An Integrated In Silico and In Vitro Approach for the Identification of Natural Products Active against SARS-CoV-2

Pennisi et al., Biomolecules, doi:10.3390/biom14010043

Dec 2023

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In Vitro and In Silico study showing SARS-CoV-2 inhibition with folic acid and luteolin-7-O-glucuronide. Enzymatic assays showed that both compounds have micromolar range inhibition of SARS-CoV-2 proteases 3CL^pro and PLpro. However, pseudovirus entry assays demonstrated much stronger antiviral effects, with both compounds significantly reducing infection of cells by alpha and omicron SARS-CoV-2 pseudoviruses. Molecular modeling revealed binding sites within the spike protein receptor binding domain of both variants, suggesting they may interfere with viral entry.

13 preclinical studies support the efficacy of vitamin B9 for COVID-19:

8 In Silico studies Chen, Eskandari, Hosseini, Kumar, Pandya, Pennisi, Serseg, Ugurel

4 In Vitro studies Chen, Moatasim, Pennisi, Zhang

1 In Vivo animal study Zhang

Vitamin B9 has been identified by the European Food Safety Authority (EFSA) as having sufficient evidence for a causal relationship between intake and optimal immune system function EFSA, Galmés, Galmés (B). Vitamin B9 inhibits SARS-CoV-2 In Silico Chen, Eskandari, Hosseini, Kumar, Moatasim, Pandya, Serseg, Ugurel, Zhang, reduces spike protein binding ability Zhang, binds with the spike protein receptor binding domain for alpha and omicron variants Pennisi, inhibits the SARS-CoV-2 nucleocapsid protein Chen, inhibits 3CLpro and PLpro in enzymatic assays Pennisi, significantly reduces infection for alpha and omicron SARS-CoV-2 pseudoviruses Pennisi, and inhibits ACE2 expression and SARS-CoV-2 infection in a mouse model Zhang.

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1. Chen et al., Folic acid: a potential inhibitor against SARS-CoV-2 nucleocapsid protein, Pharmaceutical Biology, doi:10.1080/13880209.2022.2063341.tandfonline.com, doi.org.

2. EFSA, Scientific Opinion on the substantiation of health claims related to folate and blood formation (ID 79), homocysteine metabolism (ID 80), energy-yielding metabolism (ID 90), function of the immune system (ID 91), function of blood vessels (ID 94, 175, 192), cell division (ID 193), and maternal tissue growth during pregnancy (ID 2882) pursuant to Article 13(1) of Regulation (EC) No 1924/2006, EFSA Journal, doi:10.2903/j.efsa.2009.1213.europa.eu, doi.org.

3. Eskandari, V., Repurposing the natural compounds as potential therapeutic agents for COVID-19 based on the molecular docking study of the main protease and the receptor-binding domain of spike protein, Journal of Molecular Modeling, doi:10.1007/s00894-022-05138-3.springer.com, doi.org.

4. Galmés et al., Suboptimal Consumption of Relevant Immune System Micronutrients Is Associated with a Worse Impact of COVID-19 in Spanish Populations, Nutrients, doi:10.3390/nu14112254.mdpi.com, doi.org.

5. Galmés (B) et al., Current State of Evidence: Influence of Nutritional and Nutrigenetic Factors on Immunity in the COVID-19 Pandemic Framework, Nutrients, doi:10.3390/nu12092738.mdpi.com, doi.org.

6. Hosseini et al., Computational molecular docking and virtual screening revealed promising SARS-CoV-2 drugs, Precision Clinical Medicine, doi:10.1093/pcmedi/pbab001.oup.com, doi.org.

7. Kumar et al., In silico virtual screening-based study of nutraceuticals predicts the therapeutic potentials of folic acid and its derivatives against COVID-19, VirusDisease, doi:10.1007/s13337-020-00643-6.springer.com, doi.org.

8. Moatasim et al., Potent Antiviral Activity of Vitamin B12 against Severe Acute Respiratory Syndrome Coronavirus 2, Middle East Respiratory Syndrome Coronavirus, and Human Coronavirus 229E, Microorganisms, doi:10.3390/microorganisms11112777.mdpi.com, doi.org.

9. Pandya et al., Unravelling Vitamin B12 as a potential inhibitor against SARS-CoV-2: A computational approach, Informatics in Medicine Unlocked, doi:10.1016/j.imu.2022.100951.sciencedirect.com, doi.org.

10. Pennisi et al., An Integrated In Silico and In Vitro Approach for the Identification of Natural Products Active against SARS-CoV-2, Biomolecules, doi:10.3390/biom14010043.mdpi.com, doi.org.

11. Serseg et al., Hispidin and Lepidine E: Two Natural Compounds and Folic Acid as Potential Inhibitors of 2019-novel Coronavirus Main Protease (2019-nCoVMpro), Molecular Docking and SAR Study, Current Computer-Aided Drug Design, doi:10.2174/1573409916666200422075440.eurekaselect.com, doi.org.

12. Ugurel et al., Evaluation of the potency of FDA-approved drugs on wild type and mutant SARS-CoV-2 helicase (Nsp13), International Journal of Biological Macromolecules, doi:10.1016/j.ijbiomac.2020.09.138.sciencedirect.com, doi.org.

13. Zhang et al., Folic acid restricts SARS-CoV-2 invasion by methylating ACE2, Frontiers in Microbiology, doi:10.3389/fmicb.2022.980903.frontiersin.org, doi.org.

Pennisi et al., 28 Dec 2023, Italy, peer-reviewed, 7 authors. Contact: rpennisi@unime.it (corresponding author), paola.trischitta@studenti.unime.it, anna.piperno@unime.it, mtsciortino@unime.it, arescifina@unict.it, edoardo.napoli@icb.cnr.it, davide.gentile@polimi.it.

In Vitro studies are an important part of preclinical research, however results may be very different in vivo.

This Paper Vitamin B9 All

An Integrated In Silico and In Vitro Approach for the Identification of Natural Products Active against SARS-CoV-2

Rosamaria Pennisi, Davide Gentile, Antonio Rescifina, Edoardo Napoli, Paola Trischitta, Anna Piperno, Maria Teresa Sciortino

Biomolecules, doi:10.3390/biom14010043

Coronavirus disease 2019 (COVID-19) , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has provoked a global health crisis due to the absence of a specific therapeutic agent. 3CL pro (also known as the main protease or M pro ) and PL pro are chymotrypsin-like proteases encoded by the SARS-CoV-2 genome, and play essential roles during the virus lifecycle. Therefore, they are recognized as a prospective therapeutic target in drug discovery against SARS-CoV-2 infection. Thus, this work aims to collectively present potential natural 3CL pro and PL pro inhibitors by in silico simulations and in vitro entry pseudotype-entry models. We screened luteolin-7-O-glucuronide (L7OG), cynarin (CY), folic acid (FA), and rosmarinic acid (RA) molecules against PL pro and 3CL pro through a luminogenic substrate assay. We only reported moderate inhibitory activity on the recombinant 3CL pro and PL pro by L7OG and FA. Afterward, the entry inhibitory activity of L7OG and FA was tested in cell lines transduced with the two different SARS-CoV-2 pseudotypes harboring alpha (α) and omicron (o) spike (S) protein. The results showed that both compounds have a consistent inhibitory activity on the entry for both variants. However, L7OG showed a greater degree of entry inhibition against α-SARS-CoV-2. Molecular modeling studies were used to determine the inhibitory mechanism of the candidate molecules by focusing on their interactions with residues recognized by the protease active site and receptor-binding domain (RBD) of spike SARS-CoV-2. This work allowed us to identify the binding sites of FA and L7OG within the RBD domain in the alpha and omicron variants, demonstrating how FA is active in both variants. We have confidence that future in vivo studies testing the safety and effectiveness of these natural compounds are warranted, given that they are effective against a variant of concerns.

Conflicts of Interest: The authors declare no conflict of interest.

References

Alvarado, Perez-Lemus, Menéndez, Byléhn, De Pablo, Molecular Characterization of COVID-19 Therapeutics: Luteolin as an Allosteric Modulator of the Spike Protein of SARS-CoV-2, Mol. Syst. Des. Eng, doi:10.1039/D1ME00119A

Bontempo, De Masi, Rigano, Functional Properties of Natural Products and Human Health, Nutrients, doi:10.3390/nu15132961

Chen, Wei, Qin, Hou, Zang et al., Folic Acid: A Potential Inhibitor against SARS-CoV-2 Nucleocapsid Protein, Pharm. Biol, doi:10.1080/13880209.2022.2063341

Citarella, Dimasi, Moi, Passarella, Scala et al., Recent Advances in SARS-CoV-2 Main Protease Inhibitors: From Nirmatrelvir to Future Perspectives, Biomolecules, doi:10.3390/biom13091339

Citarella, Gentile, Rescifina, Piperno, Mognetti et al., Pseudo-Dipeptide Bearing α,α-Difluoromethyl Ketone Moiety as Electrophilic Warhead with Activity against Coronaviruses, Int. J. Mol. Sci, doi:10.3390/ijms22031398

Daniloski, Jordan, Wessels, Hoagland, Kasela et al., Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells, Cell, doi:10.1016/j.cell.2020.10.030

Dissook, Umsumarng, Mapoung, Semmarath, Arjsri et al., Luteolin-Rich Fraction from Perilla Frutescens Seed Meal Inhibits Spike Glycoprotein S1 of SARS-CoV-2-Induced NLRP3 Inflammasome Lung Cell Inflammation via Regulation of JAK1/STAT3 Pathway: A Potential Anti-Inflammatory Compound against Inflammation-Induced Long-COVID, Front. Med, doi:10.3389/fmed.2022.1072056

Duan, Wu, Chowdhury, Lee, Xiong et al., A Point-charge Force Field for Molecular Mechanics Simulations of Proteins Based on Condensed-phase Quantum Mechanical Calculations, J. Comput. Chem, doi:10.1002/jcc.10349

Essmann, Perera, Berkowitz, Darden, Lee et al., A Smooth Particle Mesh Ewald Method, J. Chem. Phys, doi:10.1063/1.470117

Floresta, Amata, Barbaraci, Gentile, Turnaturi et al., A Structure-and Ligand-Based Virtual Screening of a Database of "Small" Marine Natural Products for the Identification of "Blue" Sigma-2 Receptor Ligands, Mar. Drugs, doi:10.3390/md16100384

Floresta, Dichiara, Gentile, Prezzavento, Marrazzo et al., Morphing of Ibogaine: A Successful Attempt into the Search for Sigma-2 Receptor Ligands, Int. J. Mol. Sci, doi:10.3390/ijms20030488

Floresta, Patamia, Gentile, Molteni, Santamato et al., Repurposing of FDA-Approved Drugs for Treating Iatrogenic Botulism: A Paired 3D-QSAR/Docking Approach, ChemMedChem, doi:10.1002/cmdc.201900594

Galimberti, Barbera, Guerra, Bernardi, Facile functionalization of sp2 carbon allotropes with a biobased janus molecule, Rubber Chem. Technol, doi:10.5254/rct.17.82665

Gentile, Floresta, Patamia, Chiaramonte, Mauro et al., An Integrated Pharmacophore/ Docking/3D-QSAR Approach to Screening a Large Library of Products in Search of Future Botulinum Neurotoxin a Inhibitors, Int. J. Mol. Sci, doi:10.3390/ijms21249470

Gentile, Floresta, Patamia, Nicosia, Mineo et al., Cucurbit[7]Uril as a Catalytic Nanoreactor for One-Pot Synthesis of Isoxazolidines in Water, Org. Biomol. Chem, doi:10.1039/C9OB02352F

Gentile, Patamia, Fuochi, Furneri, Rescifina, Natural Substances in the Fight of SARS-CoV-2: A Critical Evaluation Resulting from the Cross-Fertilization of Molecular Modeling Data with the Pharmacological Aspects, Curr. Med. Chem, doi:10.2174/0929867328666210614114032

Gezer, Potential Health Effects of the Popular Compound of Artichoke: Cynarin (CYN), Prog. Nutr

Heleno, Carocho, Reis, Pires, Pintado et al., Plant Extracts and SARS-CoV-2: Research and Applications, Life, doi:10.3390/life13020386

Hoffmann, Kleine-Weber, Schroeder, Krüger, Herrler et al., SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor, Cell, doi:10.1016/j.cell.2020.02.052

Hornak, Abel, Okur, Strockbine, Roitberg et al., Comparison of Multiple Amber Force Fields and Development of Improved Protein Backbone Parameters, Proteins Struct. Funct. Bioinform, doi:10.1002/prot.21123

Jakalian, Jack, Bayly, Fast, Efficient Generation of High-quality Atomic Charges. AM1-BCC Model: II. Parameterization and Validation, J. Comput. Chem, doi:10.1002/jcc.10128

Júnior, De Sousa Junior, Nze, Giozza, Júnior, Evaluation of Peppermint Leaf Flavonoids as SARS-CoV-2 Spike Receptor-Binding Domain Attachment Inhibitors to the Human ACE2 Receptor: A Molecular Docking Study, Open J. Biophys, doi:10.4236/ojbiphy.2022.122005

Karakousis, Gourgoulianis, Kotsiou, The Role of Folic Acid in SARS-CoV-2 Infection: An Intriguing Linkage under Investigation, J. Pers. Med, doi:10.3390/jpm13030561

Kaur, Sarma, Bhattacharyya, Prajapat, Kumar et al., Folic Acid as Placebo in Controlled Clinical Trials of Hydroxychloroquine Prophylaxis in COVID-19: Is It Scientifically Justifiable?, Med. Hypotheses, doi:10.1016/j.mehy.2021.110539

Krieger, Dunbrack, Hooft, Krieger, Assignment of Protonation States in Proteins and Ligands: Combining PKa Prediction with Hydrogen Bonding Network Optimization

Krieger, Nielsen, Spronk, Vriend, Fast Empirical PKa Prediction by Ewald Summation, J. Mol. Graph. Model, doi:10.1016/j.jmgm.2006.02.009

Krieger, Vriend, New Ways to Boost Molecular Dynamics Simulations, J. Comput. Chem, doi:10.1002/jcc.23899

Krüger, Kronenberger, Xie, Rocha, Pöhlmann et al., Discovery of Polyphenolic Natural Products as SARS-CoV-2 Mpro Inhibitors for COVID-19, Pharmaceuticals, doi:10.3390/ph16020190

Lv, Cano, Jia, Drag, Huang et al., Targeting SARS-CoV-2 Proteases for COVID-19 Antiviral Development, Front. Chem, doi:10.3389/fchem.2021.819165

Magi, Marini, Brenciani, Di Lodovico, Gentile et al., Chemical Composition of Pistacia vera L. Oleoresin and Its Antibacterial, Anti-Virulence and Anti-Biofilm Activities against Oral Streptococci, Including Streptococcus Mutans, Arch. Oral Biol, doi:10.1016/j.archoralbio.2018.09.013

Maier, Martinez, Kasavajhala, Wickstrom, Hauser et al., Ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from Ff99SB, J. Chem. Theory Comput, doi:10.1021/acs.jctc.5b00255

Meng, Abdullahi, Ferreira, Goonawardane, Saito et al., Altered TMPRSS2 Usage by SARS-CoV-2 Omicron Impacts Infectivity and Fusogenicity, Nature, doi:10.1038/s41586-022-04474-x

Metzdorf, Jacobsen, Greweling-Pils, Hoffmann, Lüddecke et al., TMPRSS2 Is Essential for SARS-CoV-2 Beta and Omicron Infection, Viruses, doi:10.3390/v15020271

Millet, Tang, Nathan, Jaimes, Hsu et al., Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting, J. Vis. Exp, doi:10.3791/59010

Millet, Whittaker, Murine Leukemia Virus (MLV)-Based Coronavirus Spike-Pseudotyped Particle Production and Infection, Bio Protoc, doi:10.21769/BioProtoc.2035

Mukherjee, Dikic, Proteases of SARS Coronaviruses

Munafò, Donati, Brindani, Ottonello, Armirotti et al., Quercetin and Luteolin Are Single-Digit Micromolar Inhibitors of the SARS-CoV-2 RNA-Dependent RNA Polymerase, Sci. Rep, doi:10.1038/s41598-022-14664-2

Muruganathan, Dhanapal, Baskar, Muthuramalingam, Selvaraj et al., Recent Updates on Source, Biosynthesis, and Therapeutic Potential of Natural Flavonoid Luteolin: A Review, Metabolites, doi:10.3390/metabo12111145

Musarra-Pizzo, Pennisi, Ben-Amor, Mandalari, Sciortino, Antiviral Activity Exerted by Natural Products against Human Viruses, Viruses, doi:10.3390/v13050828

Narayanan, Narwal, Majowicz, Varricchio, Toner et al., Identification of SARS-CoV-2 Inhibitors Targeting Mpro and PLpro Using in-Cell-Protease Assay, Commun. Biol, doi:10.1038/s42003-022-03090-9

Ou, Lan, Wu, Zhao, Duan et al., Tracking SARS-CoV-2 Omicron Diverse Spike Gene Mutations Identifies Multiple Inter-Variant Recombination Events, Signal Transduct. Target. Ther, doi:10.1038/s41392-022-00992-2

Pennisi, Trischitta, Tamburello, Barreca, Mandalari et al., Mechanistic Understanding of the Antiviral Properties of Pistachios and Zeaxanthin against HSV-1, Viruses, doi:10.3390/v15081651

Pišlar, Mitrović, Sabotič, Pečar Fonović, Perišić Nanut et al., The Role of Cysteine Peptidases in Coronavirus Cell Entry and Replication: The Therapeutic Potential of Cathepsin Inhibitors, PLoS Pathog, doi:10.1371/journal.ppat.1009013

Prasansuklab, Theerasri, Rangsinth, Sillapachaiyaporn, Chuchawankul et al., Anti-COVID-19 Drug Candidates: A Review on Potential Biological Activities of Natural Products in the Management of New Coronavirus Infection, J. Tradit. Complement. Med, doi:10.1016/j.jtcme.2020.12.001

Sacco, Ma, Lagarias, Gao, Townsend et al., Structure and Inhibition of the SARS-CoV-2 Main Protease Reveal Strategy for Developing Dual Inhibitors against M pro and Cathepsin L, Sci. Adv, doi:10.1126/sciadv.abe0751

Seth, Batra, Srinivasan, COVID-19: Targeting Proteases in Viral Invasion and Host Immune Response, Front. Mol. Biosci, doi:10.3389/fmolb.2020.00215

Shawan, Halder, Hasan, Luteolin and Abyssinone II as Potential Inhibitors of SARS-CoV-2: An in Silico Molecular Modeling Approach in Battling the COVID-19 Outbreak, Bull. Natl. Res. Cent, doi:10.1186/s42269-020-00479-6

Tang, Jaimes, Bidon, Straus, Daniel et al., Proteolytic Activation of SARS-CoV-2 Spike at the S1/S2 Boundary: Potential Role of Proteases beyond Furin, ACS Infect. Dis, doi:10.1021/acsinfecdis.0c00701

Toelzer, Gupta, Yadav, Borucu, Davidson et al., Free Fatty Acid Binding Pocket in the Locked Structure of SARS-CoV-2 Spike Protein, Science, doi:10.1126/science.abd3255

Ugurel, Mutlu, Sariyer, Kocer, Ugurel et al., Evaluation of the Potency of FDA-Approved Drugs on Wild Type and Mutant SARS-CoV-2 Helicase (Nsp13), Int. J. Biol. Macromol, doi:10.1016/j.ijbiomac.2020.09.138

Wang, Wolf, Caldwell, Kollman, Case, Development and Testing of a General Amber Force Field, J. Comput. Chem, doi:10.1002/jcc.20035

Wang, Yang, Liu, Guo, Zhang et al., SARS Coronavirus Entry into Host Cells through a Novel Clathrin-and Caveolae-Independent Endocytic Pathway, Cell Res, doi:10.1038/cr.2008.15

Wang, Yang, Xia, Li, Xiong, Luteolin Is a Potential Inhibitor of COVID-19: An in Silico Analysis, Medicine, doi:10.1097/MD.0000000000035029

Watanabe, Matsuyama, Shirato, Maejima, Fukushi et al., Entry from the Cell Surface of Severe Acute Respiratory Syndrome Coronavirus with Cleaved S Protein as Revealed by Pseudotype Virus Bearing Cleaved S Protein, J. Virol, doi:10.1128/JVI.01412-08

Xiang, Li, Wu, Tian, Fu, Application of Pseudovirus System in the Development of Vaccine, Antiviral-Drugs, and Neutralizing Antibodies, Microbiol. Res, doi:10.1016/j.micres.2022.126993

Zhang, Pang, Xu, Chen, Liang et al., Folic Acid Restricts SARS-CoV-2 Invasion by Methylating ACE2, Front. Microbiol, doi:10.3389/fmicb.2022.980903

Zhang, Zhao, Liu, He, Yu et al., Design of SARS-CoV-2 Mpro, PLpro Dual-Target Inhibitors Based on Deep Reinforcement Learning and Virtual Screening, Future Med. Chem, doi:10.4155/fmc-2021-0269

Zhu, Yan, Shi, Zhang, Lu et al., Luteolin Inhibits Spike Protein of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Binding to Angiotensin-converting Enzyme 2, Phytother. Res, doi:10.1002/ptr.7826

Škrbić, Travar, Stojiljković, Djuric, Suručić, Folic Acid and Leucovorin Have Potential to Prevent SARS-CoV-2-Virus Internalization by Interacting with S-Glycoprotein/Neuropilin-1 Receptor Complex, Molecules, doi:10.3390/molecules28052294

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