key: cord-1032693-ndxwg0pq
authors: Abian, Olga; Ortega-Alarcon, David; Jimenez-Alesanco, Ana; Ceballos-Laita, Laura; Vega, Sonia; Reyburn, Hugh T.; Rizzuti, Bruno; Velazquez-Campoy, Adrian
title: Structural stability of SARS-CoV-2 3CLpro and identification of quercetin as an inhibitor by experimental screening
date: 2020-08-01
journal: Int J Biol Macromol
DOI: 10.1016/j.ijbiomac.2020.07.235
sha: 313e21e8c8994a2c9c4be87521d3fe3c9443efd7
doc_id: 1032693
cord_uid: ndxwg0pq

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 (K(i) ~ 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.

J o u r n a l P r e -p r o o f INTRODUCTION December 2019 was the starting point for a global pandemic, caused by a newly identified coronavirus, SARS-CoV-2, rising to dramatic numbers of infections and deaths worldwide [1, 2] .

Much effort and many resources have been devoted to combat this health emergency on many fronts: 1) better diagnosis and patient management [3] ; 2) improving protective and palliative care [4] ; 3) reducing damage in the different organs affected by the virus and minimizing collateral harmful effects of the immune response [5] ; and 4) developing preventive vaccines and therapeutic treatments [6] , to mention the more relevant. In addition, very strict and severe social and economic measures, with the potential danger of causing a profound global crisis, have been taken [7] . In this context, even if an effective vaccine is promisingly close to becoming available, drugs are needed for infected people [8] . From experience with other pathogens, we have learnt that the best therapeutic strategy consists in administrating a combination of several drugs acting through different mechanisms, in order to minimize the probability of drug resistance appearance [9] . At the same time, cost-efficient drugs (if possible, developed by repositioning or repurposing known drugs) are urgently needed to reaching all the population [10] .

The SARS-CoV-2 genome is 82% identical to that of SARS-CoV, the causative virus of the 2002 coronavirus outbreak [11] . The genomic differences are reflected in a different infectivity and mortality rates for SARS-CoV-2 [12] . Among all the potential protein targets within coronaviruses, the main protease (Mpro, or 3C-like protease, 3CLpro) stands out as associated to a highly conserved gene (96% sequence identity between the SARS-CoV-2 and SARS-CoV), making 3CLpro a good target for developing effective drugs against SARS-CoV-2 (and other future coronavirus variants) [13, 14] . Together with PLpro, 3CLpro is responsible for the processing of the viral polyproteins synthesized from the viral RNA after infection, rendering the individual viral proteins active and functional [15] . Remarkably, 3CLpro must process at least 11 cleavage sites on polyprotein 1ab (replicase 1ab), most of them sharing a common cleavage sequence LQ(S/A/G), quite unusual for human proteases [16] . Therefore, molecules able to inhibit SARS-CoV-2 3CLpro in a specific manner would hinder viral replication and represent appropriate candidates to develop low-toxicity drugs against this devastating pathogen [17] .

Searching for drugs against a protein target always benefits from a comprehensive structural and functional characterization [18] . Experimental and computational knowledge gathered along this process is instrumental for identifying weak points and key interaction sites in the target, and developing suitable screening procedures for finding molecules interfering with or modulating its function [19] . Here we have characterized some structural and functional J o u r n a l P r e -p r o o f features of SARS-CoV-2 3CLpro by employing biophysical techniques (spectroscopy and calorimetry), and we have implemented a fast in vitro screening procedure based on 3CLpro hydrolytic activity using a Förster resonance energy transfer (FRET) substrate, which allows the ready identification of small molecules blocking the enzymatic activity of 3CLpro. Starting from a small chemical library consisting of about 150 compounds, collected from successful screening programs previously carried out in our laboratory, a natural compound was identified as an inhibitor of SARS-CoV-2 3CLpro with enough potency (inhibition constant K i  7 µM) to be considered a good candidate for further optimization and development. Quercetin, with known pharmacokinetic and ADMET properties, exhibits anti-oxidant, anti-allergic, antiinflammatory, and anti-proliferative indications, and can be directed for drug repositioning.

A pET22b plasmid containing the SARS-CoV-2 His-tagged 3CLPRO sequence was transformed into BL21 (DE3) Gold E. coli strain. Bacterial cultures were grown in 250 mL of LB/ampicillin (100 μg/mL) media at 37 °C overnight with gentle shaking. Then, 4 L of LB/ampicillin (100 μg/mL) were inoculated and incubated under the same conditions until reaching OD = 0.6 at a wavelength of 600nm. Protein expression was induced with 1 mM isopropyl 1-thio-β-Dgalactopyranoside (IPTG) at 18 °C for 5 h. Cells were harvested by centrifugation at 4 C for 10 min at 10000 rpm (using a Beckman Coulter Avanti J-26 XP Centrifuge) and then resuspended in lysis buffer (sodium phosphate 50 mM, pH 8, sodium chloride 500 mM). Cell rupture was achieved by sonication (using a Sonics Vibra-Cell Ultrasonic Liquid Processor) in ice, and 20 U/mL of benzonase (Merck-Millipore) were added to remove nucleic acids. To remove cellular debris, the extract was centrifuged at 4 °C for 30 min at 20000 rpm, and filtered through a 0.45 μm-pore membrane. After increasing imidazole concentration up to 10 mM, the protein was Circular dichroism (CD) spectra were recorded in a Chirascan spectropolarimeter (Applied Photophysics) at 25 °C. Far-UV spectrum was recorded at wavelengths between 190 and 260 nm in a 0.1 cm path-length cuvette. Near-UV spectrum was recorded at wavelengths between 250 and 310 nm in a 1-cm path-length cuvette. Protein concentration was 10 μM in all cases.

Fluorescence measurements were performed in a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies), monitoring the intrinsic tryptophan fluorescence of a protein solution at 2 μM concentration. An excitation wavelength of 290 nm was used, with excitation and emission bandwidths of 5 nm, and recording fluorescence emission between 300 and 400 nm. All spectroscopic measurements were made in sodium phosphate 50 mM, pH 8.

Thermal denaturations were monitored by CD and fluorescence, employing a protein concentration of 10 M and 2 M, respectively, and performing thermal scans with a scanning rate of 60 C/h.

Hydrodynamic radius of SARS-CoV-2 3CLpro was measured at different pH values for estimating the size of the oligomeric native state in a DynaPro NanoStar equipment (Wyatt Technology), employing a protein concentration of 3 M at pH 5, 7 and 8.

Self-association state of SARS-CoV-2 3CLpro was assessed employing an ÄKTA FPLC (GE Healthcare) using a Superdex Increase 75 10/30 chromatographic column, at pH 8 and 150 mM NaCl.

Thermal stability of SARS-CoV-2 3CLpro was assessed by temperature unfolding transitions monitored by high-precision differential scanning calorimetry (DSC). The partial molar heat capacity of the protein in solution was measured as a function of temperature in an Auto-

The first approach in DSC experimental analysis consists of applying a model-free data analysis for discriminating between different possibilities: two-state unfolding, non-two-state unfolding, and oligomer unfolding of the protein. From the thermogram (excess molar heat capacity of the protein as a function of the temperature, <C P (T)>), the calorimetric unfolding enthalpy, H cal , the unfolding temperature, T m , and the maximal unfolding heat capacity, C P,max , can be readily evaluated, from which it is possible to calculate the van't Hoff enthalpy, H vH : In the case of SARS-CoV-2 3CLpro, the thermograms under the different conditions assayed showed that H vH /H cal > 1 and, therefore, the protein is not monomeric and dissociates upon thermal unfolding. In fact, according to the crystallographic structure of the unliganded species, the protein is dimeric in its native state, although it has been reported that SARS-CoV-1 3CLpro can populate tetramers and octamers.

The conformational equilibrium is governed by the equilibrium constant, K U :

where N n is the oligomeric native state constituted of n monomeric subunits, and U is the unfolded state of each identical monomeric subunit. The total protein concentration can be expressed (per monomer) as:

from which the molar fractions of the different species can be calculated:

J o u r n a l P r e -p r o o f Given the equilibrium constant and the total protein concentration, Eq. 3 can be solved numerically for the unknown [U], and the molar fraction of both protein species can be calculated at any temperature, from which the excess average molar unfolding enthalpy, <H>(T) can be determined:

where H U is the enthalpy of the unfolded state, considering that of the native state as a reference. The temperature derivative of the excess molar unfolding enthalpy is the excess unfolding heat capacity at constant pressure, <C P >(T):

taking into account the temperature dependence of the different magnitudes:

where S U , G U and C P,U are the entropy, the Gibbs energy and the heat capacity of the unfolded state (taking the native state as a reference), respectively, and T 0 is an appropriate reference temperature. In this case, T 0 , is the temperature at which the unfolding Gibbs energy is zero, and does not coincide with the temperature for maximal heat capacity, T max , nor with the temperature for half denaturation, T 1/2 (i.e., the temperature at which F U = 0.5, or median temperature in the thermogram). This model particularized to n = 1 corresponds to the twostate model, for which T 0 is the apparent unfolding temperature, T m , which is very close to both T max and T 1/2 .

For a spectroscopically monitored unfolding of a homo-oligomeric protein, the procedure is similar, but the final observable quantity is the spectroscopic signal, S(T), which can be calculated as:

where S N (T) and S U (T) are the intrinsic spectroscopic signal values for the native and unfolded protein states, and can be considered linear functions of the temperature:

In vitro catalytic activity of 3CLpro was determined using a fluorescence resonance energy 

Screening of a small chemical library was performed based on the catalytic activity continuous 

where v is the initial slope of the enzymatic activity trace at a (free) compound concentration

[I], K m is the Michaelis-Menten constant for the enzyme-substrate interaction, [S] is the substrate concentration, and K i app is the apparent inhibition constant for the compound. If the inhibitor acts through a purely competitive mechanism, the previous equation can be substituted by the following one:

where K i is the intrinsic (i.e., substrate concentration-independent) inhibition constant. serial dilutions (ranging from 0 to 125 μM) for each compound were assayed by following the same protocol described above.

The interaction between the compound and 3CLpro was further assessed by isothermal titration calorimetry. Calorimetric titrations were performed using a high precision Auto- 

Molecular docking was performed by using the simulation software AutoDock Vina [23] and the supporting suite AutoDock Tools [24] . The structure of 3CLpro was extracted from the entries 6Y2E and 6Y2F [22] of the Protein Data Bank (PDB), which contain the crystallographic conformation of the protein in unliganded form and with an α-ketoamide inhibitor bound in the active site, respectively. The protein structures were considered in all cases free from any ligand and water molecules. A few missing residues in the entry 6Y2F were reconstructed in silico, and the structure of quercetin was also built by using the molecular editor Avogadro [25] .

A blind search was carried out on the whole protein surface, by considering a volume of size 50 Å × 60 Å × 60 Å. Extensive sampling was performed in all cases, using an exhaustiveness 16 times larger than the value normally recommended [26] .

Recombinant 3CLpro was expressed in E. coli at sufficiently high yield for initiating a biophysical characterization and an activity-based molecular screening ( Figure S1 ). The far-UV circular dichroism (CD) spectrum showed negative bands around 208 and 222 nm, typical of proteins with -helical and -sheet content ( Figure 1A ), in agreement with the crystallographic structure [22] . The near-UV CD spectrum indicated aromatic residues, especially tryptophans, are enclosed within an asymmetric environment ( Figure 1B) . The fluorescence spectrum monitoring the intrinsic emission of the tryptophans (there are three tryptophan residues, two located in the -helical domain and one in the -sheet domain) showed a maximum around 330, confirming that some of them are partially exposed to the solvent ( Figure 1C ). These results confirmed 3CLpro adopted a folded conformation with well-defined secondary and tertiary structures.

3CLpro from other coronaviruses (SARS-CoV and MERS) dimerizes, and such dimerization is known to be essential for the enzymatic activity; even higher-level oligomerization structures (tetramers and octamers) have been reported [27] . Among other experimental techniques providing information about protein oligomerization (such as analytical ultracentrifugation and gel filtration chromatography), thermal denaturation monitored by spectroscopy or calorimetry can be used to detect the self-association of proteins, since the thermal unfolding of monomeric and oligomeric proteins show markedly different features [28] .

Thermal unfolding of 3CLpro followed by far-UV CD showed a single transition with an apparent unfolding temperature close to 51 C (Figure 2A ). The same process followed by fluorescence also showed a single transition with an apparent unfolding temperature close to 48.5 C ( Figure 2B ). The small difference in the unfolding temperature could be justified by the use of a different protein concentration in CD and fluorescence assays and considering the oligomer-monomer equilibrium coupled to the unfolding process. Non-linear regression analysis of the unfolding traces according to different two-state models for a monomeric (n = 1, see equation (1)), a dimeric (n = 2), a tetrameric protein (n = 4), and an octameric protein (n = 8) were performed (see Materials & Methods section). These experiments confirmed 3CLpro adopts a well-folded conformation under native conditions and becomes unstructured upon temperature stress. However, it was not possible to discriminate which model reproduces better the experimental data, because the fitting curves from the different models overlapped.

The fitting was marginally better (according to the residual sum of squares; see Table S1 ) for the two-state monomer model in the case of CD unfolding, and for the two-state tetramer model in the fluorescence unfolding. Therefore, spectroscopic unfolding traces were not sensitive enough for discriminating the self-association nature of 3CLpro native state.

Thermal unfolding monitored by differential scanning calorimetry (DSC) also showed a single apparent unfolding transition with a marked dependence on the pH ( Figure 3A ). The lower thermal stability observed at pH 5 and 8, compared to that at pH 7, may reflect the protonation/deprotonation equilibrium of ionizable sidechains titratable around pH 6-7 (e.g., see Table S1 ). Application of a more complex model considering the coexistence of tetramers and dimers (T  2D  4U) did not improve the results (Table S1 ). In addition, other models considering a two-step unfolding process where the dimeric or tetrameric states undergo a first transition with no dissociation (N n  N n *) followed by a second transition with coupled unfolding-dissociation (N n *  nU) did not improve the results either. This indicates that any unfolding model considering more than one step lacks the sufficient cooperativity to reproduce the experimental results. Therefore, calorimetric unfolding traces were sensitive enough for discriminating the self-association nature of 3CLpro native state, and very likely there is a pH dependence of the quaternary rearrangement in 3CLpro.

The enzymatic activity of 3CLpro was measured using a FRET substrate containing Very likely, the catalytic histidine is responsible for this pH effect.

The enzymatic parameters were estimated from the initial enzymatic rate dependence on the substrate concentration at pH 8 ( Figure S3 ): K m = 11 M and k cat = 0.040 s -1 , resulting in a catalytic efficiency, k cat /K m of 3640 M -1 s -1 , comparable to reported values considering the differences in the experimental conditions [21, 22] .

The continuous FRET assay for measuring the activity of 3CLpro was adapted to an in vitro experimental screening procedure. In a first step, the screening was performed using a small chemical library consisting of 150 compounds, which also included approved drugs with known therapeutic indications. Hits consisted of compounds reducing the enzymatic activity of 3CLpro below a specified threshold, defined as the average activity for the controls (enzyme with no compound) minus twice their corresponding standard deviation ( Figure 5 ). Several hits were selected and confirmed by the inhibition curve analysis.

To assess the in vitro potency of the compounds selected through the small-scale screening, their inhibition constants was estimated from the inhibition curve. Inhibition curves were obtained by measuring the enzymatic activity, applying the previously described protocol for the continuous FRET assay, by fixing the enzyme and the substrate concentrations while varying the amount of compound ( Figure 6A ). Enzymatic activity at each compound concentration was calculated as the initial slope in each curve, and a dose-dependent effect of the compound on the enzyme activity was observed ( Figure 6B ). Non-linear regression analysis employing a simple inhibition model, according to equation (10) With a K m value of 11 µM recently reported in the literature [21, 22] , an intrinsic inhibition J o u r n a l P r e -p r o o f 14 constant K i of 7.4 M could be estimated for quercetin. This potency is similar to some of the first inhibitors found for SARS-CoV 3CLpro [30] .

In general, the interaction of a ligand with a protein results in a change in the stability of the protein against thermal or chemical denaturation. More precisely, the preferential interaction of the ligand with certain conformational state stabilizes that state, resulting in a net global stabilization effect if the preferential interaction occurs with the native state or a net destabilization if the preferential interaction occurs with the non-native state. Thermal shift assay (TSA), also called differential scanning fluorimetry, consists in detecting ligand-induced stability changes in proteins by fluorescence [31, 32] . Using an extrinsic fluorescent probe reporting the progression of the protein unfolding process, it is possible to assess the thermal stability of the protein by measuring its apparent T m in the presence of increasing concentrations of a given interacting compound.

The identified compound, quercetin, altered the thermal stability of 3CLpro causing a destabilization ( Figure 7A ). Although ligand-induced stabilizing effects on a protein are more common, it is not unusual to observe destabilizing effects [33] [34] [35] . In addition, the effect of the compounds was concentration-dependent ( Figure 7B ).

Isothermal titration calorimetry (ITC) is considered the gold-standard for binding affinity determination, as well as target engagement in drug discovery [36, 37] . Careful experimental design in ITC provides invaluable and detailed information about the drug-target interaction [38] . ITC was employed to assess target engagement for quercetin and get a direct 

The need for pharmacological agents acting against SARS-CoV-2 is urgent. The development of drugs acting through different mechanisms provides a good starting point for a combined use in a therapy with low susceptibility to drug resistance. 3CLpro (or Mpro) is an appropriate target, given its high conservation among coronaviruses, as well as a suitable experimental tractability and druggability. Drug discovery can be initiated from experimental or computational screenings, by employing small or large, highly diverse or focused (targetderived) chemical libraries. On the basis of an experimental pipeline for drug screening successfully employed in the past [35, [39] [40] [41] [42] [43] [44] [45] , we have applied the same methodology to identify small molecules blocking the activity of SARS-CoV-2 3CLpro.

The first step in this screening program was to assess the stability and the in vitro activity of SARS-CoV-2 3CLpro. According to our results, 3CLpro is well folded in solution and exhibits a large kinetic stability (activation energy barrier  200 kcal/mol), which allowed us to apply equilibrium models for unfolding. It has been established that monomeric 3CLpro is inactive [46] and the functional state of this protein is the homodimer. We observed that 3CLpro not only populates dimeric states in solution, but also tetrameric states, depending on J o u r n a l P r e -p r o o f the pH. Interestingly, even tetrameric and highly-active octameric states have been previously reported for 3CLpro [22, 27] . In addition, the effect of pH on the structural stability parallels that of the enzymatic activity, suggesting some titratable residues in the range of pH 5-8 are instrumental to guarantee both these properties. Hydrodynamic radius measurements by dynamic light scattering showed a predominant population of particles compatible with dimeric 3CLpro (hydrodynamic radius of 3.89, 3.33, and 3.94 nm at pH 5, 7, and 8, respectively), with an expected smaller size around its isoelectric point (pI  6) due to smaller net charge and lower electrostatic repulsions ( Figure S4 ). Additional experiments using sizeexclusion chromatography were also in agreement with a predominantly dimeric 3CLpro ( Figure S4 ). It may be hypothesized that, along the thermal denaturations, temperature could trigger a transient dimerization of dimers into tetramers (tetrameric intermediate) at

moderate temperatures, a temperature-driven oligomerization process that has been observed in other proteins [47, 48] , before reaching the unfolding temperature and ending in monomer unfolding/dissociation; work on this matter is in progress.

The screening test for 3CLpro could be based on detecting small molecules interfering with the enzymatic activity (which it is fairly easy for a protease, by using a continuous enzymatic assay and a peptidic FRET substrate) or based on detecting the effect of small molecules on altering the thermal stability of the protein against thermal denaturation (by using fluorescence TSA).

The TSA-based screening has the advantage of being quite general for any protein, but it has some disadvantages: 1) binding affinity does not correlate with stability changes; 2) false negatives are somewhat common, because ligands may bind to unfolded or non-native protein states besides binding to the native state; 3) stability changes are highly dependent on the binding enthalpy and binding heat capacity; and 4) the ligand binding does not always translates into an inhibitory effect [49] . In contrast, the techniques based on detection of the protein activity provide direct evidence for inhibition during the screening test, but the activity of the protein target must be measurable through a quick and appropriate procedure, which is not straightforward in general.

Through the application of a small-scale activity-based screening to SARS-CoV-2 3CLpro, the quercetin was selected and further studied. Direct evidence for target engagement was gathered by different biophysical techniques, TSA and ITC, as well as through molecular docking simulations. Quercetin showed an inhibition constant of about 7 µM, which corresponds to a binding affinity sufficiently favourable to be considered as a good candidate for optimization or preclinical studies.

The fact that quercetin exerts a destabilizing effect may be related to its physico-chemical properties and the type of interaction with the protein target. The extent of the stabilization J o u r n a l P r e -p r o o f effect induced by a ligand depends on the specific set of conformational states, within the whole conformational landscape of the protein, that interact preferentially with the ligand. If the ligand interacts with most of the conformational states with similar binding affinities, the effect on the protein stability will be small. If the ligand interacts not only with the native state, but also with partially unfolded states (which could be the case for quercetin, due to its hydrophobic moiety), a destabilization effect could be expected if the affinity (or the binding stoichiometry) for the non-native states is higher. These are some of the caveats (described above) to be born in mind when TSA is applied for in vitro molecular screening. In addition, unlike in the case of isothermal inhibition assays, where only native-like states are accessible, through TSA it is possible to gradually explore non-native states close to the native state through a temperature-driven process, and binding to non-native states can be detected. In Quercetin compares quite well to SARS-CoV-2 3CLpro inhibitors already reported. An intrinsic inhibition constant K i of 0.19 M could be estimated for an alpha-ketoamide inhibitor 13b

reported by Zhang et al [22] . In order to compare the inhibition potency of ligands, their molecular size must be considered. The ligand efficiency or binding efficiency index (BEI = pK i /MW) normalizes the inhibitor potency (pK i = -logK i ) with its molecular weight (MW)[50].

Because quercetin and inhibitor 13b have a MW of 0.302 kDa and 0.598 kDa, respectively, their ligand efficiencies are 17.0 and 11.2, respectively. Thus, although the alpha-ketoamide inhibitor has a much lower inhibition constant, which is not surprising given its larger MW of the alpha-ketoamide inhibitor; in fact, quercetin is close to be considered as a fragment (MW < 300 Da) from a screening perspective. However, BEI is significantly higher for quercetin, suggesting it is a good candidate establishing good interactions with the target and possessing high potential for optimization and development.

It is worth to note that quercetin is a natural product with well-known pharmacokinetics and 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.  SARS-CoV-2 3CLpro shows a pH-dependent hydrolytic activity that correlates with its structural stability.

 SARS-CoV-2 3CLpro is an appropriate target for drug discovery given its high sequence identity among different coronaviruses.

 Quercetin has been identified as a SARS-CoV-2 3CLpro inhibitor by an activity-based experimental screening.

 Experimental and computational evidences for quercetin target engagement have been obtained by ITC, spectroscopy, TSA, and molecular docking.

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Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas Digestivas (CIBERehd)

Institute for Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC

Writing -Original Draft Preparation

Project Administration, O.A. and A.V.C.; Funding Acquisition

Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas Digestivas (CIBERehd)

Institute for Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC

The authors declare no conflict of interest.

Supplementary Figures S1, S2, S3, S4 and S5, and Tables S1 and S2.