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. 2020 Dec 1:164:1693-1703.
doi: 10.1016/j.ijbiomac.2020.07.235. Epub 2020 Aug 1.

Structural stability of SARS-CoV-2 3CLpro and identification of quercetin as an inhibitor by experimental screening

Olga Abian  1 , David Ortega-Alarcon  2 , Ana Jimenez-Alesanco  2 , Laura Ceballos-Laita  3 , Sonia Vega  4 , Hugh T Reyburn  5 , Bruno Rizzuti  6 , Adrian Velazquez-Campoy  7
Affiliations

Structural stability of SARS-CoV-2 3CLpro and identification of quercetin as an inhibitor by experimental screening

Olga Abian et al. Int J Biol Macromol. .

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.

Keywords: Drug discovery; SARS-CoV-2 3CL protease; Structural stability.

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Conflict of interest statement

Declaration of competing interest The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Spectroscopic properties of SARS-CoV-2 3CLpro. (a) Far-UV CD at 10 μM protein concentration. (b) Near-UV CD at 10 μM protein concentration. (c) Fluorescence at 2 μM protein concentration. All the spectra were recorded at pH 8.
Fig. 2
Fig. 2
Spectroscopic thermal unfolding of SARS-CoV-2 3CLpro. (a) Far-UV CD thermal unfolding monitoring the (raw) ellipticity at 222 nm, at 10 μM protein concentration. (b) Fluorescence thermal unfolding monitoring the intrinsic tryptophan fluorescence emission at 330 nm, at 2 μM protein concentration. Both experiments were recorded at pH 8. The experimental data (circles) and the non-linear regression analysis fitting curves (continuous lines) according to two-state monomeric, dimeric and tetrameric protein are shown (indistinguishable in the plot).
Fig. 3
Fig. 3
Calorimetric thermal unfolding of SARS-CoV-2 3CLpro. (a) Molar excess heat capacity as a function of temperature at pH 5 (black), pH 7 (red), and pH 8 (green), at 14 μM protein concentration. (b) Molar excess heat capacity as a function of temperature at pH 8. The experimental data (circles) and the non-linear regression analysis fitting curves (continuous lines) according to two-state monomeric (gray), dimeric (green), tetrameric (red), and octameric (blue) protein unfolding models are shown.
Fig. 4
Fig. 4
Catalytic activity of SARS-CoV-2 3CLpro. Time evolution of the substrate fluorescence emission as a function of time at pH 5 (black), pH 7 (red), and pH 8 (green), at protein concentration of 1 μM and substrate concentration of 20 μM. The increase in fluorescence emission reflects the decrease in Dabcyl quenching on Edans emission (i.e., reduction of FRET phenomenon) due to the increase in the spatial separation of the donor-acceptor pair as a result of the catalytic cleavage of the substrate molecule.
Fig. 5
Fig. 5
Screening of a small chemical library. Enzyme activity, in the presence and the absence of compound, was quantitated as the initial slope of the fluorescence emission as a function of time. Control wells (red squares) contained enzyme and substrate in the presence of an identical concentration of DMSO as that in the wells with compounds (black squares). Hits were selected as those compounds lowering the activity below a certain threshold (average activity of controls minus twice the standard deviation of those controls, lower dotted line). The blue square corresponds to quercetin.
Fig. 6
Fig. 6
Hit confirmation through inhibition curve. (a) Substrate fluorescence emission as a function of time, fixing the enzyme concentration at 2 μM, the substrate concentration at 20 μM, and varying quercetin concentration from 0 to 125 μM (while maintaining constant the percentage of DMSO). The increase in quercetin concentration resulted in the decrease in the enzymatic activity. (b) Experimental inhibition data for quercetin (squares), together with the non-linear regression analysis fitting curve (black line) to a simple inhibition, from which the apparent inhibition constant (Kiapp = 21 μM) or the intrinsic inhibition constant (Ki = 9.6 μM) could be estimated.
Fig. 7
Fig. 7
Hit confirmation through thermal shift assay (TSA). (a) Unfolding traces of the compound-free protein (black) and the protein in the presence of 62.5 μM quercetin (red). (b) Apparent unfolding temperature for the protein at different quercetin concentrations.
Fig. 8
Fig. 8
Hit conformation through isothermal titration calorimetry (ITC). Interaction of quercetin with 3CLpro assessed by isothermal titration calorimetry in sodium phosphate 50 mM, pH 8, without (left) and with (right) NaCl 150 mM. The upper plot shows the thermogram (thermal power required to maintain a null temperature difference between sample and reference cells as a function of time) and the lower plot shows the binding isotherm (ligand-normalized heat effect per injection as a function of the molar ratio, the quotient between the ligand and protein concentrations in the cell). The fitting curve corresponds to the single ligand binding site model (continuous line). According to the data analysis, in the absence of NaCl quercetin interacts with 3CLpro with favorable enthalpic (ΔH = −3.6 kcal/mol) and entropic (−TΔS = −4.0 kcal/mol) contributions to the Gibbs energy of binding (ΔG = −7.6 kcal/mol), corresponding to a dissociation constant Kd of 2.7 μM. In the presence of NaCl quercetin interacts with 3CLpro with favorable enthalpic (ΔH = −4.3 kcal/mol) and entropic (−TΔS = −2.5 kcal/mol) contributions to the Gibbs energy of binding (ΔG = −6.8 kcal/mol), corresponding to a dissociation constant Kd of 10 μM. In both cases, the percentage of active (or binding-competent) protein is 0.75.
Fig. 9
Fig. 9
Molecular docking of quercetin to 3CLpro. Structure of (a) unliganded 3CLpro (from entry 6Y2E of the PDB; in ribbon representation, yellow) and α-ketoamide inhibitor bound in the active site (from entry 6Y2F; in stick representation, yellow) [22]. The liganded protein was superimposed by a least square fit on the Cα atoms of the liganded one, and only the latter is shown. (b) and (c) Detail of the 3CLpro active site, with the two best docking poses obtained using as receptor the structure 6Y2E (cyan) and 6Y2F (purple). Polar hydrogen atoms are explicitly shown (white) and oxygen atoms are also coloured (red).
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