RESUMO
Identifying residues critical to protein-protein binding and efficient design of stable and specific protein binders are challenging tasks. Extending beyond the direct contacts in a protein-protein binding interface, our study employs computational modeling to reveal the essential network of residue interactions and dihedral angle correlations critical in protein-protein recognition. We hypothesized that mutating residues exhibiting highly correlated dynamic motion within the interaction network could efficiently optimize protein-protein interactions to create tight and selective protein binders. We tested this hypothesis using the ubiquitin (Ub) and MERS coronaviral papain-like protease (PLpro) complex, since Ub is a central player in multiple cellular functions and PLpro is an antiviral drug target. Our designed ubiquitin variant (UbV) hosting three mutated residues displayed a â¼3,500-fold increase in functional inhibition relative to wild-type Ub. Further optimization of two C-terminal residues within the Ub network resulted in a KD of 1.5 nM and IC50 of 9.7 nM for the five-point Ub mutant, eliciting 27,500-fold and 5,500-fold enhancements in affinity and potency, respectively, as well as improved selectivity, without destabilizing the UbV structure. Our study highlights residue correlation and interaction networks in protein-protein interactions, and introduces an effective approach to design high-affinity protein binders for cell biology research and future therapeutics.
Assuntos
Proteases Semelhantes à Papaína de Coronavírus , Coronavírus da Síndrome Respiratória do Oriente Médio , Ubiquitina , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Ligação Proteica , Ubiquitina/química , Ubiquitina/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/metabolismoRESUMO
MERS-CoV main protease (Mpro) is essential for the maturation of the coronavirus; therefore, considered a potential drug target. Detailed conformational information is essential to developing antiviral therapeutics. However, the conformation of MERS-CoV Mpro under different conditions is poorly characterized. In this study, MERS-CoV Mpro was recombinantly produced in E.coli and characterized its structural stability with respect to changes in pH and temperatures. The intrinsic and extrinsic fluorescence measurements revealed that MERS-CoV Mpro tertiary structure was exposed to the polar environment due to the unfolding of the tertiary structure. However, the secondary structure of MERS-CoV Mpro was gained at low pH because of charge-charge repulsion. Furthermore, differential scanning fluorometry studies of Mpro showed a single thermal transition at all pHs except at pH 2.0; no transitions were observed. The data from the spectroscopic studies suggest that the MERS-CoV Mpro forms a molten globule-like state at pH 2.0. Insilico studies showed that the covid-19 Mpro shows 96.08% and 50.65% similarity to that of SARS-CoV Mpro and MERS-CoV Mpro, respectively. This study provides a basic understanding of the thermodynamic and structural properties of MERS-CoV Mpro.
Assuntos
Proteases 3C de Coronavírus , Coronavírus da Síndrome Respiratória do Oriente Médio , Proteases 3C de Coronavírus/genética , Proteases 3C de Coronavírus/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Conformação Proteica , Proteínas RecombinantesRESUMO
The high mutation rate of COVID-19 and the prevalence of multiple variants strongly support the need for pharmacological options to complement vaccine strategies. One region that appears highly conserved among different genera of coronaviruses is the substrate-binding site of the main protease (Mpro or 3CLpro), making it an attractive target for the development of broad-spectrum drugs for multiple coronaviruses. PF-07321332, developed by Pfizer, is the first orally administered inhibitor targeting the main protease of SARS-CoV-2, which also has shown potency against other coronaviruses. Here, we report three crystal structures of the main protease of SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome (MERS)-CoV bound to the inhibitor PF-07321332. The structures reveal a ligand-binding site that is conserved among SARS-CoV-2, SARS-CoV, and MERS-CoV, providing insights into the mechanism of inhibition of viral replication. The long and narrow cavity in the cleft between domains I and II of the main protease harbors multiple inhibitor-binding sites, where PF-07321332 occupies subsites S1, S2, and S4 and appears more restricted than other inhibitors. A detailed analysis of these structures illuminated key structural determinants essential for inhibition and elucidated the binding mode of action of the main proteases from different coronaviruses. Given the importance of the main protease for the treatment of SARS-CoV-2 infection, insights derived from this study should accelerate the design of safer and more effective antivirals. IMPORTANCE The current pandemic of multiple variants has created an urgent need for effective inhibitors of SARS-CoV-2 to complement vaccine strategies. PF-07321332, developed by Pfizer, is the first orally administered coronavirus-specific main protease inhibitor approved by the FDA. We solved the crystal structures of the main protease of SARS-CoV-2, SARS-CoV, and MERS-CoV that bound to the PF-07321332, suggesting PF-07321332 is a broad-spectrum inhibitor for coronaviruses. Structures of the main protease inhibitor complexes present an opportunity to discover safer and more effective inhibitors for COVID-19.
Assuntos
Lactamas , Leucina , Nitrilas , Peptídeo Hidrolases , Prolina , Antivirais/química , Antivirais/metabolismo , Humanos , Lactamas/química , Lactamas/metabolismo , Leucina/química , Leucina/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Nitrilas/química , Nitrilas/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Prolina/química , Prolina/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , SARS-CoV-2/química , SARS-CoV-2/enzimologia , Tratamento Farmacológico da COVID-19RESUMO
The 2',5'-oligoadenylate (2-5A)-dependent endoribonuclease, RNase L, is a principal mediator of the interferon (IFN) antiviral response. Therefore, the regulation of cellular levels of 2-5A is a key point of control in antiviral innate immunity. Cellular 2-5A levels are determined by IFN-inducible 2',5'-oligoadenylate synthetases (OASs) and by enzymes that degrade 2-5A. Importantly, many coronaviruses (CoVs) and rotaviruses encode 2-5A-degrading enzymes, thereby antagonizing RNase L and its antiviral effects. A-kinase-anchoring protein 7 (AKAP7), a mammalian counterpart, could possibly limit tissue damage from excessive or prolonged RNase L activation during viral infections or from self-double-stranded RNAs that activate OAS. We show that these enzymes, members of the two-histidine phosphoesterase (2H-PE) superfamily, constitute a subfamily referred here as 2',5'-PEs. 2',5'-PEs from the mouse CoV mouse hepatitis virus (MHV) (NS2), Middle East respiratory syndrome coronavirus (MERS-CoV) (NS4b), group A rotavirus (VP3), and mouse (AKAP7) were investigated for their evolutionary relationships and activities. While there was no activity against 3',5'-oligoribonucleotides, they all cleaved 2',5'-oligoadenylates efficiently but with variable activity against other 2',5'-oligonucleotides. The 2',5'-PEs are shown to be metal ion-independent enzymes that cleave trimer 2-5A (2',5'-p3A3) producing mono- or diadenylates with 2',3'-cyclic phosphate termini. Our results suggest that the elimination of 2-5A might be the sole function of viral 2',5'-PEs, thereby promoting viral escape from innate immunity by preventing or limiting the activation of RNase L. IMPORTANCE Viruses often encode accessory proteins that antagonize the host antiviral immune response. Here, we probed the evolutionary relationships and biochemical activities of two-histidine phosphoesterases (2H-PEs) that allow some coronaviruses and rotaviruses to counteract antiviral innate immunity. In addition, we investigated the mammalian enzyme AKAP7, which has homology and shared activities with the viral enzymes and might reduce self-injury. These viral and host enzymes, which we refer to as 2',5'-PEs, specifically degrade 2',5'-oligoadenylate activators of the antiviral enzyme RNase L. We show that the host and viral enzymes are metal ion independent and exclusively cleave 2',5'- and not 3',5'-phosphodiester bonds, producing cleavage products with cyclic 2',3'-phosphate termini. Our study defines 2',5'-PEs as enzymes that share characteristic conserved features with the 2H-PE superfamily but have specific and distinct biochemical cleavage activities. These findings may eventually lead to pharmacological strategies for developing antiviral drugs against coronaviruses, rotaviruses, and other viruses.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Nucleotídeos de Adenina/metabolismo , Endorribonucleases/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Vírus da Hepatite Murina/enzimologia , Oligorribonucleotídeos/metabolismo , Rotavirus/enzimologia , Animais , Humanos , Imunidade Inata/imunologia , Interferons/imunologia , CamundongosRESUMO
Corona Virus Disease-2019 (COVID-19), caused by the SARS CoV-2 virus, has been announced as a pandemic by the World Health Organization. COVID-19 has affected people globally, infecting more than 39.8 million people and claiming up to 1.11 million lives, yet there is no effective treatment strategy to cure this disease. As vaccine development is a time-consuming process, currently, efforts are being made to develop alternative plans for the timely and effective management of this disease. Drug repurposing always fascinated researchers and can be utilized as the most acceptable alternative to develop the therapeutics for COVID-19 using the pre-approved drugs. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has shown resemblance with distinctive enzyme targets, such as 3CLpro/Mpro, RdRp, Cathepsin L, and TMPRSS2 present in SARS CoV and MERS CoV. Therefore, the drugs that have shown efficacy in these viruses can also be used for the treatment of COVID-19. This review focuses on why repurposing could provide a better alternative in COVID- 19 treatment. The similarity in the structure and progression of infection of SARS CoV and MERS viruses offers a direction and validation to evaluate the drugs approved for SARS and MERS against COVID-19. It has been indicated that multiple therapeutic options that demonstrate efficacy against SARS CoV 2 are available to mitigate the potential emergence of COVID-19 infection.
Assuntos
Antivirais , Tratamento Farmacológico da COVID-19 , Infecções por Coronavirus/tratamento farmacológico , Reposicionamento de Medicamentos , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Antivirais/farmacologia , Antivirais/uso terapêutico , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologiaRESUMO
The papain-like protease (PLpro ) is an important enzyme for coronavirus polyprotein processing, as well as for virus-host immune suppression. Previous studies reveal that a molecular analysis of PLpro indicates the catalytic activity of viral PLpro and its interactions with ubiquitin. By using sequence comparisons, molecular models, and protein-protein interaction maps, PLpro was compared in the three recorded fatal CoV epidemics, which involved severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome CoV (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV). The pairwise sequence comparison of SARS-CoV-2 PLpro indicated similarity percentages of 82.59% and 30.06% with SARS-CoV PLpro and MERS-CoV PLpro , respectively. In comparison with SARS-CoV PLpro , in SARS-CoV-2, the PLpro had a conserved catalytic triad of C111, H278, and D293, with a slightly lower number of polar interface residues and of hydrogen bonds, a higher number of buried interface sizes, and a lower number of residues that interact with ubiquitin and PLpro . These features might contribute to a similar or slightly lower level of deubiquitinating activity in SARS-CoV-2 PLpro. It was, however, a much higher level compared to MERS-CoV, which contained amino acid mutations and a low number of polar interfaces. SARS-CoV-2 PLpro and SARS-CoV PLpro showed almost the same catalytic site profiles, interface area compositions and polarities, suggesting a general similarity in deubiquitination activity. Compared with MERS-CoV, SARS-CoV-2 had a higher potential for binding interactions with ubiquitin. These estimated parameters contribute to the knowledge gap in understanding how the new virus interacts with the immune system.
Assuntos
COVID-19/patologia , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , SARS-CoV-2/enzimologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Sequência de Aminoácidos , Domínio Catalítico/fisiologia , Humanos , Modelos Moleculares , Poliproteínas/biossíntese , Poliproteínas/genética , Alinhamento de Sequência , Síndrome Respiratória Aguda Grave/patologia , Ubiquitina/metabolismo , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
Coronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) domains. While the latter presumably supports mRNA capping, ExoN is thought to mediate proofreading during genome replication. In line with such a role, ExoN knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously reported to have crippled but viable hypermutation phenotypes. Remarkably, using reverse genetics, a large set of corresponding ExoN knockout mutations has now been found to be lethal for another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). For 13 mutants, viral progeny could not be recovered, unless-as happened occasionally-reversion had first occurred. Only a single mutant was viable, likely because its E191D substitution is highly conservative. Remarkably, a SARS-CoV-2 ExoN knockout mutant was found to be unable to replicate, resembling observations previously made for alpha- and gammacoronaviruses, but starkly contrasting with the documented phenotype of ExoN knockout mutants of the closely related SARS-CoV. Subsequently, we established in vitro assays with purified recombinant MERS-CoV nsp14 to monitor its ExoN and N7-MTase activities. All ExoN knockout mutations that proved lethal in reverse genetics were found to severely decrease ExoN activity while not affecting N7-MTase activity. Our study strongly suggests that CoV nsp14 ExoN has an additional function, which apparently is critical for primary viral RNA synthesis and thus differs from the proofreading function that, based on previous MHV and SARS-CoV studies, was proposed to boost longer-term replication fidelity.IMPORTANCE The bifunctional nsp14 subunit of the coronavirus replicase contains 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase domains. For the betacoronaviruses MHV and SARS-CoV, ExoN was reported to promote the fidelity of genome replication, presumably by mediating a form of proofreading. For these viruses, ExoN knockout mutants are viable while displaying an increased mutation frequency. Strikingly, we have now established that the equivalent ExoN knockout mutants of two other betacoronaviruses, MERS-CoV and SARS-CoV-2, are nonviable, suggesting an additional and critical ExoN function in their replication. This is remarkable in light of the very limited genetic distance between SARS-CoV and SARS-CoV-2, which is highlighted, for example, by 95% amino acid sequence identity in their nsp14 sequences. For (recombinant) MERS-CoV nsp14, both its enzymatic activities were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development.
Assuntos
Betacoronavirus/fisiologia , Exorribonucleases/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Betacoronavirus/enzimologia , Betacoronavirus/genética , Domínio Catalítico , Linhagem Celular , Exorribonucleases/química , Exorribonucleases/genética , Fluoruracila/farmacologia , Técnicas de Inativação de Genes , Genoma Viral , Humanos , Metilação , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Mutação , RNA Viral/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , SARS-CoV-2 , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Ensaio de Placa Viral , Dedos de ZincoRESUMO
Broad-spectrum antiviral drugs are urgently needed to stop the Coronavirus Disease 2019 pandemic and prevent future ones. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is related to the SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), which have caused the previous outbreaks. The papain-like protease (PLpro) is an attractive drug target due to its essential roles in the viral life cycle. As a cysteine protease, PLpro is rich in cysteines and histidines, and their protonation/deprotonation modulates catalysis and conformational plasticity. Here, we report the pKa calculations and assessment of the proton-coupled conformational dynamics of SARS-CoV-2 in comparison to SARS-CoV and MERS-CoV PLpros using the recently developed graphical processing unit (GPU)-accelerated implicit-solvent continuous constant pH molecular dynamics method with a new asynchronous replica-exchange scheme, which allows computation on a single GPU card. The calculated pKa's support the catalytic roles of the Cys-His-Asp triad. We also found that several residues can switch protonation states at physiological pH among which is C270/271 located on the flexible blocking loop 2 (BL2) of SARS-CoV-2/CoV PLpro. Simulations revealed that the BL2 can open and close depending on the protonation state of C271/270, consistent with the most recent crystal structure evidence. Interestingly, despite the lack of an analogous cysteine, BL2 in MERS-CoV PLpro is also very flexible, challenging a current hypothesis. These findings are supported by the all-atom fixed-charge simulations and provide a starting point for more detailed studies to assist the structure-based design of broad-spectrum inhibitors against CoV PLpros.
Assuntos
Antivirais/farmacologia , Betacoronavirus/enzimologia , Desenho de Fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Simulação de Dinâmica Molecular , Papaína/química , Papaína/metabolismo , Prótons , Sequência de Aminoácidos , Histidina , Concentração de Íons de Hidrogênio , Papaína/antagonistas & inibidores , Domínios Proteicos , SARS-CoV-2RESUMO
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 , COVID-19 , Domínio Catalítico , Gatos , Proteases 3C de Coronavírus , 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 , SARS-CoV-2 , Proteínas não Estruturais Virais/química , Replicação Viral/efeitos dos fármacosRESUMO
As of today, there is no antiviral for the treatment of the SARS-CoV-2 infection, and the development of a vaccine might take several months or even years. The structural superposition of the hepatitis C virus polymerase bound to sofosbuvir, a nucleoside analog antiviral approved for hepatitis C virus infections, with the SARS-CoV polymerase shows that the residues that bind to the drug are present in the latter. Moreover, a multiple alignment of several SARS-CoV-2, SARS and MERS-related coronaviruses polymerases shows that these residues are conserved in all these viruses, opening the possibility to use sofosbuvir against these highly infectious pathogens.
Assuntos
Antivirais/química , Betacoronavirus/enzimologia , Infecções por Coronavirus/virologia , Pandemias/prevenção & controle , Pneumonia Viral/virologia , RNA Polimerase Dependente de RNA/química , Sofosbuvir/química , Proteínas não Estruturais Virais/química , Antivirais/uso terapêutico , Sequência de Bases , COVID-19 , Domínio Catalítico , Simulação por Computador , Infecções por Coronavirus/tratamento farmacológico , RNA-Polimerase RNA-Dependente de Coronavírus , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Pneumonia Viral/tratamento farmacológico , Ligação Proteica , Estrutura Terciária de Proteína , RNA Polimerase Dependente de RNA/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , SARS-CoV-2 , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Síndrome Respiratória Aguda Grave/virologia , Sofosbuvir/uso terapêutico , Proteínas não Estruturais Virais/genéticaRESUMO
AIMS: In December 2019, the Coronavirus disease-2019 (COVID-19) virus has emerged in Wuhan, China. In this research, the first resolved COVID-19 crystal structure (main protease) was targeted in a virtual screening study by of FDA approved drugs dataset. In addition, a knowledge gap in relations of COVID-19 with the previously known fatal Coronaviruses (CoVs) epidemics, SARS and MERS CoVs, was covered by investigation of sequence statistics and phylogenetics. MATERIALS AND METHODS: Molecular modeling, virtual screening, docking, sequence comparison statistics and phylogenetics of the COVID-19 main protease were investigated. KEY FINDINGS: COVID-19 Mpro formed a phylogenetic group with SARS CoV that was distant from MERS CoV. The identity% was 96.061 and 51.61 for COVID-19/SARS and COVID-19/MERS CoV sequence comparisons, respectively. The top 20 drugs in the virtual screening studies comprised a broad-spectrum antiviral (ribavirin), anti-hepatitis B virus (telbivudine), two vitamins (vitamin B12 and nicotinamide) and other miscellaneous systemically acting drugs. Of special interest, ribavirin had been used in treating cases of SARS CoV. SIGNIFICANCE: The present study provided a comprehensive targeting of the first resolved COVID+19 structure of Mpro and found a suitable save drugs for repurposing against the viral Mpro. Ribavirin, telbivudine, vitamin B12 and nicotinamide can be combined and used for COVID treatment. This initiative relocates already marketed and approved safe drugs for potential use in COVID-treatment.
Assuntos
Antivirais/química , Betacoronavirus/enzimologia , Cisteína Endopeptidases/química , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Simulação de Acoplamento Molecular , Inibidores de Proteases/química , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Antivirais/farmacologia , Sítios de Ligação , Proteases 3C de Coronavírus , Curcumina/química , Curcumina/farmacologia , Aprovação de Drogas , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Modelos Moleculares , Inibidores de Proteases/farmacologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , SARS-CoV-2 , Alinhamento de Sequência , Estados Unidos , United States Food and Drug AdministrationRESUMO
An outbreak of coronavirus disease 2019 (COVID-19) occurred in Wuhan and it has rapidly spread to almost all parts of the world. For coronaviruses, RNA-dependent RNA polymerase (RdRp) is an important polymerase that catalyzes the replication of RNA from RNA template and is an attractive therapeutic target. In this study, we screened these chemical structures from traditional Chinese medicinal compounds proven to show antiviral activity in severe acute respiratory syndrome coronavirus (SARS-CoV) and the similar chemical structures through a molecular docking study to target RdRp of SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV). We found that theaflavin has a lower idock score in the catalytic pocket of RdRp in SARS-CoV-2 (-9.11 kcal/mol), SARS-CoV (-8.03 kcal/mol), and MERS-CoV (-8.26 kcal/mol) from idock. To confirm the result, we discovered that theaflavin has lower binding energy of -8.8 kcal/mol when it docks in the catalytic pocket of SARS-CoV-2 RdRp by using the Blind Docking server. Regarding contact modes, hydrophobic interactions contribute significantly in binding and additional hydrogen bonds were found between theaflavin and RdRp. Moreover, one π-cation interaction was formed between theaflavin and Arg553 from the Blind Docking server. Our results suggest that theaflavin could be a potential SARS-CoV-2 RdRp inhibitor for further study.
Assuntos
Antivirais/química , Betacoronavirus/efeitos dos fármacos , Biflavonoides/química , Catequina/química , Medicamentos de Ervas Chinesas/química , RNA Polimerase Dependente de RNA/química , Proteínas Virais/química , Sequência de Aminoácidos , Antivirais/farmacologia , Betacoronavirus/enzimologia , Betacoronavirus/genética , Biflavonoides/farmacologia , Domínio Catalítico , Catequina/farmacologia , Biologia Computacional/métodos , Medicamentos de Ervas Chinesas/farmacologia , Expressão Gênica , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , SARS-CoV-2 , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Termodinâmica , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
In the past few decades, coronaviruses have risen as a global threat to public health. Currently, the outbreak of coronavirus disease-19 (COVID-19) from Wuhan caused a worldwide panic. There are no specific antiviral therapies for COVID-19. However, there are agents that were used during the severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) epidemics. We could learn from SARS and MERS. Lopinavir (LPV) is an effective agent that inhibits the protease activity of coronavirus. In this review, we discuss the literature on the efficacy of LPV in vitro and in vivo, especially in patients with SARS and MERS, so that we might clarify the potential for the use of LPV in patients with COVID-19.
Assuntos
Antivirais/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Lopinavir/uso terapêutico , Pandemias , Pneumonia Viral/tratamento farmacológico , Ritonavir/uso terapêutico , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Animais , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Betacoronavirus/patogenicidade , COVID-19 , Linhagem Celular , Ensaios Clínicos como Assunto , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Humanos , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , SARS-CoV-2 , Síndrome Respiratória Aguda Grave/diagnóstico , Síndrome Respiratória Aguda Grave/epidemiologia , Síndrome Respiratória Aguda Grave/virologia , Resultado do TratamentoRESUMO
Antiviral drugs for managing infections with human coronaviruses are not yet approved, posing a serious challenge to current global efforts aimed at containing the outbreak of severe acute respiratory syndrome-coronavirus 2 (CoV-2). Remdesivir (RDV) is an investigational compound with a broad spectrum of antiviral activities against RNA viruses, including severe acute respiratory syndrome-CoV and Middle East respiratory syndrome (MERS-CoV). RDV is a nucleotide analog inhibitor of RNA-dependent RNA polymerases (RdRps). Here, we co-expressed the MERS-CoV nonstructural proteins nsp5, nsp7, nsp8, and nsp12 (RdRp) in insect cells as a part a polyprotein to study the mechanism of inhibition of MERS-CoV RdRp by RDV. We initially demonstrated that nsp8 and nsp12 form an active complex. The triphosphate form of the inhibitor (RDV-TP) competes with its natural counterpart ATP. Of note, the selectivity value for RDV-TP obtained here with a steady-state approach suggests that it is more efficiently incorporated than ATP and two other nucleotide analogs. Once incorporated at position i, the inhibitor caused RNA synthesis arrest at position i + 3. Hence, the likely mechanism of action is delayed RNA chain termination. The additional three nucleotides may protect the inhibitor from excision by the viral 3'-5' exonuclease activity. Together, these results help to explain the high potency of RDV against RNA viruses in cell-based assays.
Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Alanina/química , Alanina/farmacologia , Animais , Antivirais/química , Coronavirus/enzimologia , Ebolavirus/enzimologia , Expressão Gênica , Inibidores da Síntese de Ácido Nucleico/química , RNA , RNA Polimerase Dependente de RNA/genética , Células Sf9 , Proteínas não Estruturais Virais/genéticaRESUMO
Middle East respiratory syndrome coronavirus (MERS-CoV) had emerged and spread because of the worldwide travel and inefficient healthcare provided for the infected patients in several countries. Herein we investigated the anti-MERS-CoV activity of newly synthesized sixteen halogenated triazole compounds through the inhibition of helicase activity using the FRET assay. All new compounds underwent justification for their target structures via microanalytical and spectral data. SAR studies were performed. Biological results revealed that the most potent compounds were 4-(cyclopent-1-en-3-ylamino)-5-(2-(4-iodophenyl)hydrazinyl)-4H-1,2,4-triazole-3-thiol (16) and 4-(cyclopent-1-en-3-ylamino)-5-[2-(4-chlorophenyl)hydrazinyl]-4H-1,2,4-triazole-3-thiol (12). In silico molecular docking of the most potent compounds was performed to the active binding site of MERS-CoV helicase nsp13. Molecular docking results are in agreement with experimental findings.
Assuntos
Antivirais/farmacologia , Desenho Assistido por Computador , Infecções por Coronavirus/tratamento farmacológico , DNA Helicases/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Simulação de Acoplamento Molecular , Triazóis/farmacologia , Proteínas Virais/antagonistas & inibidores , Animais , Antivirais/síntese química , Antivirais/metabolismo , Infecções por Coronavirus/virologia , DNA Helicases/química , DNA Helicases/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Coronavírus da Síndrome Respiratória do Oriente Médio/crescimento & desenvolvimento , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Coronavirus papain-like proteases (PLPs or PLpro), such as the one encoded in the genome of the infectious Middle East Respiratory Syndrome (MERS) virus, have multiple enzymatic activities that promote viral infection. PLpro acts as a protease and processes the large coronavirus polyprotein for virus replication. PLpro also functions as both a deubiquitinating (DUB) and deISGylating (deISG) enzyme and removes ubiquitin (Ub) and interferon-stimulated gene 15 (ISG15) from cellular proteins. Both DUB and deISG activities are implicated in suppressing innate immune responses; however, the precise role of each activity in this process is still unclear due in part to the difficulties in separating each activity. In this study, we determine the first structure of MERS PLpro in complex with the full-length human ISG15 to a resolution of 2.3 Å. This structure and available structures of MERS PLpro-Ub complexes were used as molecular guides to design PLpro mutants that lack either or both DUB/deISG activities. We tested 13 different PLpro mutants for protease, DUB, and deISG activitites using fluorescence-based assays. Results show that we can selectively modulate DUB activity at amino acid positions 1649 and 1653 while mutation of Val1691 or His1652 of PLpro to a positive charged residue completely impairs both DUB/deISG activities. These mutant enzymes will provide new functional tools for delineating the importance of DUB versus deISG activity in virus-infected cells and may serve as potential candidates for attenuating the MERS virus in vivo for modified vaccine design efforts.
Assuntos
Infecções por Coronavirus/metabolismo , Cisteína Endopeptidases/metabolismo , Citocinas/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Ubiquitinas/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteases 3C de Coronavírus , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Citocinas/química , Citocinas/genética , Interações Hospedeiro-Parasita , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Ligação Proteica , Processamento de Proteína Pós-Traducional , Ubiquitina , Ubiquitinas/química , Ubiquitinas/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
RNA virus encodes a helicase essential for viral RNA transcription and replication when the genome size is larger than 7 kb. Coronavirus (CoV) has an exceptionally large RNA genome (~30 kb) and it encodes an essential replicase, the nonstructural protein 13 (nsp13), a member of superfamily 1 helicases. Nsp13 is among the evolutionary most conserved proteins not only in CoVs but also in nidovirales. Thus, it is considered as an important drug target. However, the high-resolution structure of CoV nsp13 remained unavailable even until more than a decade after the outbreak of the severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003, which hindered the structure-based drug design. This is in part due to the intrinsic flexibility of nsp13. Here, we describe protocols of deducing the crystal structure of Middle East respiratory syndrome coronavirus (MERS-CoV) helicase in detail, which include protein expression, purification, crystallization, enzymatic characterization, and structure determination. With these methods, catalytically active recombinant MERS-CoV nsp13 protein can be prepared and crystallized and the crystal structure can be solved.
Assuntos
Infecções por Coronavirus/virologia , Metiltransferases/química , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , RNA Helicases/química , Proteínas não Estruturais Virais/química , Cristalização , Humanos , Modelos Moleculares , RNA Viral/genética , Replicação ViralRESUMO
Middle East respiratory syndrome-coronavirus (MERS-CoV) is a zoonotic virus transmitted between animals and human beings. It causes MERS with high mortality rate. However, no vaccine or specific treatment is currently available. Since antiviral activity of some flavonoids is known, we applied a flavonoid library to probe inhibitory compounds against MERS-CoV 3C-like protease (3CLpro). Herbacetin, isobavachalcone, quercetin 3-ß-d-glucoside and helichrysetin were found to block the enzymatic activity of MERS-CoV 3CLpro. The binding of the four flavonoids was also confirmed independently using a tryptophan-based fluorescence method. The systematic comparison of the binding affinity of flavonoids made it possible to infer their scaffolds and functional groups required to bind with MERS-CoV 3CLpro. An induced-fit docking analysis revealed that S1 and S2 sites play a role in interaction with flavonoids. The experimental and computational study showed that flavonol and chalcone are favourite scaffolds to bind with the catalytic site of MERS-CoV 3CLpro. It was also deduced that some flavonoid derivatives with hydrophobic or carbohydrate attached to their core structures have a good inhibitory effect. Therefore, we suggest that flavonoids with these characteristics can be used as templates to develop potent MERS-CoV 3CLpro inhibitors.
Assuntos
Flavonoides/química , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Peptídeo Hidrolases/química , Inibidores de Proteases/química , Proteínas Virais/antagonistas & inibidores , Antivirais/química , Antivirais/metabolismo , Sítios de Ligação , Flavonoides/metabolismo , Glucosídeos/química , Glucosídeos/metabolismo , Humanos , Simulação de Acoplamento Molecular , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/metabolismo , Ligação Proteica , Quercetina/análogos & derivados , Quercetina/química , Quercetina/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The development of new therapeutic agents against the coronavirus causing Middle East Respiratory Syndrome (MERS) is a continuing imperative. The initial MERS-CoV epidemic was contained entirely through public health measures, but episodic cases continue, as there are currently no therapeutic agents effective in the treatment of MERS-CoV, although multiple strategies have been proposed. In this study, we screened 30,000 compounds from three different compound libraries against one of the essential proteases, the papain-like protease (PLpro), using a fluorescence-based enzymatic assay followed by surface plasmon resonance (SPR) direct binding analysis for hit confirmation. Mode of inhibition assays and competition SPR studies revealed two compounds to be competitive inhibitors. To improve upon the inhibitory activity of the best hit compounds, a small fragment library consisting of 352 fragments was screened in the presence of each hit compound, resulting in one fragment that enhanced the IC50 value of the best hit compound by 3-fold. Molecular docking and MM/PBSA binding energy calculations were used to predict potential binding sites, providing insight for design and synthesis of next-generation compounds.
Assuntos
Desenho de Fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Peptídeo Hidrolases/química , Inibidores de Proteases/química , Bibliotecas de Moléculas Pequenas/química , Proteínas Virais/antagonistas & inibidores , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica , Ensaios de Triagem em Larga Escala , Humanos , Simulação de Acoplamento Molecular , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/metabolismo , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-Atividade , Proteínas Virais/metabolismoRESUMO
There are currently no approved vaccines or small molecule therapeutics available for the prophylaxis or treatment of Middle East Respiratory Syndrome coronavirus (MERS-CoV) infections. MERS-CoV 3CL protease is essential for viral replication; consequently, it is an attractive target that provides a potentially effective means of developing small molecule therapeutics for combatting MERS-CoV. We describe herein the structure-guided design and evaluation of a novel class of inhibitors of MERS-CoV 3CL protease that embody a piperidine moiety as a design element that is well-suited to exploiting favorable subsite binding interactions to attain optimal pharmacological activity and PK properties. The mechanism of action of the compounds and the structural determinants associated with binding were illuminated using X-ray crystallography.