In silico screening for potential inhibitors from the phytocompounds of Carica papaya against Zika virus NS5 protein

Background The Zika virus (ZIKV) infection has emerged as a global health threat. The causal reasoning is that Zika infection is linked to the development of microcephaly in newborns and Guillain-Barré syndrome in adults. With no clinically approved antiviral treatment for ZIKV, the need for the development of potential inhibitors against the virus is essential. In this study, we aimed to screen phytochemicals from papaya ( Carica papaya L.) against NS5 protein domains of ZIKV. Methods Approximately 193 phytochemicals from an online database (IMPACT) were subjected to molecular docking using AutoDock Vina against the NS5-MTase protein domain (5WXB) and -RdRp domain (5U04). Results Our results showed that β-sitosterol, carpaine, violaxanthin, pseudocarpaine, Δ7-avenasterols, Rutin, and cis-β-carotene had the highest binding affinity to both protein domains, with β-sitosterol having the most favorable binding energy. Furthermore, ADMET analysis revealed that selected compounds had good pharmacokinetic properties and were nontoxic. Conclusions Our findings suggest that papaya-derived phytochemicals could be potential candidates for developing antiviral drugs against ZIKV. However, further experimental studies using cell lines and in vivo models are needed to validate their efficacy and safety.


Introduction
Zika virus (ZIKV), belonging to the Flaviviridae family, is a mosquito-transmitted virus that infects humans by biting Aedes mosquitos (Aedes aegypti). 1Though the ZIKV was first reported in 1947 in Uganda, the severity of this virus was globally noticed during its outbreak in the years 2015-2017 in Brazil, and later the infection spread to 46 other countries. 2,35][6] Even though the pandemic waves have subsided, sporadic detections of Zika infections continue to be reported in several parts of the world, with the virus becoming endemic to those regions. 7,8Henceforth, continuous surveillance and research are essential to develop an effective treatment.
ZIKV is an enveloped virus characterized by the presence of a single-stranded RNA genome. 5,9The genome of ZIKV encodes a single polyprotein (~3400 amino acids), which is translated to three structural proteins (capsid-C, premembrane/membrane-prM, and envelope-E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) using host and viral proteases (Figure 1). 10 Despite the significant efforts of the scientific community, Figure 1.The surface, structural, and non-structural proteins of the ZIKV are illustrated in the diagram, which highlights the virion components and the genomic RNA.The ZIKV polyprotein is composed of seven nonstructural proteins (NS1, NS2A, NS2B, NS3 protease and helicase domains, NS4A, NS4B, NS5 methyltransferase, and RNA polymerase domains) and three structural proteins (C, M, and E).In addition, NS5 methyltransferase and RNA polymerase domains' structures were retrieved from PDB (Protein Data Bank) databases.

REVISED Amendments from Version 1
We are grateful for the reviewers' comments that helped us improve the second version of the manuscript.
The changes done to this version are mentioned below: We expanded the introduction to provide a comprehensive overview of Carica papaya.Recent prevalence and incidence data on Zika virus (ZIKV) infections from 2022 and 2023 were included, and references for control inhibitors were corrected and updated.The manuscript was thoroughly reviewed for grammatical and syntax errors, and abbreviations were consistently applied throughout."Zika" was used uniformly throughout the manuscript for consistency.Higher-quality images replaced low-resolution ones, and figure captions were updated for better visualization.The conclusion highlights novel findings, impacts, and study limitations, such as the need for further experimental validation.Additionally, a typing error in the PDB structure for NS5-RdRp was corrected.The manuscript has been thoroughly checked, and the reviewer's comments have significantly improved it.
there is currently no specific therapy available for treating ZIKV infection, making developing such antivirals a critical priority. 11,12Antivirals that could target protein structures involved in genome replication, viral fusion, and RNA synthesis can be highly effective against the ZIKV.Among all the proteins expressed by ZIKV, the most significant and most conserved protein is the non-structural-5 or NS5, which is the polymerase enzyme; it consists of two major domains: RNA methyltransferase (MTase) at its N-terminus and RNA-dependent-RNA polymerase (RdRp) at its C-terminal. 13dRP is an essential protein domain for initial viral replication, whereas the MTase domain is responsible for RNA capping of the viral genome.The structure of NS5 is exclusive to ZIKV and has no similarity with the host system, which makes it a unique target for inhibitors against ZIKV. 14ntinued clinical research is necessary to discover new antivirals.8][19][20][21] Molecular modeling techniques are widely used to study the dynamics, energy, and interactions between biomolecules, including proteins.These techniques are used extensively to study protein-ligand interaction and to predict the drug's binding mode within the protein's binding site.[24][25] Henceforth, for this project, we have carried out in silico molecular docking analysis for potential anti-ZIKV compounds from Carica papaya (commonly referred to as papaya), an edible tropical fruit well-known for its many medicinal properties. 26C. papaya, belonging to the family Caricaceae, is a tropical fruit-bearing plant known for its rich content of vitamins, enzymes, and antioxidants. 27Papaya contains bioactive substances, including alkaloids, flavonoids, and phenolic acids, which have been reported to exhibit various pharmacological effects, including antioxidant, antiinflammatory, immunomodulatory, and antiviral activities. 27,28 papaya has demonstrated significant therapeutic potential and has been used in home remedies for centuries.29 In several Asian countries, its seeds and peels are utilized to treat stomach ailments, bacterial infections, and inflammations.Moreover, a few studies have reported its antiviral attributes and immunomodulatory properties, but limited research has explored the specific constituents of C. papaya that exhibit antiviral effects. Prious in silico studies have documented the potential therapeutic effects of papaya in various human diseases, [30][31][32] including an earlier study on identifying inhibitory compounds (e.g., luteolin) that targeted the dengue virus's NS2B/NS3 protease (DENV).33,34 Given that ZIKV and DENV are members of the same family, we proposed to virtually investigate small molecules from papaya with possible targeting ZIKV NS5 protein domains, and to the best of our knowledge, no prior studies have investigated this possibility.
The current study aims to conduct a virtual screening of bioactive molecules from papaya, followed by an ADMET (absorption, distribution, metabolism, elimination, and toxicity) assessment.Through molecular docking analysis using Autodoc Vina, we identified compounds that showed a promising binding affinity with the ZIKV's NS5 protein domains (MTase and RdRp).Thus, they constitute potential drug targets, and our results may contribute toward developing effective treatments against this public health priority.

Preparation of molecule database and ligand preparation
5][36][37][38] The ligands' 3-dimensional (3-D) structures were retrieved from the PubChem database.The ligands underwent a series of adjustments, such as the addition of polar hydrogens, adding charges, and conducting energy minimization using PyRx Virtual Screening Tool software (v-0.8)(RRID:SCR_018548) with the default parameters. 39

Receptor selection and preparation
The crystal structures of the ZIKV proteins were retrieved from the PDB (Protein Data Bank) database.These included the SAH-binding site of the NS5-MTase, and NS5 RNA-dependent RNA polymerase with their PDB entry 5WXB 40 and 5U04, 41,42 respectively.4][45] In accordance with standard protocol, the protein structures were treated as receptors.At the start of docking, the receptor protein was optimized by removing any unrelated substructure.Then, the side chains in the protein structure were corrected using default settings like adding hydrogens and removing water molecules.The Molprobity server evaluated selected proteins' stereo-chemical properties and Ramachandran graph. 46Chimera 1.16 (RRID SCR_004097) generated any residues missing in the selected target protein.After removing nonstandard heteroatoms, polar hydrogens and Gasteiger charge were added.Next, the structural aspects of all targets were enhanced using the steepest descent (100 steps) and conjugate gradient algorithms (0 steps) with an Amber force field (Amber Ff14SB). 46The energy-minimized proteins were then converted into 'pdbqt' format using AutoDock Tools 1.5.7 (RRID SCR_012746) by AutoDock.
Ligand and receptor molecular docking Docking was performed with Autodock Vina, 47,48 as described in a previous study. 49Briefly, the grid box's dimensions were fixed at XYZ = 30 Å Â 30 Å Â 30 Å XYZ = 30 Å Â 30 Å Â 30 Å which was found to be the best size for the default exhaustiveness (= 8), and the ligand binding site was positioned in the middle of the grid box.AutoDock Vina version 1.1.2(RRID:SCR_011958) was used to calculate each ligand's binding energy and pose against the selected protein receptors.Each ligand's best interaction energy scores (kcal/mol) were ranked and plotted against the reference inhibitor.The results obtained are limited to nine binding modes.The log file included a list with increasing binding energies and binding modes.The binding modes were viewed using the BIOVIA Discovery Studio visualizer -v21.1.0.20298 (Dassault Systemes BIOVIA, Discovery Studio, 2021, SanDiego). 50MET and drug-likeness evaluation The compounds' molecular properties and drug-like characteristics were assessed using "Lipinski's Rule of Five" as the basis of analysis. 51First, 19 phytocompounds were analyzed regarding their physicochemical properties, drug-likeness, toxicity, and ADMET properties using ADMETlab 2.0 and SwissADME.In addition, the physicochemical features of compounds, including lipophilicity (log P), solubility (log S), and polar surface area and volume (PSA), were predicted.The mentioned parameters are necessary as they influence how a drug interacts with transport proteins and enzymes involved in drug clearance.

Results molecular docking
For the molecular docking analysis, around 193 compounds found in papaya were docked against the two domains of ZIKV protein NS5 MTase (5WXB) and RdRp (5U04). 35As positive ligands, Sinefungin against the NS5-MTase and Sofosbuvir against the NS5-RdRp were docked.Sinefungin had a binding affinity of -8.1 kcal/mol, whereas the binding affinity for Sofosbuvir was -7.4 kcal/mol.Binding affinity is an essential preliminary parameter for assessing a potential candidate drug.Therefore, first of all, we assessed the binding affinity of the candidate drug and compared it with the positive controls (Sinefungin and Sofosbuvir against 5WXB and 5U04, respectively).The initial docking analysis helped narrow it down to 19 shortlisted compounds that showed higher affinity than their respective positive ligands, as shown in Figure 2. Figure 3A shows the heat map of the binding affinity of the ligands, with the lowest energy/highest affinity corresponding to red color, whereas the blue color indicates the highest binding energy or/lowest affinity.
Hydrogen bonds and hydrophobic interactions play a critical role in molecular docking, and these interactions are key components in identifying potential drugs.As mentioned in Table 1, rutin forms the highest number of hydrogen bonds involving 14 amino acid residues (ARG84, GLY86, LYS182, ARG213, CYS82, GLU218, GLU149, GLU149, GLY109, THR104, GLU111, HIS110, HIS110, LYS61) and the bonding distance ranging from 2.27 to 3.08 Å; however, rutin had no hydrophobic interaction with the MTase domain (Figure 4).In hydrophobic interactions, Δ7-Avenasterol and cis-β-Carotene form the highest number (5) bonds with the MTase domain.Ligands interaction with the RdRp domain of NS5 (Table 2, Figure 4) shows that rutin has formed the highest number of hydrogen bonds (ARG794, ARG794, THR795, THR796, GLY793, SER798) and three hydrophobic interactions.α-Carotene forms the highest number of hydrophobic interactions with the RdRp domain and has no hydrogen bonding.
Prediction of ADMET analysis ADMET and drug-likeness evaluation provide insight into the properties of drugs based on their chemical structure.The best-docked ligands (Figure 2) were analyzed for their pharmacokinetic properties.The evaluation criteria were based on solubility, gastrointestinal absorption (GI), blood-brain barrier (BBB) permeability, and violation of Lipinski's rules.
All the compounds mentioned in Figure 2 were screened through ADMET for their suitability as a drug.The screening through ADMETLab 2.0 revealed that several compounds were approved under Lipinski criteria.BBB values reflect the ability of the ligands to cross the brain barrier, with a higher score value indicating greater permeability.β-Sitosterol has the highest BBB penetration with a score of 0.84, suggesting effective crossing.Carpaine showed to have poor penetration with a BBB score of 0.01.BBB values ranged between 0.01 and 0.001, indicates poor barrier crossing.Riboflavin, rutin, and all-trans-neoxanthin displayed moderate BBB permeability values ranging from 0.111 to 0.444, while several had BBB penetrations below 0.1, suggesting the limited ability to cross the BBB (see the Underlying data, supplementary Table S2. 35ble S2 (Underlying data 35 also signifies the ligands' permeability based on Caco-2 cell permeability values, which translates to intestinal epithelium permeability.For example, β-sitosterol had the highest Caco-2 cell permeability value at -4.756, signifying improved intestinal permeability.On the other hand, carpaine showed poor intestinal epithelium permeability of -4.985.Table S2 (Underlying data 35 ) suggests ligands and their respective Lipinski's rule of five compliance.Among these ligands, β-sitosterol, violaxanthin, riboflavin, all-trans-neoxanthin, dehydrocarpaine-I, dehydrocarpaine-II, and β-carotene are Lipinski's-rule-of-five compliant and are considered to be drug-like compounds.

Discussion
Emerging viruses such as Dengue, Zika, Ebola, SARS-CoV2, and other infectious viruses demonstrate that the current antiviral therapeutic regimen is insufficient for these pathogens. 52The coronavirus 2019 (COVID-19) pandemic further highlighted this inadequacy.Vaccine development is a time-consuming and lengthy process, which also faces the challenges of large-scale administration.Moreover, the faster-evolving attribute of RNA viruses also makes it challenging to develop a particular antiviral treatment, 53 one such RNA virus, Zika, remains a global concern.Zikarelated disorders are mainly found in infants but can also affect adults.Zika-related disorders reported in adults were the cases of Guillain Barre' Syndrome, 54 Myelitis, 55 Meningoencephalitis, 56 and Uveitis. 57Currently, no approved drugs and no vaccines are available for treating ZIKV infection.
Plant-derived natural compounds are promising alternatives for treating infections with minimal side effects.Medicinal plants are the richest source of new drugs, including antivirals targeting several human ailments.Previous clinical studies have demonstrated the successful inhibition of Dengue infection by papaya extract. 58,59Phytochemical screening of papaya has been reported to constitute several compounds, which have potent therapeutic effects against several human diseases, such as inflammation, oxidative stress, antiviral and hypoglycemia. 60,61Since Zika and Dengue belong to the same family of viruses, we hypothesized that papaya could also serve as the source for identifying potential inhibitors against ZIKV infection.In this context, in the present study, we have performed molecular docking to identify possible lead compounds from papaya against NS5 protein domains of the ZIKV.
Initially, we screened 193 papaya-derived phytochemicals to know their molecular docking potential with the NS5 protein domains of the ZIKV.Our analysis revealed seven compounds (β-sitosterols, carpaine, violaxanthin, rutin, β-carotene, pseudocarpaine, and Δ7-avenasterol) that exhibited bispecific docking activity against both NS5 domains, with higher docking scores compared to their respective positive ligands (Sinefungin and Sofosbuvir against 5WXB and 5U04, respectively) (Figure 3A).The MTase activity of the ZIKV NS5 protein plays a crucial role in the replication and spread of the virus. 62Hence, inhibiting this activity can be an effective strategy to prevent the virus from spreading.Previous studies explored small molecule inhibitors and RNA-based inhibitors against ZIKV infections.For instance, Sinefungin is a small molecule that has been shown to inhibit the MTase activity of several flaviviruses. 63However, the study showed a low potency of Sinefungin on NS5 of ZIKV compared to other flaviviruses' MTase domain; additionally, the toxicity associated with the particular drug had raised concern about its use. 64drogen bonding and hydrophobic interactions are crucial in facilitating significant ligand binding at the active site residues of the receptor in docked complexes. 65In addition to hydrogen bonds and hydrophobic interactions, other noncovalent interactions such as alkyl-alkyl, pi-alkyl, and pi-pi interactions also play significant roles in ligand binding.Alkyl-alkyl interactions stabilize the complex via van der Waals forces, while pi-alkyl and pi-pi interactions enhance stability and binding affinity through aromatic ring stacking and hydrophobic effects.However, we primarily focused on hydrogen bonds and hydrophobic interactions due to their critical roles in ligand binding affinity and stability within the receptor's active site. 65,66The bioactive compounds in this study were found to form hydrogen bonds and hydrophobic interactions with MTase.In the present study, flavonoid compounds, like rutin, showed the highest binding affinity to the NS5-MTase domain (5WXB), followed by riboflavin.Rutin formed a complex that entirely occupied the protein through 14 hydrogen bonding interactions with no hydrophobic interactions, and riboflavin interacted with the NS5-MTase through nine hydrogen bonds (Table 1, Figure 4).Moreover, the interacting residues were identified as essential residues of the substrate binding of the MTase. 67The observed hydrogen bonding interactions between the selected compounds from papaya and MTase suggest that these compounds may effectively occupy the substrate binding site of MTase, making them promising lead compounds for drug development against ZIKV infection.Furthermore, the present study also indicates that phytocompounds from papaya showed several forms of strong and stable bonds like carbon-hydrogen, pi-alkyl, van der Waals, covalent, hydrophobic, and electrostatic bonds with the receptor ligands (Tables 1 and 2).
The ZIKV RdRp constitutes another important target for inhibiting Zika viral replication. 68ZIKV RdRp consists of two binding sites, the first one being the active site (formed by Asp535, Trp797, and Ile799), while the other side is the allosteric or N pocket, which contains the priming loop that is essential for stabilizing the initiation complex and releasing new dsRNA. 69In this study for docking to the RdRp domain (5U04), the result reveals that rutin, has the highest number of hydrogen bond interactions (with ARG794, ARG794, THR795, THR796, GLY793, SER798) and hydrophobic interactions (involving ARG794, MET806, ALA408 amino acids) compared to the positive ligand, Sofosbuvir (Table 2, Figure 4).
Phytosterols are naturally occurring plant molecules with a structure similar to cholesterol, 70 and they have been demonstrated to possess the antiviral activity of several sterols against the spike protein of the COVID-19 virus and influenza-A virus. 63,71Our study identified several phytosterols, including β-sitosterol and Δ7-avenasterol, demonstrating potent inhibitory affinity (Figure 3A).Earlier studies have also shown the potential health benefits, including antiviral effects of β-sitosterol, which has been shown to reduce the infectivity of the hepatitis-B virus and HIV, possibly by hindering the attachment of viruses to host cells. 72Another study on HIV patients found that the combination of β-sitosterol and β-sitosterol glycoside helped maintain stable CD4 cell counts and significantly reduced plasma viral loads. 735][76] Our docking studies identified alkaloids like carpaine and pseudocarpaine (Figure 3A) with higher binding affinity to both target protein domains compared to positive ligands.Besides, another alkaloid dehydrocarpaine-II had a good binding score (-8.8 kcal/mol) only towards the protein NS5-RdRp domain.Therefore, binding to the RdRp domain may help inhibit normal virus replication.Meanwhile, the carpaine-5U04 complex had no interaction with the protein through hydrogen or hydrophobic bonding.
Apart from it, several carotenoids had also shown good binding affinity to both domains of NS-5.Especially, carotenoids such as α-carotene, β-carotene, citroxanthin, β-cryptoxanthin, γ-carotene, violaxantin, and zeaxanthin showed higher binding affinity to the NS5-RdRp than NS5-MTase.1][82] Also, recent studies have proven the antiviral properties of carotenoids against COVID-19. 83nducting in silico analyses on compounds to assess their absorption, distribution, metabolism, and excretion (ADME) properties is critical as part of drug development. 84Failure to accurately simulate these attributes or assess any toxicities may cause inhibitors to fail the screening process and thus fall outside its criteria for approval. 85BBB penetration is important when developing drugs to treat central nervous system (CNS) conditions. 86Cytochrome P450 (CYP) isozymes metabolize drugs, fatty acids, steroids, bile acids, and carcinogens. 87Approximately 75% of phase-1 drug metabolism processes involve CYP enzymes. 88CYP inhibitor and substrate scores were calculated in this metabolism, and the result shows that the shortlisted compounds are non-substrates and non-inhibitors of CYP enzymes (Underlying data, supplementary Table S2 35 ).Our ADMET analysis (Underlying data, supplementary Table S2 35 ) demonstrated that 19 of the best-docked compounds (Figure 2) had nontoxic properties.Lipinski's rule of five was violated for ten compounds (Underlying data, supplementary Table S2 35 ).However, the remaining compounds (carpaine, dehydrocarpaine I, dehydrocarpaine II, pseudocarpaine, Δ7-avenasterol, all-trans-neoxanthin, riboflavin, β-sitosterol, and violaxanthin) were found to be acceptable candidates based on Lipinski's rule of five.
Zika virus infection remains a public health concern due to the lack of specific antiviral therapies.Our study investigated the potential of C. papaya extracts as a source of ZIKV inhibitors.In silico molecular docking identified seven compounds with favorable binding energies to both the MTase and RdRp domains of the ZIKV NS5.These findings suggest their potential as the inhibitors of viral replication.However, in silico methods require in vitro and in vivo validation to confirm their efficacy against ZIKV infection.Furthermore, these compounds' bioavailability and toxicity profiles need to be assessed to ensure their safety and effectiveness as potential drug candidates.These results offer a promising approach for developing natural, safe, and effective antiviral drugs against ZIKV, potentially filling the current therapeutic gap and contributing to global health efforts against the virus.

Luis Marquez-Dominguez
Instituto Mexicano del Seguro Social, Puebla, Mexico The quest for compounds exhibiting antiviral activity against diseases such as Zika is of paramount importance, given the absence of a definitive treatment for this ailment.However, I have several suggestions and reservations regarding the manuscript's content.Among the significant revisions required, it is observed that one of the receptors does not correspond to the Zika virus.Until the authors clarify this discrepancy, the integrity of the results, discussion, conclusions remains questionable.
1.In Figure 1, the depiction of structural and non-structural proteins aligns with the complete genome.Therefore, I recommend that the box should encompass the entire genome in the virus diagram, rather than just a segment of it.
2. Concerning the selection of receptors, the NS5 RdRp with PDB key 5UO4 corresponds to the Structure of human neuronal nitric oxide synthase heme domain in complex with 3-[(2-amino-4methylquinolin-7-yl)methoxy]-5-(2-(methylamino)ethyl) benzonitrile, and not to the NS5 RdRp of the Zika virus.This discrepancy is also evident in the supplementary data.Was all docking and figure rendering performed using the 5UO4 structure, which does not represent the Zika NS5 RdRp?If so, a substantial portion of the results may not be reflective.I recommend a thorough verification.When compared with NS5 RdRp structures with PDB codes 5Wz3 or 5U0B, there appears to be no similarity.
3. The methodology mentions obtaining 9 coupling results, yet it fails to specify the criteria used to select the optimal molecular coupling model.Please provide clarity on the selection process.
4. In the section on "Ligand and receptor molecular docking," the size of the grid is redundantly stated.
5. In the "Result Molecular Docking" section, the references for the positive ligands (controls) do not support their selection.For example, concerning sinefungin, it is mentioned that they were designed by HVC, but there is no information related to flavivirus or Zika.The same applies to sofosbuvir.I suggest modifying the references to accurately reflect their relevance.
6.The images exhibit low resolution, hindering effective structure visualization.Therefore, I recommend enhancing clarity for better interpretation.Specifically, in Figure 4, the sharpness is insufficient for clear visualization of 2D diagrams, and the residues interacting with molecules are not distinguishable from the representation of alpha helices and beta sheets.The legend of interactions is unreadable, and the figure caption lacks information on the meaning of each color.For improved comparison, I suggest including controls (sofosbuvir and sinefungin) in Figure 4.
Additionally, consider incorporating a surface representation with electrostatic charges to highlight hydrophobic regions or regions with electrostatic charges interacting with molecules.
7. In Figure 3, in the 5UO4 table, the control -7.40 should be white as it serves as the reference.It appears blue, possibly due to incorrectly taking the positive ligand of 5WXB as a reference.This is inaccurate, as it is a receptor and a distinct molecule.
8. Table 1 in the docking results mentions different types of non-covalent interactions without justifying why only hydrogen bond bonds and hydrophobic interactions were considered.The discussion also lacks insights into the differences or importance of various interactions such as alkyl-alkyl, pi-alkyl, etc.I recommend ordering the amino acids in ascending order for clarity.9.In the discussion of interactions between molecules (rutin) versus sofosbuvir, there is mention of a greater number of interactions, but the explanation on how these interactions lead to blocking the active site is lacking.Consider discussing whether this blocking results from size or other characteristics inherent to enzymatic activity inhibition.A more in-depth discussion of the results is suggested.Reviewer Expertise: virology, antiviral design, rational drug design, and enzyme kinetics, molecular biology.

References
I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.
5. In the "Result Molecular Docking" section, the references for the positive ligands (controls) do not support their selection.For example, concerning sinefungin, it is mentioned that they were designed by HVC, but there is no information related to flavivirus or Zika.The same applies to sofosbuvir.I suggest modifying the references to accurately reflect their relevance.

Answer:
Thank you for your careful review and for highlighting the need to ensure the references for our positive control ligands are accurate and relevant.We apologize for any oversight in the current references and appreciate your suggestion to provide more relevant citations.
In response to your concern and to support our selection, we have updated the manuscript and have included the following references: Sinefungin and Sofosbuvir were the reference inhibitors for NS5-MTase and NS5 RdRp, respectively.References:  4, the sharpness is insufficient for clear visualization of 2D diagrams, and the residues interacting with molecules are not distinguishable from the representation of alpha helices and beta sheets.The legend of interactions is unreadable, and the figure caption lacks information on the meaning of each color.For improved comparison, I suggest including controls (sofosbuvir and sinefungin) in Figure 4. Additionally, consider incorporating a surface representation with electrostatic charges to highlight hydrophobic regions or regions with electrostatic charges interacting with molecules.

Answer:
Thank you for your constructive feedback regarding the visualization quality of the images in Figure 4. We have considered your recommendations and replaced the images with higher-resolution versions to enhance clarity and facilitate better interpretation.However, at this stage, we have not incorporated surface representations with electrostatic charges to highlight hydrophobic regions and regions with electrostatic interactions.We will consider including these enhancements in future revisions to improve the visualization quality further.
Additionally, we have included controls (sofosbuvir and sinefungin) in Figure 4 for improved comparison.We appreciate your valuable feedback, which has significantly improved the quality and clarity of our manuscript.Thank you for your continued support in refining our research.
7. In Figure 3, in the 5UO4 table, the control -7.40 should be white as it serves as the reference.It appears blue, possibly due to incorrectly taking the positive ligand of 5WXB as a reference.This is inaccurate, as it is a receptor and a distinct molecule.

Answer:
Thank you for pointing out the issue with the color assignment in Figure 3.We have redone the color mapping for the complexes 5U04 and 5WXB table in Figure 3.The new figure has been added to the manuscript.
8. Table 1 in the docking results mentions different types of non-covalent interactions without justifying why only hydrogen bond bonds and hydrophobic interactions were considered.The discussion also lacks insights into the differences or importance of various interactions such as alkyl-alkyl, pi-alkyl, etc.I recommend ordering the amino acids in ascending order for clarity.

Answer:
Thank you for your insightful feedback regarding the non-covalent interactions mentioned in Table 1 of the docking results.
In our study, we focused on hydrogen bonds and hydrophobic interactions due to their significant roles in stabilizing ligand binding within the receptor's active site.Hydrogen bonds are essential for providing specificity and strong directional interactions, while hydrophobic interactions contribute to binding affinity by facilitating the burial of non-polar surfaces and reducing desolvation penalties.Although other non-covalent interactions such as alkyl-alkyl, pi-alkyl, and pi-pi interactions also play important roles in ligand-receptor binding, we prioritized hydrogen bonds and hydrophobic interactions due to their dominant influence in many docking studies.We have also included an explanation in the manuscript's discussion section.Furthermore, we have arranged the amino acids in Table 1 in their chronological order of occurrence within the protein sequence and structure, reflecting the sequential interaction pattern and spatial context, which is crucial for understanding the binding interactions.This arrangement aids in visualizing the interactions as they occur along the protein's active site and aligns with standard conventions in protein interaction studies.9.In the discussion of interactions between molecules (rutin) versus sofosbuvir, there is mention of a greater number of interactions, but the explanation on how these interactions lead to blocking the active site is lacking.Consider discussing whether this blocking results from size or other characteristics inherent to enzymatic activity inhibition.A more in-depth discussion of the results is suggested.

Answer:
Thank you for your valuable feedback and suggestion for a more in-depth discussion of the interactions between rutin and sofosbuvir To address the interactions between rutin and sofosbuvir with the active site, we provide a more detailed discussion on how these interactions contribute to the inhibition of enzymatic activity.Rutin exhibits a greater number of interactions compared to sofosbuvir, which enhances its ability to effectively block the active site.This blocking is not solely a function of the number of interactions but also their nature and strength.The larger size of rutin allows it to span a broader area of the active site, facilitating multiple points of contact through hydrogen bonds, hydrophobic interactions, and aromatic stacking.These interactions collectively stabilize the binding of rutin within the active site, thereby preventing substrate access and subsequent enzymatic activity.In contrast, while sofosbuvir also forms stable interactions, the fewer number and potentially weaker nature of these interactions may result in less effective blocking.Thus, rutin's increased binding affinity and comprehensive interaction profile contribute to its potential as a more effective inhibitor of the enzyme's active site.
Competing Interests: No competing interests were disclosed.
Reviewer Report 18 October 2023 https://doi.org/10.5256/f1000research.148054.r214337 1.The English need improvement since there are some grammatical and syntax errors in the manuscript.For example, the words "subjected for" may be as "subjected to"; "NS5-MTase" as "the NS5-MTase"; "by ADMET" as "by an ADMET"; "addition of" as "the addition of"; "removal of" as "the removal of"; "ranging 0.111" as "ranging from 0.111"; "zeaxanthin" as "and zeaxanthin".The grammar mistakes which are not mentioned here are also to be checked and corrected properly.
2. There are some typing mistakes as well, and authors are advised to carefully proof-read the text.For example, the words "encode a" may be as "encodes a"; "decent" as "descent"; "Ligands" as "Ligand"; "best docked" as "best-docked"; "nor vaccines" as "no vaccines"; "other site" as "other side"; "rutin, has" as "rutin has"; "β-sitsterol" as "β-sitosterol or check correct spelling"; "caretenoids" as "carotenoids".The typos not mentioned here are also to be checked and corrected properly.
3. Check the abbreviations throughout the manuscript and introduce the abbreviation when the full word appears the first time in the abstract (The use of abbreviations in the abstract (ADME) section may distract readers who wish to quickly skim through several publications before deciding to read one in full) and the remaining part of the manuscript (For example, PDP, FDA, etc.,).Make a word abbreviated in the article that is repeated at least three times in the text, not all words to be abbreviated.
4. The full form of the species should be given when the first time appears in both the abstract and in the remaining part of the manuscript and it should be followed by only the first letter of the genus (For example, Carica papaya when the first time appear and followed by C. papaya).
5. The authors should use uniformly either "ZIKA" or "Zika" all over the manuscript for better understanding.
6.The authors may cite recent prevalence or incidence data Zika virus (ZIKV) infection and it should be at-least of 2022 or 2023.
7. The introduction part appears less informative about Carica papaya, thus this section should be indicated as detailed to understand the manuscript in clear since the main objectives is focused on Carica papaya.The botanical description, family name and different plants of the plant with it medicinal uses.
8. The authors should rewrite the following "The paper aims" as "The present work or study aims".9.The authors should avoid the repetition some content in the manuscript and also it affect the flow of reading.It should be carefully checked and removed.For example, the family name "Flaviviridae" of ZIKA has been mentioned in three times.
10.The conclusion seems in general.All conclusions must be convincing statements on what was found to be novel, impact based on the strong support of the review.Moreover, the authors may also be included the limitation of the present findings for a better understanding of the manuscript.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
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Figure 2 .
Figure 2. Structural representation (2D) of the ligands shortlisted for having greater binding affinity to the receptor than the positive ligands used (Sofosbuvir and Sinefungin for 5U04 and 5WXB, respectively).

Figure 3 .
Figure 3. Docking results of phytocompounds from C. papaya against target NS5 protein domains NS5-MTase (5WXB) and NS5-RdRp (5U04) of Zika virus (A) The Heatmap showing the binding affinities of best-docked compounds with target protein domains.Blue indicates low binding affinity and red indicates high binding affinity.(B) Venn diagram representing the commonly shared best-docked ligands (compared to respective positive control ligands) with the target protein domains.

Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly
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