Reframing quercetin as a promiscuous inhibitor against SARS-CoV-2 main protease

Yan et al., Proceedings of the National Academy of Sciences, doi:10.1073/pnas.2309289120

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.

Yan et al., 5 Sep 2023, peer-reviewed, 5 authors.

Contact: yanchang.wang@med.fsu.edu, chenyunyu1984@163.com.

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

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Abstract: LETTER Reframing quercetin as a promiscuous inhibitor against SARS-CoV-2 main protease Haohao Yana,1, Rui Zhanga,1, Xiaoping Liua, Yanchang Wangb,2 , and Yunyu Chena,2 Fig. 1. Inhibitory effect of quercetin on SARS-CoV-2 Mpro in vitro. (A) The chemical structure of quercetin. (B) Inhibition of Mpro by quercetin using FRET assay. (C) The fluorescence quenching effect of quercetin on MCA-AVLQ fragment in the FRET assay. MCA: 7-methoxycoumarin-4-acetic acid. (D) The comparison between Mpro inhibition and fluorescence quenching effect of quercetin in the FRET assay. (E) Inhibition of Mpro by quercetin using FP assay. The FRET and FP assays were carried out as previously described (4, 5, 7). The IC50 value of quercetin was shown. Nirmatrelvir (PF-332, 1 μM) and DMSO served as the positive and negative controls, respectively. (F and G) Inhibition of Mpro by quercetin using ddRFP assay. The time course trajectories of ddRFP biosensor in the presence of quercetin at the indicated concentrations were recorded every minute for 30 min by a microplate reader (BioTek). A fluorescent ddRFP biosensor produces a high RFU value in the presence of Mpro inhibitors, whereas the cleavage by Mpro generates two separate RFP fragments with a low RFU signal. In the gel-based assay, the ddRFP biosensor (55 kDa) can be cleaved by Mpro (34 kDa) to generate RFP-A1 (top band, 29 kDa) and RFP-B1 fragments (bottom band, 26 kDa). The testing concentration of quercetin was 50, 100, or 200 μM. Nirmatrelvir (PF-332, 10 μM) and DMSO were used as the positive and negative controls, respectively. The ddRFP assay was performed based on our previous publications (6, 7). Traditional Chinese medicine has made contributions to the treatment of coronavirus disease 2019 (COVID-19) because of its favorable efficacy, such as Huashi Baidu decoction (Q-14) (1, 2). Recently, quercetin, a main component of Q-14, has been identified as a potent inhibitor against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) using an integrative pharmacological strategy, and its inhibitory effect on Mpro is examined by the fluorescence resonance energy transfer (FRET) assay with the half- maximal inhibitory concentration (IC50) value of 22.47 μM (3). Considering the potential of quercetin in COVID-19 treatment, a rigorous validation for its Mpro inhibition is necessary. We have developed a systematic high-throughput screening (HTS) platform for the discovery and assessment of Mpro inhibitors, including FRET, fluorescence polarization (FP), and dimerization-dependent red fluorescent protein (ddRFP) assays (4–7). With these assays, we previously demonstrated that baicalein is a nonspecific Mpro inhibitor (7). Herein, we rigorously evaluated the inhibition of Mpro by quercetin in vitro using these HTS assays (Fig. 1A). To ensure the PNAS 2023 Vol. 120 No. 37 e2309289120 reliability of these assays, nirmatrelvir (PF-07321332, PF-332) served as a positive control in the presence of dithiothreitol (DTT) (7). Using FRET assay, our results showed that quercetin exhibits apparent inhibition against Mpro (IC50 = 42.81 μM) (Fig. 1B). However, the presence of quercetin at the testing concentrations was able to quench the fluorescence signal of MCA-AVLQ fragment, which is generated by the cleaved FRET substrate (Fig. 1C). Importantly, this quenching effect Author affiliations: aInstitute for Drug Screening and Evaluation, Wannan Medical..

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