RESUMO
The development of specific antiviral therapies targeting SARS-CoV-2 remains fundamental because of the continued high incidence of COVID-19 and limited accessibility to antivirals in some countries. In this context, dark chemical matter (DCM), a set of drug-like compounds with outstanding selectivity profiles that have never shown bioactivity despite being extensively assayed, appears to be an excellent starting point for drug development. Accordingly, in this study, we performed a high-throughput screening to identify inhibitors of the SARS-CoV-2 main protease (Mpro) using DCM compounds as ligands. Multiple receptors and two different docking scoring functions were employed to identify the best molecular docking poses. The selected structures were subjected to extensive conventional and Gaussian accelerated molecular dynamics. From the results, four compounds with the best molecular behavior and binding energy were selected for experimental testing, one of which presented inhibitory activity with a Ki value of 48 ± 5 µM. Through virtual screening, we identified a significant starting point for drug development, shedding new light on DCM compounds.
Assuntos
Antivirais , Proteases 3C de Coronavírus , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases , SARS-CoV-2 , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Antivirais/farmacologia , Antivirais/química , Humanos , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , COVID-19/virologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Ligação Proteica , LigantesRESUMO
MeCP2 is a general regulator of transcription involved in the repression/activation of genes depending on the local epigenetic context. It acts as a chromatin regulator and binds with exquisite specificity to gene promoters. The set of epigenetic marks recognized by MeCP2 has been already established (mainly, cytosine modifications in CpG and CpA), as well as many of the constituents of its interactome. We unveil a new set of interactions for MeCP2 with the four canonical nucleosomal histones. MeCP2 interacts with high affinity with H2A, H2B, H3 and H4. In addition, Rett syndrome associated mutations in MeCP2 and histone epigenetic marks modulate these interactions. Given the abundance and the structural/functional relevance of histones and their involvement in epigenetic regulation, this new set of interactions and its modulating elements provide a new addition to the 'alphabet' for this epigenetic reader.
Assuntos
Epigênese Genética , Histonas , Proteína 2 de Ligação a Metil-CpG , Nucleossomos , Proteína 2 de Ligação a Metil-CpG/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Nucleossomos/metabolismo , Histonas/metabolismo , Humanos , Ligação Proteica , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Mutação , AnimaisRESUMO
Over 750 million cases of COVID-19, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), have been reported since the onset of the global outbreak. The need for effective treatments has spurred intensive research for therapeutic agents based on pharmaceutical repositioning or natural products. In light of prior studies asserting the bioactivity of natural compounds of the autochthonous Peruvian flora, the present study focuses on the identification SARS-CoV-2 Mpro main protease dimer inhibitors. To this end, a target-based virtual screening was performed over a representative set of Peruvian flora-derived natural compounds. The best poses obtained from the ensemble molecular docking process were selected. These structures were subjected to extensive molecular dynamics steps for the computation of binding free energies along the trajectory and evaluation of the stability of the complexes. The compounds exhibiting the best free energy behaviors were selected for in vitro testing, confirming the inhibitory activity of Hyperoside against Mpro, with a Ki value lower than 20 µM, presumably through allosteric modulation.
RESUMO
Hydroxymethylated cytosine (5hmC) is a stable DNA epigenetic mark recognized by methyl-CpG binding protein 2 (MeCP2), which acts as a transcriptional regulator and a global chromatin-remodeling element. Because 5hmC triggers a gene regulation response markedly different from that produced by methylated cytosine (5mC), both modifications must affect DNA structure and/or DNA interaction with MeCP2 differently. MeCP2 is a six-domain intrinsically disordered protein (IDP) with two domains responsible for dsDNA binding: methyl-CpG binding domain (MBD) and intervening domain (ID). Here we report the detailed thermodynamic characterization of the interaction of hmCpG-DNA with MeCP2. We find that hmCpG-DNA interacts with MeCP2 in a distinctly different mode with a particular thermodynamic signature, compared to methylated or unmethylated DNA. In addition, we find evidence for Rett syndrome-associated mutations altering the interaction of MeCP2 with dsDNA in a cytosine modification-specific manner which may correlate with disease onset time and clinical severity score.
Assuntos
Cromatina , DNA , Citosina , Epigenômica , TermodinâmicaRESUMO
Isoprenoids are a very large and diverse family of metabolites required by all living organisms. All isoprenoids derive from the double-bond isomers isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are produced by the methylerythritol 4-phosphate (MEP) pathway in bacteria and plant plastids. It has been reported that IPP and DMAPP feedback-regulate the activity of deoxyxylulose 5-phosphate synthase (DXS), a dimeric enzyme that catalyzes the main flux-controlling step of the MEP pathway. Here we provide experimental insights into the underlying mechanism. Isothermal titration calorimetry and dynamic light scattering approaches showed that IPP and DMAPP can allosterically bind to DXS in vitro, causing a size shift. In silico ligand binding site analysis and docking calculations identified a potential allosteric site in the contact region between the two monomers of the active DXS dimer. Modulation of IPP and DMAPP contents in vivo followed by immunoblot analyses confirmed that high IPP/DMAPP levels resulted in monomerization and eventual aggregation of the enzyme in bacterial and plant cells. Loss of the enzymatically active dimeric conformation allows a fast and reversible reduction of DXS activity in response to a sudden increase or decrease in IPP/DMAPP supply, whereas aggregation and subsequent removal of monomers that would otherwise be available for dimerization appears to be a more drastic response in the case of persistent IPP/DMAPP overabundance (e.g., by a blockage in their conversion to downstream isoprenoids). Our results represent an important step toward understanding the regulation of the MEP pathway and rational design of biotechnological endeavors aimed at increasing isoprenoid contents in microbial and plant systems.
Assuntos
Plantas , Terpenos , Retroalimentação , Terpenos/metabolismo , Plantas/metabolismo , FosfatosRESUMO
Transthyretin amyloidosis (ATTR) is a group of fatal diseases described by the misfolding and amyloid deposition of transthyretin (TTR). Discovering small molecules that bind and stabilize the TTR tetramer, preventing its dissociation and subsequent aggregation, is a therapeutic strategy for these pathologies. Departing from the crystal structure of TTR in complex with tolcapone, a potent binder in clinical trials for ATTR, we combined rational design and molecular dynamics (MD) simulations to generate a series of novel halogenated kinetic stabilizers. Among them, M-23 displays one of the highest affinities for TTR described so far. The TTR/M-23 crystal structure confirmed the formation of unprecedented protein-ligand contacts, as predicted by MD simulations, leading to an enhanced tetramer stability both in vitro and in whole serum. We demonstrate that MD-assisted design of TTR ligands constitutes a new avenue for discovering molecules that, like M-23, hold the potential to become highly potent drugs to treat ATTR.
Assuntos
Neuropatias Amiloides Familiares , Pré-Albumina , Humanos , Pré-Albumina/química , Amiloide/metabolismo , Neuropatias Amiloides Familiares/tratamento farmacológico , Neuropatias Amiloides Familiares/metabolismo , Tolcapona/uso terapêutico , CinéticaRESUMO
SARS-CoV-2 is a type of coronavirus responsible for the international outbreak of respiratory illness termed COVID-19 that forced the World Health Organization to declare a pandemic infectious disease situation of international concern at the beginning of 2020. The need for a swift response against COVID-19 prompted to consider different sources to identify bioactive compounds that can be used as therapeutic agents, including available drugs and natural products. Accordingly, this work reports the results of a virtual screening process aimed at identifying antiviral natural product inhibitors of the SARS-CoV-2 Mpro viral protease. For this purpose, ca. 2000 compounds of the Selleck database of Natural Compounds were the subject of an ensemble docking process targeting the Mpro protease. Molecules that showed binding to most of the protein conformations were retained for a further step that involved the computation of the binding free energy of the ligand-Mpro complex along a molecular dynamics trajectory. The compounds that showed a smooth binding free energy behavior were selected for in vitro testing. From the resulting set of compounds, five compounds exhibited an antiviral profile, and they are disclosed in the present work.
Assuntos
Produtos Biológicos , COVID-19 , Antivirais/farmacologia , Produtos Biológicos/farmacologia , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeo Hidrolases , Inibidores de Proteases/farmacologia , SARS-CoV-2RESUMO
The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr9-30), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr9-30 is mainly disordered. We attempted to improve the affinity of HPr9-30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr9-30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. Therefore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Histidina/metabolismo , Mutação , Fragmentos de Peptídeos/farmacologia , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Domínio Catalítico , Histidina/química , Fragmentos de Peptídeos/química , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Staphylococcus aureus/metabolismoRESUMO
The COVID-19 pandemic outbreak prompts an urgent need for efficient therapeutics, and repurposing of known drugs has been extensively used in an attempt to get to anti-SARS-CoV-2 agents in the shortest possible time. The glycoside rutin shows manifold pharmacological activities and, despite its use being limited by its poor solubility in water, it is the active principle of many pharmaceutical preparations. We herein report our in silico and experimental investigations of rutin as a SARS-CoV-2 Mpro inhibitor and of its water solubility improvement obtained by mixing it with l-arginine. Tests of the rutin/l-arginine mixture in a cellular model of SARS-CoV-2 infection highlighted that the mixture still suffers from unfavorable pharmacokinetic properties, but nonetheless, the results of this study suggest that rutin might be a good starting point for hit optimization.
Assuntos
Antivirais/farmacologia , Arginina/farmacologia , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Rutina/farmacologia , SARS-CoV-2/efeitos dos fármacos , Células A549 , Proteases 3C de Coronavírus/metabolismo , Humanos , Simulação de Acoplamento Molecular , Inibidores de Proteases/farmacologia , SARS-CoV-2/metabolismo , SolubilidadeRESUMO
Inhibiting the main protease 3CLpro is the most common strategy in the search for antiviral drugs to fight the infection from SARS-CoV-2. We report that the natural compound eugenol is able to hamper in vitro the enzymatic activity of 3CLpro, the SARS-CoV-2 main protease, with an inhibition constant in the sub-micromolar range (Ki = 0.81 µM). Two phenylpropene analogs were also tested: the same effect was observed for estragole with a lower potency (Ki = 4.1 µM), whereas anethole was less active. The binding efficiency index of these compounds is remarkably favorable due also to their small molecular mass (MW < 165 Da). We envision that nanomolar inhibition of 3CLpro is widely accessible within the chemical space of simple natural compounds.
RESUMO
Intrinsic disorder plays an important functional role in proteins. Disordered regions are linked to posttranslational modifications, conformational switching, extra/intracellular trafficking, and allosteric control, among other phenomena. Disorder provides proteins with enhanced plasticity, resulting in a dynamic protein conformational/functional landscape, with well-structured and disordered regions displaying reciprocal, interdependent features. Although lacking well-defined conformation, disordered regions may affect the intrinsic stability and functional properties of ordered regions. MeCP2, methyl-CpG binding protein 2, is a multifunctional transcriptional regulator associated with neuronal development and maturation. MeCP2 multidomain structure makes it a prototype for multidomain, multifunctional, intrinsically disordered proteins (IDP). The methyl-binding domain (MBD) is one of the key domains in MeCP2, responsible for DNA recognition. It has been reported previously that the two disordered domains flanking MBD, the N-terminal domain (NTD) and the intervening domain (ID), increase the intrinsic stability of MBD against thermal denaturation. In order to prove unequivocally this stabilization effect, ruling out any artifactual result from monitoring the unfolding MBD with a local fluorescence probe (the single tryptophan in MBD) or from driving the protein unfolding by temperature, we have studied the MBD stability by differential scanning calorimetry (reporting on the global unfolding process) and chemical denaturation (altering intramolecular interactions by a different mechanism compared to thermal denaturation).
Assuntos
DNA/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , Sítios de Ligação , Regulação da Expressão Gênica , Humanos , Ligação Proteica , Domínios Proteicos , Desdobramento de ProteínaRESUMO
The development of new antiviral drugs against SARS-CoV-2 is a valuable long-term strategy to protect the global population from the COVID-19 pandemic complementary to the vaccination. Considering this, the viral main protease (Mpro) is among the most promising molecular targets in light of its importance during the viral replication cycle. The natural flavonoid quercetin 1 has been recently reported to be a potent Mpro inhibitor in vitro, and we explored the effect produced by the introduction of organoselenium functionalities in this scaffold. In particular, we report here a new synthetic method to prepare previously inaccessible C-8 seleno-quercetin derivatives. By screening a small library of flavonols and flavone derivatives, we observed that some compounds inhibit the protease activity in vitro. For the first time, we demonstrate that quercetin (1) and 8-(p-tolylselenyl)quercetin (2d) block SARS-CoV-2 replication in infected cells at non-toxic concentrations, with an IC50 of 192 µM and 8 µM, respectively. Based on docking experiments driven by experimental evidence, we propose a non-covalent mechanism for Mpro inhibition in which a hydrogen bond between the selenium atom and Gln189 residue in the catalytic pocket could explain the higher Mpro activity of 2d and, as a result, its better antiviral profile.
Assuntos
Antivirais/química , Quercetina/química , SARS-CoV-2/metabolismo , Selênio/química , Proteínas da Matriz Viral/antagonistas & inibidores , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico , Chlorocebus aethiops , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Quercetina/metabolismo , Quercetina/farmacologia , SARS-CoV-2/isolamento & purificação , Selênio/metabolismo , Células Vero , Proteínas da Matriz Viral/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.
Assuntos
Benchmarking , Laboratórios , Calorimetria , Reprodutibilidade dos Testes , TemperaturaRESUMO
BACKGROUND: Plakophilin 1 (PKP1) is a component of desmosomes, which are key structural components for cell-cell adhesion, and can also be found in other cell locations. The p53, p63 and p73 proteins belong to the p53 family of transcription factors, playing crucial roles in tumour suppression. The α-splice variant of p73 (p73α) has at its C terminus a sterile alpha motif (SAM); such domain, SAMp73, is involved in the interaction with other macromolecules. METHODS: We studied the binding of SAMp73 with the armadillo domain of PKP1 (ARM-PKP1) in the absence and the presence of 100 mM NaCl, by using several biophysical techniques, namely fluorescence, far-ultraviolet circular dichroism (CD), nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), and molecular docking and simulations. RESULTS: Association was observed between the two proteins, with a dissociation constant of ~5 µM measured by ITC and fluorescence in the absence of NaCl. The binding region of SAMp73 involved residues of the so-called "middle-loop-end-helix" binding region (i.e., comprising the third helix, together with the C terminus of the second one, and the N-cap of the fourth), as shown by 15N, 1H- HSQC-NMR spectra. Molecular modelling provided additional information on the possible structure of the binding complex. CONCLUSIONS: This newly-observed interaction could have potential therapeutic relevance in the tumour pathways where PKP1 is involved, and under conditions when there is a possible inactivation of p53. GENERAL SIGNIFICANCE: The discovery of the binding between SAMp73 and ARM-PKP1 suggests a functional role for their interaction, including the possibility that SAMp73 could assist PKP1 in signalling pathways.
Assuntos
Proteínas do Domínio Armadillo/metabolismo , Placofilinas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Motivo Estéril alfa , Proteína Tumoral p73/metabolismo , Proteínas do Domínio Armadillo/química , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Placofilinas/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteína Tumoral p73/químicaRESUMO
The pandemic, due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has stimulated the search for antivirals to tackle COVID-19 infection. Molecules with known pharmacokinetics and already approved for human use have been demonstrated or predicted to be suitable to be used either directly or as a base for a scaffold-based drug design. Among these substances, quercetin is known to be a potent in vitro inhibitor of 3CLpro, the SARS-CoV-2 main protease. However, its low in vivo bioavailability calls for modifications to its molecular structure. In this work, this issue is addressed by using rutin, a natural flavonoid that is the most common glycosylated conjugate of quercetin, as a model. Combining experimental (spectroscopy and calorimetry) and simulation techniques (docking and molecular dynamics simulations), we demonstrate that the sugar adduct does not hamper rutin binding to 3CLpro, and the conjugated compound preserves a high potency (inhibition constant in the low micromolar range, Ki = 11 µM). Although showing a disruption of the pseudo-symmetry in the chemical structure, a larger steric volume and molecular weight, and a higher solubility compared to quercetin, rutin is able to associate in the active site of 3CLpro, interacting with the catalytic dyad (His41/Cys145). The overall results have implications in the drug-design of quercetin analogs, and possibly other antivirals, to target the catalytic site of the SARS-CoV-2 3CLpro.
RESUMO
Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-associated structural protein. MeCP2 deregulation results in two neurodevelopmental disorders: MeCP2 dysfunction is associated with Rett syndrome, while excess of activity is associated with MeCP2 duplication syndrome. MeCP2 is an intrinsically disordered protein (IDP) constituted by six structural domains with variable, small percentage of well-defined secondary structure. Two domains, methyl-CpG binding domain (MBD) and transcription repressor domain (TRD), are the elements responsible for dsDNA binding ability and recruitment of the gene transcription/silencing machinery, respectively. Previously we studied the influence of the completely disordered, MBD-flanking domains (N-terminal domain, NTD, and intervening domain, ID) on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41,635). Here we report the biophysical study of the influence of the remaining domains (transcriptional repressor domain, TRD, and C-terminal domains, CTDα and CTDß) on the structural stability of MBD and the dsDNA binding capabilities of MBD and ID. The influence of distant disordered domains on MBD properties makes it necessary to consider the NTD-MBD-ID variant as the minimal protein construct for studying dsDNA/chromatin binding properties, while the full-length protein should be considered for transcriptional regulation studies.
Assuntos
Proteína 2 de Ligação a Metil-CpG/química , Proteína 2 de Ligação a Metil-CpG/metabolismo , Cromatina/química , DNA/química , Metilação de DNA/fisiologia , Proteínas de Ligação a DNA/química , Humanos , Mutação , Ligação Proteica/fisiologia , Domínios Proteicos/fisiologia , Estabilidade Proteica , Estrutura Secundária de Proteína/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-binding protein involved in neuronal development and maturation. Loss-of-function mutations in MeCP2 result in Rett syndrome (RTT), a neurodevelopmental disorder that is the main cause of mental retardation in females. MeCP2 is an intrinsically disordered protein (IDP) constituted by six domains. Two domains are the main responsible elements for DNA binding (methyl-CpG binding domain, MBD) and recruitment of gene transcription/silencing machinery (transcription repressor domain, TRD). These two domains concentrate most of the RTT-associated mutations. R106W and R133C are associated with severe and mild RTT phenotype, respectively. We have performed a comprehensive characterization of the structural and functional impact of these substitutions at molecular level. Because we have previously shown that the MBD-flanking disordered domains (N-terminal domain, NTD, and intervening domain, ID) exert a considerable influence on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41635), here we report the biophysical study of the influence of the protein scaffold on the structural and functional effect induced by these two RTT-associated mutations. These results represent an example of how a given mutation may show different effects (sometimes opposing effects) depending on the molecular context.
Assuntos
Proteína 2 de Ligação a Metil-CpG/genética , Mutação , Síndrome de Rett/genética , Feminino , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Masculino , Proteína 2 de Ligação a Metil-CpG/química , Proteína 2 de Ligação a Metil-CpG/metabolismo , Fenótipo , Domínios Proteicos , Estabilidade Proteica , Síndrome de Rett/metabolismoRESUMO
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.