Reply to Yan et al.: Quercetin possesses a fluorescence quenching effect but is a weak inhibitor against SARS-CoV-2 main protease

Xu et al., Proceedings of the National Academy of Sciences, doi:10.1073/pnas.2309870120

Sep 2023

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In Vitro study Yan and associated response from the original authors Xu, collectively showing that quercetin and echinatin had weak SARS-CoV-2 protease inhibition in SDS-PAGE assays Xu, despite false positive FRET results from MCA-AVLQ quenching Xu, Yan. Authors note that the compounds may act via other targets to achieve reported anti-COVID-19 effects Xu, and underscore the importance of meticulous validation with multiple assays when identifying SARS-CoV-2 protease inhibitors Xu, Yan.

23 preclinical studies support the efficacy of quercetin for COVID-19:

14 In Silico studies Alavi, Azmi, Chellasamy, Corbo, Ibeh, Kandeil, Mandal, Nguyen, Sai Ramesh, Sekiou, Thapa, Wang, Yang, Şimşek

7 In Vitro studies Aguado, Bahun, Goc, Kandeil, Munafò, Singh, Xu (B)

2 In Vivo animal studies Aguado, Wu

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1. Aguado et al., Senolytic therapy alleviates physiological human brain aging and COVID-19 neuropathology, bioRxiv, doi:10.1101/2023.01.17.524329.biorxiv.org, doi.org.

2. Alavi et al., Interaction of Epigallocatechin Gallate and Quercetin with Spike Glycoprotein (S-Glycoprotein) of SARS-CoV-2: In Silico Study, Biomedicines, doi:10.3390/biomedicines10123074.mdpi.com, doi.org.

3. Azmi et al., Utilization of quercetin flavonoid compounds in onion (Allium cepa L.) as an inhibitor of SARS-CoV-2 spike protein against ACE2 receptors, 11th International Seminar on New Paradigm and Innovation on Natural Sciences and its Application, doi:10.1063/5.0140285.scitation.org, doi.org.

4. Bahun et al., Inhibition of the SARS-CoV-2 3CLpro main protease by plant polyphenols, Food Chemistry, doi:10.1016/j.foodchem.2021.131594.sciencedirect.com, doi.org.

5. Chellasamy et al., Docking and molecular dynamics studies of human ezrin protein with a modelled SARS-CoV-2 endodomain and their interaction with potential invasion inhibitors, Journal of King Saud University - Science, doi:10.1016/j.jksus.2022.102277.sciencedirect.com, doi.org.

6. Corbo et al., Inhibitory potential of phytochemicals on five SARS-CoV-2 proteins: in silico evaluation of endemic plants of Bosnia and Herzegovina, Biotechnology & Biotechnological Equipment, doi:10.1080/13102818.2023.2222196.tandfonline.com, doi.org.

7. Goc et al., Inhibitory effects of specific combination of natural compounds against SARS-CoV-2 and its Alpha, Beta, Gamma, Delta, Kappa, and Mu variants, European Journal of Microbiology and Immunology, doi:10.1556/1886.2021.00022.akjournals.com, doi.org.

8. Ibeh et al., Computational studies of potential antiviral compounds from some selected Nigerian medicinal plants against SARS-CoV-2 proteins, Informatics in Medicine Unlocked, doi:10.1016/j.imu.2023.101230.sciencedirect.com, doi.org.

9. Kandeil et al., Bioactive Polyphenolic Compounds Showing Strong Antiviral Activities against Severe Acute Respiratory Syndrome Coronavirus 2, Pathogens, doi:10.3390/pathogens10060758.mdpi.com, doi.org.

10. Mandal et al., In silico anti-viral assessment of phytoconstituents in a traditional (Siddha Medicine) polyherbal formulation – Targeting Mpro and pan-coronavirus post-fusion Spike protein, Journal of Traditional and Complementary Medicine, doi:10.1016/j.jtcme.2023.07.004.sciencedirect.com, doi.org.

11. Munafò et al., Quercetin and Luteolin Are Single-digit Micromolar Inhibitors of the SARS-CoV-2 RNA-dependent RNA Polymerase, Research Square, doi:10.21203/rs.3.rs-1149846/v1.researchsquare.com, doi.org.

12. Nguyen et al., The Potential of Ameliorating COVID-19 and Sequelae From Andrographis paniculata via Bioinformatics, Bioinformatics and Biology Insights, doi:10.1177/11779322221149622.sagepub.com, doi.org.

13. Sai Ramesh et al., Computational analysis of the phytocompounds of Mimusops elengi against spike protein of SARS CoV2 – An Insilico model, International Journal of Biological Macromolecules, doi:10.1016/j.ijbiomac.2023.125553.sciencedirect.com, doi.org.

14. Sekiou et al., In-Silico Identification of Potent Inhibitors of COVID-19 Main Protease (Mpro) and Angiotensin Converting Enzyme 2 (ACE2) from Natural Products: Quercetin, Hispidulin, and Cirsimaritin Exhibited Better Potential Inhibition than Hydroxy-Chloroquine Against COVID-19 Main Protease Active Site and ACE2, ChemRxiv, doi:10.26434/chemrxiv.12181404.v1.chemrxiv.org, doi.org.

15. Şimşek et al., In silico identification of SARS-CoV-2 cell entry inhibitors from selected natural antivirals, Journal of Molecular Graphics and Modelling, doi:10.1016/j.jmgm.2021.108038.sciencedirect.com, doi.org.

16. Singh et al., The spike protein of SARS-CoV-2 virus induces heme oxygenase-1: Pathophysiologic implications, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, doi:10.1016/j.bbadis.2021.166322.sciencedirect.com, doi.org.

17. Thapa et al., In-silico Approach for Predicting the Inhibitory Effect of Home Remedies on Severe Acute Respiratory Syndrome Coronavirus-2, Makara Journal of Science, doi:10.7454/mss.v27i3.1609.ac.id, doi.org.

18. Wang et al., Computational Analysis of Lianhua Qingwen as an Adjuvant Treatment in Patients with COVID-19, Society of Toxicology Conference, 2023, www.researchgate.net/publication/370491709_Y_Wang_A_E_Tan_O_Chew_A_Hsueh_and_D_E_Johnson_2023_Computational_Analysis_of_Lianhua_Qingwen_as_an_Adjuvant_Treatment_in_Patients_with_COVID-19_Toxicologist_1921_507.researchgate.net.

19. Wu et al., Treatment with Quercetin inhibits SARS-CoV-2 N protein-induced acute kidney injury by blocking Smad3-dependent G1 cell cycle arrest, Molecular Therapy, doi:10.1016/j.ymthe.2022.12.002.sciencedirect.com, doi.org.

20. Xu et al., Reply to Yan et al.: Quercetin possesses a fluorescence quenching effect but is a weak inhibitor against SARS-CoV-2 main protease, Proceedings of the National Academy of Sciences, doi:10.1073/pnas.2309870120.pnas.org, doi.org.

21. Xu (B) et al., Bioactive compounds from Huashi Baidu decoction possess both antiviral and anti-inflammatory effects against COVID-19, Proceedings of the National Academy of Sciences, doi:10.1073/pnas.2301775120.pnas.org, doi.org.

22. Yan et al., Reframing quercetin as a promiscuous inhibitor against SARS-CoV-2 main protease, Proceedings of the National Academy of Sciences, doi:10.1073/pnas.2309289120.pnas.org, doi.org.

23. Yang et al., In silico evidence implicating novel mechanisms of Prunella vulgaris L. as a potential botanical drug against COVID-19-associated acute kidney injury, Frontiers in Pharmacology, doi:10.3389/fphar.2023.1188086.frontiersin.org, doi.org.

Xu et al., 5 Sep 2023, peer-reviewed, 7 authors.

Contact: huangluqi01@126.com.

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

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Reply to Yan et al.: Quercetin possesses a fluorescence quenching effect but is a weak inhibitor against SARS-CoV-2 main protease

Haiyu Xu, Yute Zhong, Jinyue Yang, Lifeng Fu, Yi Shi, Luqi Huang, George F Gao

Proceedings of the National Academy of Sciences, doi:10.1073/pnas.2309870120

Recently, we published an article (1) showing that several bioactive compounds were identified from Huashi Baidu decoction (Q-14) that showed both antiviral activity and antiinflammatory effects against COVID-19. The main protease (M pro ) of SARS-CoV-2 is an attractive target for the development of antiviral drugs. Our results showed that quercetin and echinatin from Q-14 displayed moderate inhibition activities against SARS-CoV-2 M pro at submicromolar levels by the fluorescence resonance energy transfer (FRET)-based protease activity assay. However, Yan et al. suggested that quercetin is a promiscuous M pro inhibitor because of its fluorescence quenching effect (2). We took this suspicion very seriously. Therefore, we repeated the FRET-based protease activity assay and tested the fluorescence quenching effects with the MCA-AVLQ-Lys (Dnp)-Lys-NH2 (MCA-AVLQ) fragment. The FRET assay results showed that both quercetin and echinatin have M pro inhibition activity (Fig. 1 A and E ), but both quercetin and echinatin displayed a fluorescence quenching effect on MCA-AVLQ fragment (Fig. 1 B and F ). The inhibition ratio and quenching ratio were further calculated (Fig. 1 C and G). We found that the inhibition ratio is significantly greater than the quenching rate, especially at low concentrations, which indicates that quercetin and echinatin should possess certain M pro inhibition activity. To further validate the M pro inhibition activity of

References

Xu, Bioactive compounds from Huashi Baidu decoction possess both antiviral and anti-inflammatory effects against COVID-19, Proc. Natl. Acad. Sci. U.S.A

Yan, Production of a versatile SARS-CoV-2 main protease biosensor based on a dimerization-dependent red fluorescent protein, J. Med. Virol

Yan, Zhang, Liu, Wang, Chen, Reframing quercetin as a promiscuous inhibitor against SARS-CoV-2 main protease, Proc. Natl. Acad. Sci. U.S.A

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