Insights for Future Pharmacology: Exploring Phytochemicals as Potential Inhibitors Targeting SARS-CoV-2 Papain-like Protease

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors did a very significant job performing a detailed literature review to search for phytochemicals (plant secondary metabolites) that exhibit potential SARS-CoV-2 Papain-like protease inhibitory activity. They evaluated the activity of about 100 phytochemicals and showed that only 6 of them exhibit particularly potent biological activity, being potential candidates as drugs to combat COVID-19. The analysis of the manuscript’s content also reveals a weakness of the cited studies in that only for a small number of phytochemicals are the results of in-vivo studies known, and only on the basis of these studies will it be possible to assess the efficacy of phytochemicals in medical practice. The paper is eligible for publication after the clarification of several issues indicated below.

Lines 547-559, the meaning of the term the selectivity index (SI) is explained, but no SI values are given for the phytochemicals examined. If SI values are not known for all the secondary metabolites described, perhaps they are known for phytochemicals categorized as good inhibitors/lead compounds?

Table 1: Since the sixth column (In-vitro/Ex-vivo/In vivo ...), lacks any data for all rows, this column should be removed from Table 1.

Tables 1-4, Columns three and four in all Tables should have the names “Plant Scientific Name” and “Plant Family”. Also, why in the last column of all Tables, all cited publications are in semicircular brackets?

References – titles of journals are given in different ways (abbreviations or full names) - this should be standardized. Also, for refs. 32 and 70 the correct bibliographic data should be given.

Author Response

ID: FCP-OA-07-24-0261.

Title

Insights for Future Pharmacology: Exploring Phytochemicals as Potential Inhibitors Targeting SARS-CoV-2 Papain-like Protease

 

Reviewer 1

 

Comment 1: Lines 547-559, the meaning of the term the selectivity index (SI) is explained, but no SI values are given for the phytochemicals examined. If SI values are not known for all the secondary metabolites described, perhaps they are known for phytochemicals categorized as good inhibitors/lead compounds?

Response 1: Thank you for pointing this out. The SI represents a ratio of CC₅₀ (50% cytotoxic concentration) to EC₅₀(50% effective concentration). It is an in-vitro test measure. Only in-silico studies have been reported on virtual inhibitors. Hence, SI can’t reported/calculated. We also didn’t report the SIs for weak PLpro inhibitors (IC50 > 20uM) since the review's objective was to report potential PLpro inhibitors with significant IC50 values or in-vivo performance. The SI status of the ‘modest inhibitors (IC50 10-20 uM)’ and good inhibitors (IC50 < 10) has been described in Tables 3 and 4, respectively.

Comment 2: Table 1: Since the sixth column (In-vitro/Ex-vivo/In vivo ...), lacks any data for all rows, this column should be removed from Table 1.

Response 2: Thank you for pointing this out. The column has been removed from Table 1.

 

Comment 3: Tables 1-4, Columns three and four in all Tables should have the names “Plant Scientific Name” and “Plant Family”. Also, why in the last column of all Tables, all cited publications are in semicircular brackets?

Response 3: Thank you. The column names have been changed for Tables 1-4. The semicircular brackets were not added with a specific purpose and have been removed in the revised manuscript.

 

Comment 4: References – titles of journals are given in different ways (abbreviations or full names) - this should be standardized. Also, for refs. 32 and 70 the correct bibliographic data should be given.

Response 4: Thank you. We’ve updated the entire reference section according to the journal’s guidelines, including references 32 (Goswami et al. 2020) and 70 (Montone et al. 2021).

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript "Insights for Future Pharmacology: Exploring Phytochemicals as Potential Inhibitors Targeting SARS-CoV-2 Papain-like Protease" presents relevant data on potential inhibitors targeting the SARS-CoV-2 papain-like protease. However, the authors need to address the following aspects:

1.     What is the specific molecular structure of the phytochemicals identified as potential inhibitors of SARS-CoV-2 PLpro and how does it relate to their inhibitory activity?

2.     Which in vivo studies have evaluated the efficacy of the identified phytochemicals in animal models to validate their therapeutic potential against COVID-19?

3.     What are the underlying molecular mechanisms by which phytochemicals such as schaftoside, tanshinones, hypericin, and methyl 3,4-dihydroxybenzoate (HE9) exert their inhibitory effects on PLpro, and how can these mechanisms be leveraged for the rational design of new inhibitors?

4.     How do the pharmacological profiles of the identified phytochemicals compare with those of currently approved antiviral treatments in terms of efficacy, selectivity, and toxicity

5.     What are the most relevant methodological limitations in in vitro and in vivo studies that could affect the extrapolation of results to clinical practice?

6.     What strategies could be employed to enhance the bioaccessibility and bioavailability of the identified phytochemicals, thereby increasing their therapeutic effectiveness against SARS-CoV-2?

7.     What is the potential for viral resistance to the identified phytochemicals as PLpro inhibitors, and how could potential resistance mechanisms be mitigated?

8.        What evidence exists regarding the interaction of the phytochemicals with other complementary therapies or concomitant medications used in the treatment of COVID-19?

Author Response

ID: FCP-OA-07-24-0261.

Title

Insights for Future Pharmacology: Exploring Phytochemicals as Potential Inhibitors Targeting SARS-CoV-2 Papain-like Protease

 

Response to Reviewer 2

 

Comment 1: What is the specific molecular structure of the phytochemicals identified as potential inhibitors of SARS-CoV-2 PLpro, and how does it relate to their inhibitory activity?

Response 1: Thank you for the comment. We added the chemical structures of the six compounds identified as potential inhibitors. For the rest of the phytochemicals, we have added a new column in all tables to show the PubChem CID of the compounds presented in the study that can be used as a reference to the specific molecular structure of the compounds. Regarding the structure-activity relationships, we have reviewed the reported studies and added information on the structure-activity relationships where provided. Some examples are given below:

Lines 150-157: Biflavones with C-O-C connections show better activity.

Lines 174-178: The aromatic system of glycyrrhizoflavone plays a critical role in its inhibitory activity.

Lines 227-236: Discussion about the structure-activity relationship of tanshinones

Lines 280-283: Role of -OH groups in the inhibitory activity of AGP3

Lines 301-304: α-methylene-γ-lactone (aMyL) ring involvement in the inhibitory activity of sesquiterpene lactone (STLs).

Lines 425-428: Role of C6-glucosyl and C8-arabinosyl moieties of Schaftoside in inhibiting SARS-CoV-2 PLpro.

Comment 2: Which in vivo studies have evaluated the efficacy of the identified phytochemicals in animal models to validate their therapeutic potential against COVID-19?

Response 2: Thank you. We have reported this limitation about the inadequate in-vivo evidence (animal models and clinical studies) regarding the reported phytochemicals in the manuscript on several occasions (Lines 25-26, Lines 562-565, 569-572, 637-639). However, the authors have endeavored to provide evidence from clinical studies, where available, to present data on the therapeutic potential of phytochemicals against COVID-19.

Lines 394-395: Information on clinical trials of Hesperidin for COVID-19 treatment.

Lines 602-604: The use of tanshinones in Chinese patent “Xuebijing” for the treatment of COVID-19.

Comment 3: What are the underlying molecular mechanisms by which phytochemicals such as schaftoside, tanshinones, hypericin, and methyl 3,4-dihydroxybenzoate (HE9) exert their inhibitory effects on PLpro, and how can these mechanisms be leveraged for the rational design of new inhibitors?

Response 3: We have added additional information in the revised manuscript about the detailed molecular mechanisms of inhibition of the mentioned phytochemicals (please refer to the lines given below). Moreover, we have provided details on the specific moieties involved in making those interactions which can be used to formulate/improve new/existing inhibitors.

Schaftoside: Lines 425-428

Tanshinones: Lines 219-220; 227-236

Hypericin: Line 523 onwards

HE9: Lines 627-629

Comment 4: How do the pharmacological profiles of the identified phytochemicals compare with those of currently approved antiviral treatments in terms of efficacy, selectivity, and toxicity?

Response 4: Thank you for the comment. Most of the phytochemicals (70%) reported in the review are derived from in-silico approaches, and the rest are from in-vitro experiments. Therefore, at this stage, it is too early to compare the efficacy of the reported phytochemicals against the approved treatments. However, we expect some good results of the reported phytochemicals, particularly the ‘good inhibitors’, in clinical studies based on this initial data.

In addition, we have reported the results of positive controls for all phytochemicals to make a comparison of the in-vitro/in-silico efficacy of the reported phytochemicals. These positive controls were obtained from the respective studies of the phytochemicals and include established anti-viral drugs such as Remdesivir, Ivermectin, Chloroquine, Darunavir, Lopinavir, Disulfiram, Procainamide, and a number of other PLpro inhibitors such as GRL-0617.

Comment 5: What are the most relevant methodological limitations in in vitro and in vivo studies that could affect the extrapolation of results to clinical practice?

Response 5: Thank you. We have addressed these limitations in the revised manuscript in the conclusion and perspective section (Lines 645-654).

Comment 6: What strategies could be employed to enhance the bioaccessibility and bioavailability of the identified phytochemicals, thereby increasing their therapeutic effectiveness against SARS-CoV-2?

Response 6: While several strategies exist to improve the pharmacokinetic profiles of the identified compounds during the development stage, currently, there’s limited knowledge about the ADME data of these phytochemicals in in-vivo models. Therefore, we can’t be sure if the phytochemicals need improvement in their bioaccessibility or bioavailability.

Comment 7: What is the potential for viral resistance to the identified phytochemicals as PLpro inhibitors, and how could potential resistance mechanisms be mitigated?

Response 7: Thank you. We agree that viral resistance is a real phenomenon, and the reason for conducting this review was the emerging resistance of approved treatments against the novel variants of SARS-CoV-2. To the best of our knowledge, no study reported in this review shared insights on the resistance potential of the phytochemicals. Our introduction highlighted that the currently approved treatments do not contain a single PLpro inhibitor. Moreover, of the approved treatments, almost five candidates became resistant, whose mechanisms were related to spike protein. Therefore, we propose these phytochemicals as inhibitors of an underexplored target and urge the scientific community to perform clinical experiments. Only then can we determine the potential and extent of resistance.

Comment 8: What evidence exists regarding the interaction of the phytochemicals with other complementary therapies or concomitant medications used in the treatment of COVID-19?

Response 8: Thank you for the comment. We have added additional information on the evidence regarding phytochemicals in treating COVID-19 (Lines 93-114). While several studies have reported the concomitant use of phytochemicals with complementary therapies or other treatments, we have only discussed a few of them to support our argument.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is very well-written and provides a wealth of information for the readers. However, I have one concern that needs to be addressed before publication. In Figure 1, the author refers to "virtual inhibitors." What does this term mean? If these inhibitors were identified through an in silico study, the authors should provide a detailed description of the source and relevant information.

Comments on the Quality of English Language

Minor English revision. 

Author Response

ID: FCP-OA-07-24-0261.

Title

Insights for Future Pharmacology: Exploring Phytochemicals as Potential Inhibitors Targeting SARS-CoV-2 Papain-like Protease

 

Response to Reviewer 3

 

Comment 1: The manuscript is very well-written and provides a wealth of information for the readers. However, I have one concern that needs to be addressed before publication. In Figure 1, the author refers to "virtual inhibitors." What does this term mean? If these inhibitors were identified through an in-silico study, the authors should provide a detailed description of the source and relevant information.

Response 1: Thank you for the comment. The term ‘virtual inhibitors’ indeed refers to inhibitors identified in in-silico studies. The term has been explained in the methodology (section 2.3, lines 118-119). Moreover, a detailed description of the source and the relevant information for these inhibitors is provided in Table 1 of the manuscript.  

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of manuscript “Insights for Future Pharmacology: Exploring Phytochemicals as Potential Inhibitors Targeting SARS-CoV-2 Papain-like Protease” have argued and responded to each of the questions raised. The manuscript in its current form can be published on the journal's platform.