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1.
Phys Chem Chem Phys ; 22(43): 25335-25343, 2020 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-33140777

RESUMO

Coronavirus disease 2019 (COVID-19) is an ongoing global pandemic with very limited specific treatments. To fight COVID-19, various traditional antiviral medicines have been prescribed in China to infected patients with mild to moderate symptoms and received unexpected success in controlling the disease. However, the molecular mechanisms of how these herbal medicines interact with the SARS-CoV-2 virus that causes COVID-19 have remained elusive. It is well known that the main protease (Mpro) of SARS-CoV-2 plays an important role in maturation of many viral proteins such as the RNA-dependent RNA polymerase. Here, we explore the underlying molecular mechanisms of the computationally determined top candidate, namely, rutin which is a key component in many traditional antiviral medicines such as Lianhuaqinwen and Shuanghuanlian, for inhibiting the viral target-Mpro. Using in silico methods (docking and molecular dynamics simulations), we revealed the dynamics and energetics of rutin when interacting with the Mpro of SARS-CoV-2, suggesting that the highly hydrophilic rutin molecule can be bound inside the Mpro's pocket (active site) and possibly inhibit its biological functions. In addition, we optimized the structure of rutin and designed two more hydrophobic analogs, M1 and M2, which satisfy the rule of five for western medicines and demonstrated that they (M2 in particular) possess much stronger binding affinities to the SARS-COV-2s Mpro than rutin, due to the enhanced hydrophobic interaction as well as more hydrogen bonds. Therefore, our results provide invaluable insights into the mechanism of a ligand's binding inside the Mpro and shed light on future structure-based designs of high-potent inhibitors for SARS-CoV-2 Mpro.


Assuntos
Betacoronavirus/enzimologia , Cisteína Endopeptidases/metabolismo , Inibidores de Proteases/química , Rutina/química , Proteínas não Estruturais Virais/metabolismo , Betacoronavirus/isolamento & purificação , Sítios de Ligação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Medicina Herbária , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Inibidores de Proteases/metabolismo , Domínios Proteicos , Rutina/metabolismo , Termodinâmica , Proteínas não Estruturais Virais/química
2.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 10): 483-487, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33006576

RESUMO

The replication of SARS-CoV-2 produces two large polyproteins, pp1a and pp1ab, that are inactive until cleavage by the viral chymotrypsin-like cysteine protease enzyme (3CL Mpro) into a series of smaller functional proteins. At the heart of 3CL Mpro is an unusual catalytic dyad formed by the side chains of His41 and Cys145 and a coordinated water molecule. The catalytic mechanism by which the enzyme operates is still unknown, as crucial information on the protonation states within the active site is unclear. To experimentally determine the protonation states of the catalytic site and of the other residues in the substrate-binding cavity, and to visualize the hydrogen-bonding networks throughout the enzyme, room-temperature neutron and X-ray data were collected from a large H/D-exchanged crystal of ligand-free (apo) 3CL Mpro.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Pneumonia Viral/virologia , Proteínas não Estruturais Virais/química , Betacoronavirus/química , Betacoronavirus/genética , Domínio Catalítico , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Humanos , Modelos Moleculares , Difração de Nêutrons , Pandemias , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Temperatura , Proteínas não Estruturais Virais/genética
3.
PLoS One ; 15(10): e0240079, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33022015

RESUMO

The Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) pandemic represents a global challenge. SARS-CoV-2's ability to replicate in host cells relies on the action of its non-structural proteins, like its main protease (Mpro). This cysteine protease acts by processing the viruses' precursor polyproteins. As proteases, together with polymerases, are main targets of antiviral drug design, we here have performed biochemical high throughput screening (HTS) with recombinantly expressed SARS-CoV-2 Mpro. A fluorescent assay was used to identify inhibitors in a compound library containing known drugs, bioactive molecules and natural products. These screens led to the identification of 13 inhibitors with IC50 values ranging from 0.2 µM to 23 µM. The screens confirmed several known SARS-CoV Mpro inhibitors as inhibitors of SARS-CoV-2 Mpro, such as the organo-mercuric compounds thimerosal and phenylmercuric acetate. Benzophenone derivatives could also be identified among the most potent screening hits. Additionally, Evans blue, a sulfonic acid-containing dye, could be identified as an Mpro inhibitor. The obtained compounds could be of interest as lead compounds for the development of future SARS-CoV-2 drugs.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos/métodos , Pneumonia Viral/virologia , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Cisteína Endopeptidases/química , Desenho de Fármacos , Escherichia coli/genética , Concentração Inibidora 50 , Modelos Moleculares , Pandemias , Proteínas não Estruturais Virais/química
4.
Nat Commun ; 11(1): 5047, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028810

RESUMO

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.


Assuntos
Betacoronavirus/química , Cisteína Endopeptidases/química , Fragmentos de Peptídeos/química , Proteínas não Estruturais Virais/química , Betacoronavirus/enzimologia , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Cisteína Endopeptidases/metabolismo , Desenho de Fármacos , Espectrometria de Massas , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Eletricidade Estática , Proteínas não Estruturais Virais/metabolismo
5.
J Comput Aided Mol Des ; 34(12): 1237-1259, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33034007

RESUMO

Computational protein-ligand docking is well-known to be prone to inaccuracies in input receptor structures, and it is challenging to obtain good docking results with computationally predicted receptor structures (e.g. through homology modeling). Here we introduce a fragment-based docking method and test if it reduces requirements on the accuracy of an input receptor structures relative to non-fragment docking approaches. In this method, small rigid fragments are docked first using AutoDock Vina to generate a large number of favorably docked poses spanning the receptor binding pocket. Then a graph theory maximum clique algorithm is applied to find combined sets of docked poses of different fragment types onto which the complete ligand can be properly aligned. On the basis of these alignments, possible binding poses of complete ligand are determined. This docking method is first tested for bound docking on a series of Cytochrome P450 (CYP450) enzyme-substrate complexes, in which experimentally determined receptor structures are used. For all complexes tested, ligand poses of less than 1 Å root mean square deviations (RMSD) from the actual binding positions can be recovered. Then the method is tested for unbound docking with modeled receptor structures for a number of protein-ligand complexes from different families including the very recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protease. For all complexes, poses with RMSD less than 3 Å from actual binding positions can be recovered. Our results suggest that for docking with approximately modeled receptor structures, fragment-based methods can be more effective than common complete ligand docking approaches.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/efeitos dos fármacos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Proteínas não Estruturais Virais/efeitos dos fármacos , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Ligantes , Modelos Químicos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
6.
Molecules ; 25(20)2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-33050360

RESUMO

The current pandemic, caused by SARS-CoV-2 virus, is a severe challenge for human health and the world economy. There is an urgent need for development of drugs that can manage this pandemic, as it has already infected 19 million people and led to the death of around 711,277 people worldwide. At this time, in-silico studies are providing lots of preliminary data about potential drugs, which can be a great help in further in-vitro and in-vivo studies. Here, we have selected three polyphenolic compounds, mangiferin, glucogallin, and phlorizin. These compounds are isolated from different natural sources but share structural similarities and have been reported for their antiviral activity. The objective of this study is to analyze and predict the anti-protease activity of these compounds on SARS-CoV-2main protease (Mpro) and TMPRSS2 protein. Both the viral protein and the host protein play an important role in the viral life cycle, such as post-translational modification and viral spike protein priming. This study has been performed by molecular docking of the compounds using PyRx with AutoDock Vina on the two aforementioned targets chosen for this study, i.e., SARS-CoV-2 Mpro and TMPRSS2. The compounds showed good binding affinity and are further analyzed by (Molecular dynamic) MD and Molecular Mechanics Poisson-Boltzmann Surface Area MM-PBSA study. The MD-simulation study has predicted that these natural compounds will have a great impact on the stabilization of the binding cavity of the Mpro of SARS-CoV-2. The predicted pharmacokinetic parameters also show that these compounds are expected to have good solubility and absorption properties. Further predictions for these compounds also showed no involvement in drug-drug interaction and no toxicity.


Assuntos
Betacoronavirus/isolamento & purificação , Produtos Biológicos/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/química , Pneumonia Viral/tratamento farmacológico , Polifenóis/farmacologia , Inibidores de Proteases/farmacologia , Serina Endopeptidases/química , Proteínas não Estruturais Virais/química , Antivirais/farmacologia , Simulação por Computador , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/metabolismo , Humanos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/virologia , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/metabolismo
7.
Comput Biol Med ; 126: 104046, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33065388

RESUMO

Coronavirus Disease 2019 (COVID-19) is an infectious illness caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), originally identified in Wuhan, China (December 2019) and has since expanded into a pandemic. Here, we investigate metabolites present in several common spices as possible inhibitors of COVID-19. Specifically, 32 compounds isolated from 14 cooking seasonings were examined as inhibitors for SARS-CoV-2 main protease (Mpro), which is required for viral multiplication. Using a drug discovery approach to identify possible antiviral leads, in silico molecular docking studies were performed. Docking calculations revealed a high potency of salvianolic acid A and curcumin as Mpro inhibitors with binding energies of -9.7 and -9.2 kcal/mol, respectively. Binding mode analysis demonstrated the ability of salvianolic acid A and curcumin to form nine and six hydrogen bonds, respectively with amino acids proximal to Mpro's active site. Stabilities and binding affinities of the two identified natural spices were calculated over 40 ns molecular dynamics simulations and compared to an antiviral protease inhibitor (lopinavir). Molecular mechanics-generalized Born surface area energy calculations revealed greater salvianolic acid A affinity for the enzyme over curcumin and lopinavir with energies of -44.8, -34.2 and -34.8 kcal/mol, respectively. Using a STRING database, protein-protein interactions were identified for salvianolic acid A included the biochemical signaling genes ACE, MAPK14 and ESR1; and for curcumin, EGFR and TNF. This study establishes salvianolic acid A as an in silico natural product inhibitor against the SARS-CoV-2 main protease and provides a promising inhibitor lead for in vitro enzyme testing.


Assuntos
Betacoronavirus/enzimologia , Ácidos Cafeicos/química , Infecções por Coronavirus/tratamento farmacológico , Curcumina/química , Cisteína Endopeptidases , Descoberta de Drogas , Lactatos/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/química , Proteínas não Estruturais Virais , Ácidos Cafeicos/uso terapêutico , Infecções por Coronavirus/enzimologia , Curcumina/uso terapêutico , Cisteína Endopeptidases/química , Humanos , Lactatos/uso terapêutico , Pandemias , Pneumonia Viral/enzimologia , Inibidores de Proteases/uso terapêutico , Termodinâmica , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química
8.
J Mol Graph Model ; 101: 107758, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33007575

RESUMO

A novel strain of coronavirus, namely, SARS-CoV-2 identified in Wuhan city of China in December 2019, continues to spread at a rapid rate worldwide. There are no specific therapies available and investigations regarding the treatment of this disease are still lacking. In order to identify a novel potent inhibitor, we performed blind docking studies on the main virus protease Mpro with eight approved drugs belonging to four pharmacological classes such as: anti-malarial, anti-bacterial, anti-infective and anti-histamine. Among the eight studied compounds, Lymecycline and Mizolastine appear as potential inhibitors of this protease. When docked against Mpro crystal structure, these two compounds revealed a minimum binding energy of -8.87 and -8.71 kcal/mol with 168 and 256 binding modes detected in the binding substrate pocket, respectively. Further, to study the interaction mechanism and conformational dynamics of protein-ligand complexes, Molecular dynamic simulation and MM/PBSA binding free calculations were performed. Our results showed that both Lymecycline and Mizolastine bind in the active site. And exhibited good binding affinities towards target protein. Moreover, the ADMET analysis also indicated drug-likeness properties. Thus it is suggested that the identified compounds can inhibit Chymotrypsin-like protease (3CLpro) of SARS-CoV-2.


Assuntos
Cisteína Endopeptidases/química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Animais , Antibacterianos/química , Antivirais/química , Antivirais/farmacocinética , Antivirais/farmacologia , Sítios de Ligação , Simulação por Computador , Cisteína Endopeptidases/metabolismo , Bases de Dados de Produtos Farmacêuticos , Aprovação de Drogas , Reposicionamento de Medicamentos , Antagonistas dos Receptores Histamínicos/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/farmacocinética , Proteínas não Estruturais Virais/metabolismo
9.
J Mol Graph Model ; 101: 107762, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33022569

RESUMO

Since the emergence of SARS-CoV2, to date, no effective antiviral drug has been approved to treat the disease, and no vaccine against SARS-CoV2 is available. Under this scenario, the combination of two HIV-1 protease inhibitors, lopinavir and ritonavir, has attracted attention since they have been previously employed against the SARS-CoV main proteinase (Mpro) and exhibited some signs of effectiveness. Recently, the 3D structure of SARS-CoV2 Mpro was constructed based on the monomeric SARS-CoV Mpro and employed to identify potential approved small inhibitors against SARS-CoV2 Mpro, allowing the selection of 15 drugs among 1903 approved drugs to be employed. In this study, we performed docking of these 15 approved drugs against the recently solved X-ray crystallography structure of SARS-CoV2 Mpro in the monomeric and dimeric states; the latter is the functional state that was determined in a biological context, and these were submitted to molecular dynamics (MD) simulations coupled with the molecular mechanics generalized Born surface area (MM/GBSA) approach to obtain insight into the inhibitory activity of these compounds. Similar studies were performed with lopinavir and ritonavir coupled to monomeric and dimeric SARS-CoV Mpro and SARS-CoV2 Mpro to compare the inhibitory differences. Our study provides the structural and energetic basis of the inhibitory properties of lopinavir and ritonavir on SARS-CoV Mpro and SARS-CoV2 Mpro, allowing us to identify two FDA-approved drugs that can be used against SARS-CoV2 Mpro. This study also demonstrated that drug discovery requires the dimeric state to obtain good results.


Assuntos
Antivirais/farmacologia , Cisteína Endopeptidases/química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Antivirais/química , Cisteína Endopeptidases/metabolismo , Lopinavir/química , Lopinavir/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Análise de Componente Principal , Conformação Proteica , Multimerização Proteica , Ritonavir/química , Ritonavir/farmacologia , Proteínas não Estruturais Virais/metabolismo
10.
Molecules ; 25(19)2020 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-33036293

RESUMO

A novel series of some hydrazones bearing thiazole moiety were generated via solvent-drop grinding of thiazole carbohydrazide 2 with various carbonyl compounds. Also, dehydrative-cyclocondensation of 2 with active methylene compounds or anhydrides gave the respective pyarzole or pyrazine derivatives. The structures of the newly synthesized compounds were established based on spectroscopic evidences and their alternative syntheses. Additionally, the anti-viral activity of all the products was tested against SARS-CoV-2 main protease (Mpro) using molecular docking combined with molecular dynamics simulation (MDS). The average binding affinities of the compounds 3a, 3b, and 3c (-8.1 ± 0.33 kcal/mol, -8.0 ± 0.35 kcal/mol, and -8.2 ± 0.21 kcal/mol, respectively) are better than that of the positive control Nelfinavir (-6.9 ± 0.51 kcal/mol). This shows the possibility of these three compounds to effectively bind to SARS-CoV-2 Mpro and hence, contradict the virus lifecycle.


Assuntos
Antivirais/síntese química , Betacoronavirus/enzimologia , Hidrazonas/síntese química , Inibidores de Proteases/síntese química , Pirazinas/síntese química , Pirazóis/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/farmacologia , Betacoronavirus/química , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Descoberta de Drogas , Humanos , Hidrazonas/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pirazinas/farmacologia , Pirazóis/farmacologia , Termodinâmica , Interface Usuário-Computador , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
11.
Molecules ; 25(17)2020 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-32867349

RESUMO

Three types of new coronaviruses (CoVs) have been identified recently as the causative viruses for the severe pneumonia-like respiratory illnesses, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and corona-virus disease 2019 (COVID-19). Neither therapeutic agents nor vaccines have been developed to date, which is a major drawback in controlling the present global pandemic of COVID-19 caused by SARS coronavirus 2 (SARS-CoV-2) and has resulted in more than 20,439,814 cases and 744,385 deaths. Each of the 3C-like (3CL) proteases of the three CoVs is essential for the proliferation of the CoVs, and an inhibitor of the 3CL protease (3CLpro) is thought to be an ideal therapeutic agent against SARS, MERS, or COVID-19. Among these, SARS-CoV is the first corona-virus isolated and has been studied in detail since the first pandemic in 2003. This article briefly reviews a series of studies on SARS-CoV, focusing on the development of inhibitors for the SARS-CoV 3CLpro based on molecular interactions with the 3CL protease. Our recent approach, based on the structure-based rational design of a novel scaffold for SARS-CoV 3CLpro inhibitor, is also included. The achievements summarized in this short review would be useful for the design of a variety of novel inhibitors for corona-viruses, including SARS-CoV-2.


Assuntos
Antivirais/química , Betacoronavirus/química , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Inibidores de Proteases/química , Vírus da SARS/patogenicidade , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/classificação , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Domínio Catalítico , Infecções por Coronavirus/tratamento farmacológico , Cristalografia por Raios X , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Humanos , Cinética , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/classificação , Inibidores de Proteases/uso terapêutico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Vírus da SARS/genética , Vírus da SARS/metabolismo , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Especificidade por Substrato , Termodinâmica , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
12.
Molecules ; 25(17)2020 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-32872217

RESUMO

A pandemic caused by the novel coronavirus (SARS-CoV-2 or COVID-19) began in December 2019 in Wuhan, China, and the number of newly reported cases continues to increase. More than 19.7 million cases have been reported globally and about 728,000 have died as of this writing (10 August 2020). Recently, it has been confirmed that the SARS-CoV-2 main protease (Mpro) enzyme is responsible not only for viral reproduction but also impedes host immune responses. The Mpro provides a highly favorable pharmacological target for the discovery and design of inhibitors. Currently, no specific therapies are available, and investigations into the treatment of COVID-19 are lacking. Therefore, herein, we analyzed the bioactive phytocompounds isolated by gas chromatography-mass spectroscopy (GC-MS) from Tinospora crispa as potential COVID-19 Mpro inhibitors, using molecular docking study. Our analyses unveiled that the top nine hits might serve as potential anti-SARS-CoV-2 lead molecules, with three of them exerting biological activity and warranting further optimization and drug development to combat COVID-19.


Assuntos
Antivirais/química , Betacoronavirus/química , Compostos Fitoquímicos/química , Inibidores de Proteases/química , Tinospora/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/classificação , Antivirais/isolamento & purificação , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Domínio Catalítico , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Descoberta de Drogas , Cromatografia Gasosa-Espectrometria de Massas , Expressão Gênica , Humanos , Cinética , Simulação de Acoplamento Molecular , Pandemias , Compostos Fitoquímicos/classificação , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/classificação , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Especificidade por Substrato , Termodinâmica , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
13.
Molecules ; 25(17)2020 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-32899354

RESUMO

Peptidyl fluoromethyl ketones occupy a pivotal role in the current scenario of synthetic chemistry, thanks to their numerous applications as inhibitors of hydrolytic enzymes. The insertion of one or more fluorine atoms adjacent to a C-terminal ketone moiety greatly modifies the physicochemical properties of the overall substrate, especially by increasing the reactivity of this functionalized carbonyl group toward nucleophiles. The main application of these peptidyl α-fluorinated ketones in medicinal chemistry relies in their ability to strongly and selectively inhibit serine and cysteine proteases. These compounds can be used as probes to study the proteolytic activity of the aforementioned proteases and to elucidate their role in the insurgence and progress on several diseases. Likewise, if the fluorinated methyl ketone moiety is suitably connected to a peptidic backbone, it may confer to the resulting structure an excellent substrate peculiarity and the possibility of being recognized by a specific subclass of human or pathogenic proteases. Therefore, peptidyl fluoromethyl ketones are also currently highly exploited for the target-based design of compounds for the treatment of topical diseases such as various types of cancer and viral infections.


Assuntos
Clorometilcetonas de Aminoácidos/síntese química , Fenilalanina/análogos & derivados , Vírus da SARS/efeitos dos fármacos , Inibidores de Serino Proteinase/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Clorometilcetonas de Aminoácidos/farmacologia , Química Farmacêutica/métodos , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , HIV/efeitos dos fármacos , HIV/enzimologia , Protease de HIV/química , Protease de HIV/metabolismo , Humanos , Cinética , Fenilalanina/síntese química , Fenilalanina/farmacologia , Vírus da SARS/enzimologia , Inibidores de Serino Proteinase/farmacologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
14.
Nat Commun ; 11(1): 4417, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887884

RESUMO

COVID-19 was declared a pandemic on March 11 by WHO, due to its great threat to global public health. The coronavirus main protease (Mpro, also called 3CLpro) is essential for processing and maturation of the viral polyprotein, therefore recognized as an attractive drug target. Here we show that a clinically approved anti-HCV drug, Boceprevir, and a pre-clinical inhibitor against feline infectious peritonitis (corona) virus (FIPV), GC376, both efficaciously inhibit SARS-CoV-2 in Vero cells by targeting Mpro. Moreover, combined application of GC376 with Remdesivir, a nucleotide analogue that inhibits viral RNA dependent RNA polymerase (RdRp), results in sterilizing additive effect. Further structural analysis reveals binding of both inhibitors to the catalytically active side of SARS-CoV-2 protease Mpro as main mechanism of inhibition. Our findings may provide critical information for the optimization and design of more potent inhibitors against the emerging SARS-CoV-2 virus.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Prolina/análogos & derivados , Inibidores de Proteases/farmacologia , Pirrolidinas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/farmacologia , Betacoronavirus/enzimologia , Sítios de Ligação/efeitos dos fármacos , Domínio Catalítico , Chlorocebus aethiops , Cristalografia por Raios X , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Modelos Animais de Doenças , Ensaios de Triagem em Larga Escala , Modelos Moleculares , Pandemias , Prolina/farmacologia , RNA Replicase/antagonistas & inibidores , RNA Replicase/química , RNA Replicase/metabolismo , Células Vero , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
15.
J Chem Theory Comput ; 16(10): 6383-6396, 2020 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-32905698

RESUMO

Molecular dynamics simulations are a popular means to study biomolecules, but it is often difficult to gain insights from the trajectories due to their large size, in both time and number of features. The Sapphire (States And Pathways Projected with HIgh REsolution) plot allows a direct visual inference of the dominant states visited by high-dimensional systems and how they are interconnected in time. Here, we extend this visual inference into a clustering algorithm. Specifically, the automatic procedure derives from the Sapphire plot states that are kinetically homogeneous, structurally annotated, and of tunable granularity. We provide a relative assessment of the kinetic fidelity of the Sapphire-based partitioning in comparison to popular clustering methods. This assessment is carried out on trajectories of n-butane, a ß-sheet peptide, and the small protein BPTI. We conclude with an application of our approach to a recent 100 µs trajectory of the main protease of SARS-CoV-2.


Assuntos
Butanos/química , Simulação de Dinâmica Molecular , Peptídeos/química , Proteínas/química , Algoritmos , Betacoronavirus/química , Análise por Conglomerados , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Humanos , Cinética , Pandemias , Pneumonia Viral/virologia , Conformação Proteica , Proteínas não Estruturais Virais/química
16.
Antiviral Res ; 182: 104924, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32896566

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic that began in 2019. The coronavirus 3-chymotrypsin-like cysteine protease (3CLpro) controls replication and is therefore considered a major target for antiviral discovery. This study describes the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay. Compared with a traditional FRET readout, the label-free SAMDI-MS assay offers greater sensitivity and eliminates false positive inhibition from compound interference with the optical signal. The SAMDI-MS assay was optimized and validated with known inhibitors of coronavirus 3CLpro such as GC376 (IC50 = 0.060 µM), calpain inhibitors II and XII (IC50 ~20-25 µM). The FDA-approved drugs shikonin, disulfiram, and ebselen did not inhibit SARS-CoV-2 3CLpro activity in the SAMDI-MS assay under physiologically relevant reducing conditions. The three drugs did not directly inhibit human ß-coronavirus OC-43 or SARS-CoV-2 in vitro, but instead induced cell death. In conclusion, the SAMDI-MS 3CLpro assay, combined with antiviral and cytotoxic assessment, provides a robust platform to evaluate antiviral agents directed against SARS-CoV-2.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas não Estruturais Virais/antagonistas & inibidores , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Glicoproteínas/farmacologia , Células HeLa , Humanos , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
17.
Antiviral Res ; 182: 104927, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32910955

RESUMO

Feline infectious peritonitis (FIP) which is caused by feline infectious peritonitis virus (FIPV), a variant of feline coronavirus (FCoV), is a member of family Coronaviridae, together with severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. So far, neither effective vaccines nor approved antiviral therapeutics are currently available for the treatment of FIPV infection. Both human and animal CoVs shares similar functional proteins, particularly the 3CL protease (3CLpro), which plays the pivotal role on viral replication. We investigated the potential drug-liked compounds and their inhibitory interaction on the 3CLpro active sites of CoVs by the structural-bases virtual screening. Fluorescence resonance energy transfer (FRET) assay revealed that three out of twenty-eight compounds could hamper FIPV 3CLpro activities with IC50 of 3.57 ± 0.36 µM to 25.90 ± 1.40 µM, and Ki values of 2.04 ± 0.08 to 15.21 ± 1.76 µM, respectively. Evaluation of antiviral activity using cell-based assay showed that NSC629301 and NSC71097 could strongly inhibit the cytopathic effect and also reduced replication of FIPV in CRFK cells in all examined conditions with the low range of EC50 (6.11 ± 1.90 to 7.75 ± 0.48 µM and 1.99 ± 0.30 to 4.03 ± 0.60 µM, respectively), less than those of ribavirin and lopinavir. Analysis of FIPV 3CLpro-ligand interaction demonstrated that the selected compounds reacted to the crucial residues (His41 and Cys144) of catalytic dyad. Our investigations provide a fundamental knowledge for the further development of antiviral agents and increase the number of anti-CoV agent pools for feline coronavirus and other related CoVs.


Assuntos
Antivirais/farmacologia , Coronavirus Felino/efeitos dos fármacos , Coronavirus Felino/enzimologia , Inibidores de Cisteína Proteinase/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Domínio Catalítico , Gatos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Avaliação Pré-Clínica de Medicamentos/métodos , Peritonite Infecciosa Felina/tratamento farmacológico , Peritonite Infecciosa Felina/virologia , Humanos , Concentração Inibidora 50 , Cinética , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Modelos Moleculares , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Proteínas não Estruturais Virais/química , Replicação Viral/efeitos dos fármacos
18.
Clin Sci (Lond) ; 134(17): 2235-2241, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32869854

RESUMO

Human serine protease inhibitors (serpins) are the main inhibitors of serine proteases, but some of them also have the capability to effectively inhibit cysteine proteases. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) main protease (Mpro) is a chymotrypsin-type cysteine protease that is needed to produce functional proteins essential for virus replication and transcription. Serpin traps its target proteases by presenting a reactive center loop (RCL) as protease-specific cleavage site, resulting in protease inactivation. Mpro target sites with its active site serine and other flanking residues can possibly interact with serpins. Alternatively, RCL cleavage site of serpins with known evidence of inhibition of cysteine proteases can be replaced by Mpro target site to make chimeric proteins. Purified chimeric serpin can possibly inhibit Mpro that can be assessed indirectly by observing the decrease in ability of Mpro to cleave its chromogenic substrate. Chimeric serpins with best interaction and active site binding and with ability to form 1:1 serpin-Mpro complex in human plasma can be assessed by using SDS/PAGE and Western blot analysis with serpin antibody. Trapping SARS-CoV-2 Mpro cysteine protease using cross-class serpin cysteine protease inhibition activity is a novel idea with significant therapeutic potential.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Serpinas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/uso terapêutico , Betacoronavirus/enzimologia , Western Blotting , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Eletroforese em Gel de Poliacrilamida , Humanos , Pandemias , Pneumonia Viral/virologia , Serpinas/uso terapêutico , Proteínas não Estruturais Virais/química
19.
PLoS One ; 15(9): e0238344, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881907

RESUMO

A novel severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) causing COVID-19 pandemic in humans, recently emerged and has exported in more than 200 countries as a result of rapid spread. In this study, we have made an attempt to investigate the SARS-CoV-2 genome reported from 13 different countries, identification of mutations in major coronavirus proteins of these different SARS-CoV-2 genomes and compared with SARS-CoV. These thirteen complete genome sequences of SARS-CoV-2 showed high identity (>99%) to each other, while they shared 82% identity with SARS-CoV. Here, we performed a very systematic mutational analysis of SARS-CoV-2 genomes from different geographical locations, which enabled us to identify numerous unique features of this viral genome. This includes several important country-specific unique mutations in the major proteins of SARS-CoV-2 namely, replicase polyprotein, spike glycoprotein, envelope protein and nucleocapsid protein. Indian strain showed mutation in spike glycoprotein at R408I and in replicase polyprotein at I671T, P2144S and A2798V,. While the spike protein of Spain & South Korea carried F797C and S221W mutation, respectively. Likewise, several important country specific mutations were analyzed. The effect of mutations of these major proteins were also investigated using various in silico approaches. Main protease (Mpro), the therapeutic target protein of SARS with maximum reported inhibitors, was thoroughly investigated and the effect of mutation on the binding affinity and structural dynamics of Mpro was studied. It was found that the R60C mutation in Mpro affects the protein dynamics, thereby, affecting the binding of inhibitor within its active site. The implications of mutation on structural characteristics were determined. The information provided in this manuscript holds great potential in further scientific research towards the design of potential vaccine candidates/small molecular inhibitor against COVID19.


Assuntos
Betacoronavirus/genética , Cisteína Endopeptidases/genética , Genoma Viral , Mutação , Proteínas do Nucleocapsídeo/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Betacoronavirus/classificação , Cisteína Endopeptidases/química , Variação Genética , Simulação de Dinâmica Molecular , Proteínas do Nucleocapsídeo/química , Filogenia , Glicoproteína da Espícula de Coronavírus/química , Proteínas do Envelope Viral/química , Proteínas não Estruturais Virais/química
20.
J Mol Graph Model ; 101: 107730, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32920239

RESUMO

The SARS-CoV-2 main protease (Mpro) is an attractive target towards discovery of drugs to treat COVID-19 because of its key role in virus replication. The atomic structure of Mpro in complex with an α-ketoamide inhibitor (Lig13b) is available (PDB ID:6Y2G). Using 6Y2G and the prior knowledge that protease inhibitors could eradicate COVID-19, we designed a computational study aimed at identifying FDA-approved drugs that could interact with Mpro. We searched the DrugBank and PubChem for analogs and built a virtual library containing ∼33,000 conformers. Using high-throughput virtual screening and ligand docking, we identified Isavuconazonium, a ketoamide inhibitor (α-KI) and Pentagastrin as the top three molecules (Lig13b as the benchmark) based on docking energy. The ΔGbind of Lig13b, Isavuconazonium, α-KI, Pentagastrin was -28.1, -45.7, -44.7, -34.8 kcal/mol, respectively. Molecular dynamics simulation revealed that these ligands are stable within the Mpro active site. Binding of these ligands is driven by a variety of non-bonded interaction, including polar bonds, H-bonds, van der Waals and salt bridges. The overall conformational dynamics of the complexed-Mpro was slightly altered relative to apo-Mpro. This study demonstrates that three distinct classes molecules, Isavuconazonium (triazole), α-KI (ketoamide) and Pentagastrin (peptide) could serve as potential drugs to treat patients with COVID-19.


Assuntos
Cisteína Endopeptidases/química , Nitrilos/farmacologia , Pentagastrina/farmacologia , Inibidores de Proteases/farmacologia , Piridinas/farmacologia , Triazóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Antivirais/química , Antivirais/farmacologia , Domínio Catalítico , Simulação por Computador , Cisteína Endopeptidases/metabolismo , Bases de Dados de Produtos Farmacêuticos , Aprovação de Drogas , Descoberta de Drogas , Reposicionamento de Medicamentos , Ensaios de Triagem em Larga Escala/métodos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Nitrilos/química , Pentagastrina/química , Inibidores de Proteases/química , Piridinas/química , Triazóis/química , Estados Unidos , United States Food and Drug Administration , Proteínas não Estruturais Virais/metabolismo
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