limitations of their use, and potential strategies to overcome these constraints.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsI have no further comments or suggestions about the revised version of the manuscript.
