Structural stability of SARS-CoV-2 3CLpro and identification of quercetin as an inhibitor by experimental screening
Authors: Bruno Rizzuti
David Ortega-Alarcon
Sonia Vega
Adrian Velazquez-Campoy
Laura Ceballos-Laita
Ana Jimenez-Alesanco
Hugh T Reyburn
Olga Abian
Journal: International Journal of Biological Macromolecules (2020)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Drug discovery; SARS-CoV-2 3CL protease; Structural stability.
Affiliations: Instituto Aragonés de Ciencias de la Salud (IACS), 50009 Zaragoza, Spain; Instituto de Investigación Sanitaria de Aragón (IIS Aragon), Zaragoza, Spain; Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas Digestivas (CIBERehd), Madrid, Spain; Institute for Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, 50009 Zaragoza, Spain; Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, 50009 Zaragoza, Spain., Institute for Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, 50009 Zaragoza, Spain; Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, 50009 Zaragoza, Spain., Instituto de Investigación Sanitaria de Aragón (IIS Aragon), Zaragoza, Spain; Institute for Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, 50009 Zaragoza, Spain., Institute for Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, 50009 Zaragoza, Spain., Department of Immunology and Oncology, National Centre for Biotechnology (CNB), CSIC, 28049 Madrid, Spain., CNR-NANOTEC, Licryl-UOS Cosenza and CEMIF.Cal, Department of Physics, University of Calabria, 87036 Rende, Italy., Instituto de Investigación Sanitaria de Aragón (IIS Aragon), Zaragoza, Spain; Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas Digestivas (CIBERehd), Madrid, Spain; Institute for Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, 50009 Zaragoza, Spain; Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, 50009 Zaragoza, Spain; Fundación ARAID, Gobierno de Aragón, 50018 Zaragoza, Spain. Electronic address: .

Abstract:  The global health emergency generated by coronavirus disease 2019 (COVID-19) has prompted the search for preventive and therapeutic treatments for its pathogen, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). There are many potential targets for drug discovery and development to tackle this disease. One of these targets is the main protease, Mpro or 3CLpro, which is highly conserved among coronaviruses. 3CLpro is an essential player in the viral replication cycle, processing the large viral polyproteins and rendering the individual proteins functional. We report a biophysical characterization of the structural stability and the catalytic activity of 3CLpro from SARS-CoV-2, from which a suitable experimental in vitro molecular screening procedure has been designed. By screening of a small chemical library consisting of about 150 compounds, the natural product quercetin was identified as reasonably potent inhibitor of SARS-CoV-2 3CLpro (Ki ~ 7 μM). Quercetin could be shown to interact with 3CLpro using biophysical techniques and bind to the active site in molecular simulations. Quercetin, with well-known pharmacokinetic and ADMET properties, can be considered as a good candidate for further optimization and development, or repositioned for COVID-19 therapeutic treatment. 

Retracted: No

PubPeer comments:
 Quercetina anc cathecols in general are well known to be IMPS and PAINS. PAINS are Pan Assay Interference Compounds and IMPS are Invalid Metabolic Panaceas. These compounds share the ability to interfere aspecifically with many biological assay in vitro, and they show major issues when administer in vivo: poor pharmacokinetic pharmacodynamic (PK PD) properties, low efficacy in several disease models, and toxic effects under certain testing conditions 1-2. PAINS are described as u201cnonprogressable moleculesu201d 3 , since they suffer of u201clittle or no medicinal chemistry optimization, unconvincing structureu2212activity relationships (SAR), relative lack of improvement in biological activity to meaningful levels that often hover around the micromolar marku201d 3. Finally, proposed compounds are not orally bioavailable 4.5. In conclusion it is not possible to claim any specific or suppose any pharma use 1.tBaell, J. B. Holloway, G. A. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J. Med. Chem. 53, 2719u20132740 (2010). 2.tBisson, J. et al. Can Invalid Bioactives Undermine Natural Product-Based Drug Discovery? J. Med. Chem. 59, 1671u20131690 (2016). 3.tBaell, J. B. Screening-based translation of public research encounters painful problems. ACS Med. Chem. Lett. 6, 229u2013234 (2015). 4.tDel Rio, D. et al. Dietary (poly)phenolics in human health: structures, bioavailability, and evidence of protective effects against chronic diseases. Antioxid. Redox Signal. 18, 1818u20131892 (2013). 5.tBaell, J. B. Feeling Natureu2019s PAINS: Natural Products, Natural Product Drugs, and Pan Assay Interference Compounds (PAINS). J. Nat. Prod. 79, 6 , 
 Dear Dictyostelium Montium, I completely understand your comments. Quercetin is not an appropriate drug provided that its pharmacokinetic properties are quite poor. Even if quercetin is highly abundant in Nature, it appears in plants is glycosylated, inactive, low-bioavailable forms. In addition, it is rather unstable, suffering from oxidation and other processes. When taken orally, it is quickly metabolized into other derivatives, and its plasma concentration is very low. Surely, if any trial is to be organized, the administration should be different from oral administration. Anyway, I am sure there many drawbacks in employing quercetin for many purposes. We did not claim quercetin is the solution. We thought it would be interesting to show that quercetin is able to inhibit SARS-CoV-2 3CLpro. We did a blind screening at small scale, and quercetin came up from that screening. We then assessed the inhibition activity in more detail, and tested the interaction using several biophysical techniques (ITC, TSA, fluorescence, circular dichroism). Even if quercetin may not arrive to the pharmacy, it is a good starting point for developing improved drugs: 1) it is small (like a molecule fragment); 2) it is abundant; 3) it is easily chemically modified or derivatized; 4) it has a reasonable potency (Ki 7 microM), considering its molecular size, with an efficacy index (for binding or inhibition) much better than other specifically designed SARS-CoV-2 3CLpro inhibitors. Anyway... never let anyone tell you that you can t do something... just do it, and prove it right or wrong. ,