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1.
Sci Rep ; 11(1): 17748, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34493768

RESUMO

Based on WHO reports the new SARS-CoV-2 coronavirus is currently widespread all over the world. So far > 162 million cases have been confirmed, including > 3 million deaths. Because of the pandemic still spreading across the globe the accomplishment of computational methods to find new potential mechanisms of virus inhibitions is necessary. According to the fact that C60 fullerene (a sphere-shaped molecule consisting of carbon) has shown inhibitory activity against various protein targets, here the analysis of the potential binding mechanism between SARS-CoV-2 proteins 3CLpro and RdRp with C60 fullerene was done; it has resulted in one and two possible binding mechanisms, respectively. In the case of 3CLpro, C60 fullerene interacts in the catalytic binding pocket. And for RdRp in the first model C60 fullerene blocks RNA synthesis pore and in the second one it prevents binding with Nsp8 co-factor (without this complex formation, RdRp can't perform its initial functions). Then the molecular dynamics simulation confirmed the stability of created complexes. The obtained results might be a basis for other computational studies of 3CLPro and RdRp potential inhibition ways as well as the potential usage of C60 fullerene in the fight against COVID-19 disease.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Fulerenos/farmacologia , Antivirais/uso terapêutico , COVID-19/epidemiologia , COVID-19/virologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/ultraestrutura , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologia , Inibidores de Protease de Coronavírus/uso terapêutico , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , RNA-Polimerase RNA-Dependente de Coronavírus/ultraestrutura , Cristalografia por Raios X , Fulerenos/química , Fulerenos/uso terapêutico , Humanos , Simulação de Dinâmica Molecular , Inibidores da Síntese de Ácido Nucleico/química , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores da Síntese de Ácido Nucleico/uso terapêutico , Pandemias/prevenção & controle , RNA Viral/biossíntese , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , SARS-CoV-2/ultraestrutura
2.
Molecules ; 26(17)2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34500664

RESUMO

This study aims to identify and isolate the secondary metabolites of Zingiber officinale using GC-MS, preparative TLC, and LC-MS/MS methods, to evaluate the inhibitory potency on SARS-CoV-2 3 chymotrypsin-like protease enzyme, as well as to study the molecular interaction and stability by using docking and molecular dynamics simulations. GC-MS analysis suggested for the isolation of terpenoids compounds as major compounds on methanol extract of pseudostems and rhizomes. Isolation and LC-MS/MS analysis identified 5-hydro-7, 8, 2'-trimethoxyflavanone (9), (E)-hexadecyl-ferulate (1), isocyperol (2), N-isobutyl-(2E,4E)-octadecadienamide (3), and nootkatone (4) from the rhizome extract, as well as from the leaves extract with the absence of 9. Three known steroid compounds, i.e., spinasterone (7), spinasterol (8), and 24-methylcholesta-7-en-3ß-on (6), were further identified from the pseudostem extract. Molecular docking showed that steroids compounds 7, 8, and 6 have lower predictive binding energies (MMGBSA) than other metabolites with binding energy of -87.91, -78.11, and -68.80 kcal/mole, respectively. Further characterization on the single isolated compound by NMR showed that 6 was identified and possessed 75% inhibitory activity on SARS-CoV-2 3CL protease enzyme that was slightly different with the positive control GC376 (77%). MD simulations showed the complex stability with compound 6 during 100 ns simulation time.


Assuntos
COVID-19/tratamento farmacológico , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Protease de Coronavírus/farmacologia , Gengibre/química , Extratos Vegetais/farmacologia , Proteases 3C de Coronavírus/metabolismo , Proteases 3C de Coronavírus/ultraestrutura , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/isolamento & purificação , Inibidores de Protease de Coronavírus/uso terapêutico , Cristalografia por Raios X , Ensaios Enzimáticos , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/uso terapêutico , Pirrolidinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Relação Estrutura-Atividade , Ácidos Sulfônicos/farmacologia
3.
Chem Commun (Camb) ; 57(72): 9096-9099, 2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-34498651

RESUMO

We present a detailed computational analysis of the binding mode and reactivity of the novel oral inhibitor PF-07321332 developed against the SARS-CoV-2 3CL protease. Alchemical free energy calculations suggest that positions P3 and P4 could be susceptible to improvement in order to get a larger binding strength. QM/MM simulations unveil the reaction mechanism for covalent inhibition, showing that the nitrile warhead facilitates the recruitment of a water molecule for the proton transfer step.


Assuntos
Proteases 3C de Coronavírus/antagonistas & inibidores , Simulação de Dinâmica Molecular , Nitrilas/química , Inibidores de Proteases/química , SARS-CoV-2/enzimologia , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico , Proteases 3C de Coronavírus/metabolismo , Humanos , Lactamas/química , Lactamas/metabolismo , Leucina/química , Leucina/metabolismo , Nitrilas/metabolismo , Prolina/química , Prolina/metabolismo , Inibidores de Proteases/metabolismo , Teoria Quântica , SARS-CoV-2/isolamento & purificação , Termodinâmica
4.
Int J Mol Sci ; 22(17)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34502033

RESUMO

The novel coronavirus disease, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), rapidly spreading around the world, poses a major threat to the global public health. Herein, we demonstrated the binding mechanism of PF-07321332, α-ketoamide, lopinavir, and ritonavir to the coronavirus 3-chymotrypsin-like-protease (3CLpro) by means of docking and molecular dynamic (MD) simulations. The analysis of MD trajectories of 3CLpro with PF-07321332, α-ketoamide, lopinavir, and ritonavir revealed that 3CLpro-PF-07321332 and 3CLpro-α-ketoamide complexes remained stable compared with 3CLpro-ritonavir and 3CLpro-lopinavir. Investigating the dynamic behavior of ligand-protein interaction, ligands PF-07321332 and α-ketoamide showed stronger bonding via making interactions with catalytic dyad residues His41-Cys145 of 3CLpro. Lopinavir and ritonavir were unable to disrupt the catalytic dyad, as illustrated by increased bond length during the MD simulation. To decipher the ligand binding mode and affinity, ligand interactions with SARS-CoV-2 proteases and binding energy were calculated. The binding energy of the bespoke antiviral PF-07321332 clinical candidate was two times higher than that of α-ketoamide and three times than that of lopinavir and ritonavir. Our study elucidated in detail the binding mechanism of the potent PF-07321332 to 3CLpro along with the low potency of lopinavir and ritonavir due to weak binding affinity demonstrated by the binding energy data. This study will be helpful for the development and optimization of more specific compounds to combat coronavirus disease.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Protease de Coronavírus/farmacologia , Lactamas/farmacologia , Leucina/farmacologia , Nitrilas/farmacologia , Prolina/farmacologia , Antivirais/uso terapêutico , Domínio Catalítico/efeitos dos fármacos , Proteases 3C de Coronavírus/metabolismo , Inibidores de Protease de Coronavírus/uso terapêutico , Humanos , Lactamas/uso terapêutico , Leucina/uso terapêutico , Lopinavir/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Nitrilas/uso terapêutico , Prolina/uso terapêutico , Ritonavir/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia
5.
Biomolecules ; 11(9)2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34572579

RESUMO

SARS-CoV-2 virus mutations might increase its virulence, and thus the severity and duration of the ongoing pandemic. Global drug discovery campaigns have successfully developed several vaccines to reduce the number of infections by the virus. However, finding a small molecule pharmaceutical that is effective in inhibiting SARS-CoV-2 remains a challenge. Natural products are the origin of many currently used pharmaceuticals and, for this reason, a library of in-house fungal extracts were screened to assess their potential to inhibit the main viral protease Mpro in vitro. The extract of Penicillium citrinum, TDPEF34, showed potential inhibition and was further analysed to identify potential Mpro inhibitors. Following bio-guided isolation, a series of benzodiazepine alkaloids cyclopenins with good-to-moderate activity against SARS-CoV-2 Mpro were identified. The mode of enzyme inhibition of these compounds was predicted by docking and molecular dynamic simulation. Compounds 1 (isolated as two conformers of S- and R-isomers), 2, and 4 were found to have promising in vitro inhibitory activity towards Mpro, with an IC50 values range of 0.36-0.89 µM comparable to the positive control GC376. The in silico investigation revealed compounds to achieve stable binding with the enzyme active site through multiple H-bonding and hydrophobic interactions. Additionally, the isolated compounds showed very good drug-likeness and ADMET properties. Our findings could be utilized in further in vitro and in vivo investigations to produce anti-SARS-CoV-2 drug candidates. These findings also provide critical structural information that could be used in the future for designing potent Mpro inhibitors.


Assuntos
Proteases 3C de Coronavírus , Inibidores de Cisteína Proteinase , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Penicillium/química , SARS-CoV-2/enzimologia , Benzodiazepinonas/química , Benzodiazepinonas/isolamento & purificação , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/isolamento & purificação
6.
Int J Mol Sci ; 22(18)2021 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-34576002

RESUMO

Recently, inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) have been proposed as potential therapeutic agents for COVID-19. Studying effects of amino acid mutations in the conformation of drug targets is necessary for anticipating drug resistance. In this study, with the structure of the SARS-CoV-2 Mpro complexed with a non-covalent inhibitor, we performed molecular dynamics (MD) simulations to determine the conformation of the complex when single amino acid residue in the active site is mutated. As a model of amino acid mutation, we constructed mutant proteins with one residue in the active site mutated to alanine. This method is called virtual alanine scan. The results of the MD simulations showed that the conformation and configuration of the ligand was changed for mutants H163A and E166A, although the structure of the whole protein and of the catalytic dyad did not change significantly, suggesting that mutations in His163 and Glu166 may be linked to drug resistance.


Assuntos
COVID-19 , Proteases 3C de Coronavírus , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , SARS-CoV-2 , Alanina , Substituição de Aminoácidos , COVID-19/enzimologia , COVID-19/genética , Domínio Catalítico/genética , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/genética , Humanos , SARS-CoV-2/enzimologia , SARS-CoV-2/genética
7.
Bioorg Med Chem ; 47: 116393, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34509862

RESUMO

The continued toll of COVID-19 has halted the smooth functioning of civilization on a global scale. With a limited understanding of all the essential components of viral machinery and the lack of structural information of this new virus, initial drug discovery efforts had limited success. The availability of high-resolution crystal structures of functionally essential SARS-CoV-2 proteins, including 3CLpro, supports the development of target-specific therapeutics. 3CLpro, the main protease responsible for the processing of viral polypeptide, plays a vital role in SARS-CoV-2 viral replication and translation and is an important target in other coronaviruses. Additionally, 3CLpro is the target of repurposed drugs, such as lopinavir and ritonavir. In this study, target proteins were retrieved from the protein data bank (PDB IDs: 6 M03, 6LU7, 2GZ7, 6 W63, 6SQS, 6YB7, and 6YVF) representing different open states of the main protease to accommodate macromolecular substrate. A hydroxyethylamine (HEA) library was constructed from harvested chemical structures from all the series being used in our laboratories for screening against malaria and Leishmania parasites. The database consisted of ∼1000 structure entries, of which 70% were new to ChemSpider at the time of screening. This in-house library was subjected to high throughput virtual screening (HTVS), followed by standard precision (SP) and then extra precision (XP) docking (Schrodinger LLC 2021). The ligand strain and complex energy of top hits were calculated by Molecular Mechanics Generalized Born Surface Area (MM/GBSA) method. Promising hit compounds (n = 40) specifically binding to 3CLpro with high energy and average MM/GBSA scores were then subjected to (100-ns) MD simulations. Using this sequential selection followed by an in-silico validation approach, we found a promising HEA-based compound (N,N'-((3S,3'S)-piperazine-1,4-diylbis(3-hydroxy-1-phenylbutane-4,2-diyl))bis(2-(5-methyl-1,3-dioxoisoindolin-2-yl)-3-phenylpropanamide)), which showed high in vitro antiviral activity against SARS-CoV-2. Further to reduce the size of the otherwise larger ligand, a pharmacophore-based predicted library of âˆ¼42 derivatives was constructed, which were added to the previous compound library and rescreened virtually. Out of several hits from the predicted library, two compounds were synthesized, tested against SARS-CoV-2 culture, and found to have markedly improved antiviral activity.


Assuntos
Antivirais/química , Proteases 3C de Coronavírus/antagonistas & inibidores , Etilaminas/química , Inibidores de Proteases/química , SARS-CoV-2/enzimologia , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Proteases 3C de Coronavírus/metabolismo , Etilaminas/metabolismo , Etilaminas/farmacologia , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , SARS-CoV-2/isolamento & purificação , Termodinâmica , Células Vero
8.
Chem Commun (Camb) ; 57(78): 10083-10086, 2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34514483

RESUMO

Zinc deficiency is linked to poor prognosis in COVID-19 patients while clinical trials with zinc demonstrate better clinical outcomes. The molecular targets and mechanistic details of the anti-coronaviral activity of zinc remain obscure. We show that zinc not only inhibits the SARS-CoV-2 main protease (Mpro) with nanomolar affinity, but also viral replication. We present the first crystal structure of the Mpro-Zn2+ complex at 1.9 Å and provide the structural basis of viral replication inhibition. We show that Zn2+ coordinates with the catalytic dyad at the enzyme active site along with two previously unknown water molecules in a tetrahedral geometry to form a stable inhibited Mpro-Zn2+ complex. Further, the natural ionophore quercetin increases the anti-viral potency of Zn2+. As the catalytic dyad is highly conserved across SARS-CoV, MERS-CoV and all variants of SARS-CoV-2, Zn2+ mediated inhibition of Mpro may have wider implications.


Assuntos
Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Proteases/química , SARS-CoV-2/enzimologia , Zinco/química , Animais , Sítios de Ligação , COVID-19/patologia , Domínio Catalítico , Chlorocebus aethiops , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Proteases 3C de Coronavírus/metabolismo , Cristalografia por Raios X , Humanos , Íons/química , Cinética , Simulação de Dinâmica Molecular , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , SARS-CoV-2/isolamento & purificação , Ressonância de Plasmônio de Superfície , Termodinâmica , Células Vero , Replicação Viral/efeitos dos fármacos
9.
Drug Res (Stuttg) ; 71(8): 462-472, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34517419

RESUMO

BACKGROUND: Replication of SARS-CoV-2 depends on viral RNA-dependent RNA-polymerase (RdRp). Remdesivir, the broad-spectrum RdRp inhibitor acts as nucleoside-analogues (NAs). Remdesivir has initially been repurposed as a promising drug against SARS-CoV-2 infection with some health hazards like liver damage, allergic reaction, low blood-pressure, and breathing-shortness, throat-swelling. In comparison, theaflavin-3'-O-gallate (TFMG), the abundant black tea component has gained importance in controlling viral infection. TFMG is a non-toxic, non-invasive, antioxidant, anticancer and antiviral molecule. RESULTS: Here, we analyzed the inhibitory effect of theaflavin-3'-O-gallate on SARS CoV-2 RdRp in comparison with remdesivir by molecular-docking study. TFMG has been shown more potent in terms of lower Atomic-Contact-Energy (ACE) and higher occupancy of surface area; -393.97 Kcal/mol and 771.90 respectively, favoured with lower desolvation-energy; -9.2: Kcal/mol. TFMG forms more rigid electrostatic and H-bond than remdesivir. TFMG showed strong affinity to RNA primer and template and RNA passage-site of RdRp. CONCLUSIONS: TFMG can block the catalytic residue, NTP entry site, cation binding site, nsp7-nsp12 junction with binding energy of -6. 72 Kcal/mol with Ki value of 11.79, and interface domain with binding energy of -7.72 and -6.16 Kcal/mol with Ki value of 2.21 and 30.71 µM. And most importantly, TFMG shows antioxidant/anti-inflammatory/antiviral effect on human studies.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , Biflavonoides/farmacologia , COVID-19/tratamento farmacológico , Catequina/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Ácido Gálico/análogos & derivados , Simulação de Acoplamento Molecular , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Alanina/química , Alanina/farmacologia , Antivirais/química , Biflavonoides/química , COVID-19/virologia , Domínio Catalítico , Catequina/química , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Inibidores Enzimáticos/química , Ácido Gálico/química , Ácido Gálico/farmacologia , Conformação Proteica , SARS-CoV-2/enzimologia , Relação Estrutura-Atividade
10.
Phys Chem Chem Phys ; 23(36): 20117-20128, 2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34514487

RESUMO

The ongoing pandemic caused by SARS-CoV-2 emphasizes the need for effective therapeutics. Inhibition of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) by nucleotide analogs provides a promising antiviral strategy. One common group of RdRp inhibitors, 2'-modified nucleotides, are reported to exhibit different behaviors in the SARS-CoV-2 RdRp transcription assay. Three of these analogs, 2'-O-methyl UTP, Sofosbuvir, and 2'-methyl CTP, act as effective inhibitors in previous biochemical experiments, while Gemcitabine and ara-UTP show no inhibitory activity. To understand the impact of the 2'-modification on their inhibitory effects, we conducted extensive molecular dynamics simulations and relative binding free energy calculations using the free energy perturbation method on SARS-CoV-2 replication-transcription complex (RTC) with these five nucleotide analogs. Our results reveal that the five nucleotide analogs display comparable binding affinities to SARS-CoV-2 RdRp and they can all be added to the nascent RNA chain. Moreover, we examine how the incorporation of these nucleotide triphosphate (NTP) analogs will impact the addition of the next nucleotide. Our results indicate that 2'-O-methyl UTP can weaken the binding of the subsequent NTP and consequently lead to partial chain termination. Additionally, Sofosbuvir and 2'-methyl CTP can cause immediate termination due to the strong steric hindrance introduced by their bulky 2'-methyl groups. In contrast, nucleotide analogs with smaller substitutions, such as the fluorine atoms and the ara-hydroxyl group in Gemcitabine and ara-UTP, have a marginal impact on the polymerization process. Our findings are consistent with experimental observations, and more importantly, shed light on the detailed molecular mechanism of SARS-CoV-2 RdRp inhibition by 2'-substituted nucleotide analogs, and may facilitate the rational design of antiviral agents to inhibit SARS-CoV-2 RdRp.


Assuntos
Antivirais/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Nucleotídeos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Antivirais/química , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Inibidores Enzimáticos/química , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleotídeos/química , SARS-CoV-2/enzimologia
11.
Nat Commun ; 12(1): 5493, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535675

RESUMO

Macromolecular dynamics manifest as disorder in structure determination, which is subsequently accounted for by displacement parameters (also called temperature factors, or B-factors) or alternate conformations. Though B-factors contain detailed information about structural dynamics, they are the total of multiple sources of disorder, making them difficult to interpret and thus little-used in structural analysis. We report here an analytical approach for decomposing molecular disorder into a parsimonious hierarchical series of contributions, providing an intuitive basis for quantitative structural-dynamics analysis. We demonstrate the decomposition of disorder on example SARS-CoV-2 and STEAP4 structures, from both crystallographic and cryo-electron microscopy data, and reveal how understanding of the macromolecular disorder leads to deeper understanding of molecular motions and flexibility, and suggests hypotheses for molecular mechanisms.


Assuntos
Proteases 3C de Coronavírus/química , Substâncias Macromoleculares/química , Simulação de Dinâmica Molecular , SARS-CoV-2/enzimologia , COVID-19 , Microscopia Crioeletrônica , Humanos , Proteínas de Membrana/química , Oxirredutases/química , Conformação Proteica
12.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502340

RESUMO

The SARS-CoV-2 main protease (Mpro) is one of the molecular targets for drug design. Effective vaccines have been identified as a long-term solution but the rate at which they are being administered is slow in several countries, and mutations of SARS-CoV-2 could render them less effective. Moreover, remdesivir seems to work only with some types of COVID-19 patients. Hence, the continuous investigation of new treatments for this disease is pivotal. This study investigated the inhibitory role of natural products against SARS-CoV-2 Mpro as repurposable agents in the treatment of coronavirus disease 2019 (COVID-19). Through in silico approach, selected flavonoids were docked into the active site of Mpro. The free energies of the ligands complexed with Mpro were computationally estimated using the molecular mechanics-generalized Born surface area (MM/GBSA) method. In addition, the inhibition process of SARS-CoV-2 Mpro with these ligands was simulated at 100 ns in order to uncover the dynamic behavior and complex stability. The docking results showed that the selected flavonoids exhibited good poses in the binding domain of Mpro. The amino acid residues involved in the binding of the selected ligands correlated well with the residues involved with the mechanism-based inhibitor (N3) and the docking score of Quercetin-3-O-Neohesperidoside (-16.8 Kcal/mol) ranked efficiently with this inhibitor (-16.5 Kcal/mol). In addition, single-structure MM/GBSA rescoring method showed that Quercetin-3-O-Neohesperidoside (-87.60 Kcal/mol) is more energetically favored than N3 (-80.88 Kcal/mol) and other ligands (Myricetin 3-Rutinoside (-87.50 Kcal/mol), Quercetin 3-Rhamnoside (-80.17 Kcal/mol), Rutin (-58.98 Kcal/mol), and Myricitrin (-49.22 Kcal/mol). The molecular dynamics simulation (MDs) pinpointed the stability of these complexes over the course of 100 ns with reduced RMSD and RMSF. Based on the docking results and energy calculation, together with the RMSD of 1.98 ± 0.19 Å and RMSF of 1.00 ± 0.51 Å, Quercetin-3-O-Neohesperidoside is a better inhibitor of Mpro compared to N3 and other selected ligands and can be repurposed as a drug candidate for the treatment of COVID-19. In addition, this study demonstrated that in silico docking, free energy calculations, and MDs, respectively, are applicable to estimating the interaction, energetics, and dynamic behavior of molecular targets by natural products and can be used to direct the development of novel target function modulators.


Assuntos
Produtos Biológicos/metabolismo , SARS-CoV-2/enzimologia , Proteínas da Matriz Viral/metabolismo , Sítios de Ligação , Produtos Biológicos/química , Produtos Biológicos/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico , Desenho de Fármacos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Inibidores de Proteases/uso terapêutico , Quercetina/análogos & derivados , Quercetina/química , Quercetina/metabolismo , Quercetina/uso terapêutico , SARS-CoV-2/isolamento & purificação , Proteínas da Matriz Viral/química
13.
Biochemistry ; 60(39): 2925-2931, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34506130

RESUMO

Rupintrivir targets the 3C cysteine proteases of the picornaviridae family, which includes rhinoviruses and enteroviruses that cause a range of human diseases. Despite being a pan-3C protease inhibitor, rupintrivir activity is extremely weak against the homologous 3C-like protease of SARS-CoV-2. In this study, the crystal structures of rupintrivir were determined bound to enterovirus 68 (EV68) 3C protease and the 3C-like main protease (Mpro) from SARS-CoV-2. While the EV68 3C protease-rupintrivir structure was similar to previously determined complexes with other picornavirus 3C proteases, rupintrivir bound in a unique conformation to the active site of SARS-CoV-2 Mpro splitting the catalytic cysteine and histidine residues. This bifurcation of the catalytic dyad may provide a novel approach for inhibiting cysteine proteases.


Assuntos
Antivirais/metabolismo , Proteases 3C de Coronavírus/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Isoxazóis/metabolismo , Fenilalanina/análogos & derivados , Pirrolidinonas/metabolismo , SARS-CoV-2/enzimologia , Valina/análogos & derivados , Antivirais/química , Domínio Catalítico , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/química , Enterovirus Humano D/enzimologia , Ligação de Hidrogênio , Isoxazóis/química , Fenilalanina/química , Fenilalanina/metabolismo , Ligação Proteica , Pirrolidinonas/química , Eletricidade Estática , Valina/química , Valina/metabolismo
14.
Pharmacol Res ; 172: 105820, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34403732

RESUMO

Coronavirus Disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which enter the host cells through the interaction between its receptor binding domain (RBD) of spike glycoprotein with angiotensin-converting enzyme 2 (ACE2) receptor on the plasma membrane of host cell. Neutralizing antibodies and peptide binders of RBD can block viral infection, however, the concern of accessibility and affordability of viral infection inhibitors has been raised. Here, we report the identification of natural compounds as potential SARS-CoV-2 entry inhibitors using the molecular docking-based virtual screening coupled with bilayer interferometry (BLI). From a library of 1871 natural compounds, epigallocatechin gallate (EGCG), 20(R)-ginsenoside Rg3 (RRg3), 20(S)-ginsenoside Rg3 (SRg3), isobavachalcone (Ibvc), isochlorogenic A (IscA) and bakuchiol (Bkc) effectively inhibited pseudovirus entry at concentrations up to 100 µM. Among these compounds, four compounds, EGCG, Ibvc, salvianolic acid A (SalA), and isoliensinine (Isl), were effective in inhibiting SARS-CoV-2-induced cytopathic effect and plaque formation in Vero E6 cells. The EGCG was further validated with no observable animal toxicity and certain antiviral effect against SARS-CoV-2 pseudovirus mutants (D614G, N501Y, N439K & Y453F). Interestingly, EGCG, Bkc and Ibvc bind to ACE2 receptor in BLI assay, suggesting a dual binding to RBD and ACE2. Current findings shed some insight into identifications and validations of SARS-CoV-2 entry inhibitors from natural compounds.


Assuntos
Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Antivirais/química , Produtos Biológicos/química , COVID-19/tratamento farmacológico , Inibidores Enzimáticos/química , SARS-CoV-2/enzimologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Antivirais/farmacologia , Ligação Competitiva , Produtos Biológicos/farmacologia , Catequina/análogos & derivados , Catequina/farmacologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Chalconas/farmacologia , Ácido Clorogênico/análogos & derivados , Ácido Clorogênico/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Ginsenosídeos/farmacologia , Humanos , Interferometria , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Fenóis/farmacologia , Ligação Proteica
15.
mBio ; 12(4): e0209421, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34399606

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), encodes two proteases required for replication. The main protease (Mpro), encoded as part of two polyproteins, pp1a and pp1ab, is responsible for 11 different cleavages of these viral polyproteins to produce mature proteins required for viral replication. Mpro is therefore an attractive target for therapeutic interventions. Certain proteins in cells under oxidative stress undergo modification of reactive cysteines. We show Mpro is susceptible to glutathionylation, leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that modification of a single cysteine with glutathione is sufficient to block dimerization and inhibit its activity. Gel filtration studies as well as analytical ultracentrifugation confirmed that glutathionylated Mpro exists as a monomer. Tryptic and chymotryptic digestions of Mpro as well as experiments using a C300S Mpro mutant revealed that Cys300, which is located at the dimer interface, is a primary target of glutathionylation. Moreover, Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of a non-active site cysteine, Cys300, which itself is not required for Mpro activity, and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to the pathophysiology of SARS-CoV-2 and related bat coronaviruses. IMPORTANCE SARS-CoV-2 is responsible for the devastating COVID-19 pandemic. Therefore, it is imperative that we learn as much as we can about the biochemistry of the coronavirus proteins to inform development of therapy. One attractive target is the main protease (Mpro), a dimeric enzyme necessary for viral replication. Most work thus far developing Mpro inhibitors has focused on the active site. Our work has revealed a regulatory mechanism for Mpro activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits Mpro activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. Moreover, this process may have implications for disease pathophysiology in humans and bats. It may be a mechanism by which SARS-CoV-2 has evolved to limit replication and avoid killing host bats when they are under oxidative stress during flight.


Assuntos
Proteases 3C de Coronavírus/metabolismo , Cisteína/química , Glutationa/química , Multimerização Proteica , SARS-CoV-2/metabolismo , Animais , COVID-19/patologia , Quirópteros/virologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Dimerização , Glutarredoxinas/metabolismo , Humanos , SARS-CoV-2/enzimologia
16.
Dalton Trans ; 50(35): 12226-12233, 2021 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-34396374

RESUMO

Numerous organic molecules are known to inhibit the main protease of SARS-CoV-2, (SC2Mpro), a key component in viral replication of the 2019 novel coronavirus. We explore the hypothesis that zinc ions, long used as a medicinal supplement and known to support immune function, bind to the SC2Mpro enzyme in combination with lipophilic tropolone and thiotropolone ligands, L, block substrate docking, and inhibit function. This study combines synthetic inorganic chemistry, in vitro protease activity assays, and computational modeling. While the ligands themselves have half maximal inhibition concentrations, IC50, for SC2Mpro in the 8-34 µM range, the IC50 values are ca. 100 nM for Zn(NO3)2 which are further enhanced in Zn-L combinations (59-97 nM). Isolation of the Zn(L)2 binary complexes and characterization of their ability to undergo ligand displacement is the basis for computational modeling of the chemical features of the enzyme inhibition. Blind docking onto the SC2Mpro enzyme surface using a modified Autodock4 protocol found preferential binding into the active site pocket. Such Zn-L combinations orient so as to permit dative bonding of Zn(L)+ to basic active site residues.


Assuntos
COVID-19/tratamento farmacológico , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , Tropolona/farmacologia , Zinco/farmacologia , Antivirais/química , Antivirais/farmacologia , COVID-19/virologia , Domínio Catalítico/efeitos dos fármacos , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Inibidores de Proteases/química , SARS-CoV-2/enzimologia , Tropolona/análogos & derivados , Zinco/química
17.
Commun Biol ; 4(1): 999, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429502

RESUMO

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Previous structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the enzyme and resolve its structure at 5.5 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 each and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs.


Assuntos
SARS-CoV-2/enzimologia , Dimerização , Humanos , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo
18.
J Org Chem ; 86(18): 13104-13110, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34459196

RESUMO

An intermediate in the synthesis of numerous antiviral protease inhibitors is the glutamine analogue, (3S)-pyrrolid-2-one-3-yl-l-alanine. Preparations of compounds based on this pharmacophore are hindered by the lack of a reliably high yielding synthesis of protected forms of this amino acid. We describe an improved scalable route with readily available reagents and facile purification. This methodology employs γ-allylation of dimethyl N-BocGlu, further Boc N-protection, OsO4-periodate oxidation, O-Me oxime formation, and RaNi-catalyzed hydrogenolysis with concomitant cyclization under basic conditions.


Assuntos
Antivirais , COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Proteases , Antivirais/farmacologia , Glutamina , Humanos , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia
19.
Biophys Chem ; 278: 106677, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34428682

RESUMO

The SARS-CoV-2 pandemic has accelerated the study of existing drugs. The mixture of homologs called ivermectin (avermectin-B1a [HB1a] + avermectin-B1b [HB1b]) has shown antiviral activity against SARS-CoV-2 in vitro. However, there are few reports on the behavior of each homolog. We investigated the interaction of each homolog with promising targets of interest associated with SARS-CoV-2 infection from a biophysical and computational-chemistry perspective using docking and molecular dynamics. We observed a differential behavior for each homolog, with an affinity of HB1b for viral structures, and of HB1a for host structures considered. The induced disturbances were differential and influenced by the hydrophobicity of each homolog and of the binding pockets. We present the first comparative analysis of the potential theoretical inhibitory effect of both avermectins on biomolecules associated with COVID-19, and suggest that ivermectin through its homologs, has a multiobjective behavior.


Assuntos
Antivirais/química , Proteases 3C de Coronavírus/antagonistas & inibidores , DNA Helicases/antagonistas & inibidores , Ivermectina/análogos & derivados , alfa Carioferinas/antagonistas & inibidores , beta Carioferinas/antagonistas & inibidores , Animais , Antivirais/farmacologia , Sítios de Ligação , COVID-19/tratamento farmacológico , COVID-19/virologia , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Humanos , Ivermectina/química , Ivermectina/farmacologia , Cinética , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2/química , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Termodinâmica , alfa Carioferinas/química , alfa Carioferinas/metabolismo , beta Carioferinas/química , beta Carioferinas/metabolismo
20.
Bioorg Med Chem Lett ; 50: 128333, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34418570

RESUMO

Specific anti-coronaviral drugs complementing available vaccines are urgently needed to fight the COVID-19 pandemic. Given its high conservation across the betacoronavirus genus and dissimilarity to human proteases, the SARS-CoV-2 main protease (Mpro) is an attractive drug target. SARS-CoV-2 Mpro inhibitors have been developed at unprecedented speed, most of them being substrate-derived peptidomimetics with cysteine-modifying warheads. In this study, Mpro has proven resistant towards the identification of high-affinity short substrate-derived peptides and peptidomimetics without warheads. 20 cyclic and linear substrate analogues bearing natural and unnatural residues, which were predicted by computational modelling to bind with high affinity and designed to establish structure-activity relationships, displayed no inhibitory activity at concentrations as high as 100 µM. Only a long linear peptide covering residues P6 to P5' displayed moderate inhibition (Ki = 57 µM). Our detailed findings will inform current and future drug discovery campaigns targeting Mpro.


Assuntos
COVID-19/patologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Proteases/química , SARS-CoV-2/enzimologia , COVID-19/virologia , Proteases 3C de Coronavírus/metabolismo , Cisteína/química , Cisteína/metabolismo , Humanos , Lactamas/química , Lactamas/metabolismo , Leucina/química , Leucina/metabolismo , Nitrilas/química , Nitrilas/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Prolina/química , Prolina/metabolismo , Inibidores de Proteases/metabolismo , SARS-CoV-2/isolamento & purificação , Relação Estrutura-Atividade , Especificidade por Substrato
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