RESUMO
N-terminal autoprocessing from its polyprotein precursor enables creating the mature-like stable dimer interface of SARS-CoV-2 main protease (MPro), concomitant with the active site oxyanion loop equilibrium transitioning to the active conformation (E*) and onset of catalytic activity. Through mutagenesis of critical interface residues and evaluating noncovalent inhibitor (ensitrelvir, ESV) facilitated dimerization through its binding to MPro, we demonstrate that residues extending from Ser1 through Glu14 are critical for dimerization. Combined mutations G11A, E290A and R298A (MPro™) restrict dimerization even upon binding of ESV to monomeric MPro™ with an inhibitor dissociation constant of 7.4 ± 1.6 µM. Contrasting the covalent inhibitor NMV or GC373 binding to monomeric MPro, ESV binding enabled capturing the transition of the oxyanion loop conformations in the absence of a reactive warhead and independent of dimerization. Characterization of complexes by room-temperature X-ray crystallography reveals ESV bound to the E* state of monomeric MPro as well as an intermediate approaching the inactive state (E). It appears that the E* to E equilibrium shift occurs initially from G138-F140 residues, leading to the unwinding of the loop and formation of the 310-helix. Finally, we describe a transient dimer structure of the MPro precursor held together through interactions of residues A5-G11 with distinct states of the active sites, E and E*, likely representing an intermediate in the autoprocessing pathway.
Assuntos
Domínio Catalítico , Proteases 3C de Coronavírus , Inibidores de Protease de Coronavírus , Indazóis , Multimerização Proteica , SARS-CoV-2 , Triazinas , Triazóis , Humanos , Proteases 3C de Coronavírus/metabolismo , Proteases 3C de Coronavírus/química , Indazóis/química , Indazóis/farmacologia , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , SARS-CoV-2/enzimologia , SARS-CoV-2/metabolismo , Triazinas/química , Triazinas/farmacologia , Triazóis/química , Triazóis/farmacologia , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologiaRESUMO
Since the emergence of SARS-CoV-2, many genetic variations within its genome have been identified, but only a few mutations have been found in nonstructural proteins (NSPs). Among this class of viral proteins, NSP3 is a multidomain protein with 16 different domains, and its largest domain is known as the macrodomain or Mac1 domain. In this study, we present a virtual screening campaign in which we computationally evaluated the NCI anticancer library against the NSP3 Mac1 domain, using Molegro Virtual Docker. The top hits with the best MolDock and Re-Rank scores were selected. The physicochemical analysis and drug-like potential of the top hits were analyzed using the SwissADME data server. The binding stability and affinity of the top NSC compounds against the NSP3 Mac1 domain were analyzed using molecular dynamics (MD) simulation, using Desmond software, and their interaction energies were analyzed using the MM/GBSA method. In particular, by applying subsequent computational filters, we identified 10 compounds as possible NSP3 Mac1 domain inhibitors. Among them, after the assessment of binding energies (ΔGbind) on the whole MD trajectories, we identified the four most interesting compounds that acted as strong binders of the NSP3 Mac1 domain (NSC-358078, NSC-287067, NSC-123472, and NSC-142843), and, remarkably, it could be further characterized for developing innovative antivirals against SARS-CoV-2.
Assuntos
COVID-19 , Inibidores de Protease de Coronavírus , Simulação de Dinâmica Molecular , Humanos , COVID-19/prevenção & controle , SARS-CoV-2/química , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologia , Tratamento Farmacológico da COVID-19/métodosRESUMO
Viral infection in cells triggers a cascade of molecular defense mechanisms to maintain host-cell homoeostasis. One of these mechanisms is ADP-ribosylation, a fundamental post-translational modification (PTM) characterized by the addition of ADP-ribose (ADPr) on substrates. Poly(ADP-ribose) polymerases (PARPs) are implicated in this process and they perform ADP-ribosylation on host and pathogen proteins. Some viral families contain structural motifs that can reverse this PTM. These motifs known as macro domains (MDs) are evolutionarily conserved protein domains found in all kingdoms of life. They are divided in different classes with the viral belonging to Macro-D-type class because of their properties to recognize and revert the ADP-ribosylation. Viral MDs are potential pharmaceutical targets, capable to counteract host immune response. Sequence and structural homology between viral and human MDs are an impediment for the development of new active compounds against their function. Remdesivir, is a drug administrated in viral infections inhibiting viral replication through RNA-dependent RNA polymerase (RdRp). Herein, GS-441524, the active metabolite of the remdesivir, is tested as a hydrolase inhibitor for several viral MDs and for its binding to human homologs found in PARPs. This study presents biochemical and biophysical studies, which indicate that GS-441524 selectively modifies SARS-CoV-2 MD de-MARylation activity, while it does not interact with hPARP14 MD2 and hPARP15 MD2. The structural investigation of MDâ¢GS-441524 complexes, using solution NMR and X-ray crystallography, discloses the impact of certain amino acids in ADPr binding cavity suggesting that F360 and its adjacent residues tune the selective binding of the inhibitor to SARS-CoV-2 MD.
Assuntos
ADP-Ribosilação , Adenosina/análogos & derivados , Inibidores de Protease de Coronavírus , Poli(ADP-Ribose) Polimerases , SARS-CoV-2 , ADP-Ribosilação/efeitos dos fármacos , Adenosina/química , Adenosina/farmacologia , Adenosina Difosfato Ribose/química , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologia , Humanos , Poli(ADP-Ribose) Polimerases/química , Ligação Proteica , Domínios Proteicos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologiaRESUMO
To control the COVID-19 pandemic, antivirals that specifically target the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently required. The 3-chymotrypsin-like protease (3CLpro) is a promising drug target since it functions as a catalytic dyad in hydrolyzing polyprotein during the viral life cycle. Bioactive peptides, especially food-derived peptides, have a variety of functional activities, including antiviral activity, and also have a potential therapeutic effect against COVID-19. In this study, the hemp seed trypsinized peptidome was subjected to computer-aided screening against the 3CLpro of SARS-CoV-2. Using predictive trypsinized products of the five major proteins in hemp seed (i.e., edestin 1, edestin 2, edestin 3, albumin, and vicilin), the putative hydrolyzed peptidome was established and used as the input dataset. To select the Cannabis sativa antiviral peptides (csAVPs), a predictive bioinformatic analysis was performed by three webserver screening programs: iAMPpred, AVPpred, and Meta-iAVP. The amino acid composition profile comparison was performed by COPid to screen for the non-toxic and non-allergenic candidates, ToxinPred and AllerTOP and AllergenFP, respectively. GalaxyPepDock and HPEPDOCK were employed to perform the molecular docking of all selected csAVPs to the 3CLpro of SARS-CoV-2. Only the top docking-scored candidate (csAVP4) was further analyzed by molecular dynamics simulation for 150 nanoseconds. Molecular docking and molecular dynamics revealed the potential ability and stability of csAVP4 to inhibit the 3CLpro catalytic domain with hydrogen bond formation in domain 2 with short bonding distances. In addition, these top ten candidate bioactive peptides contained hydrophilic amino acid residues and exhibited a positive net charge. We hope that our results may guide the future development of alternative therapeutics against COVID-19.
Assuntos
Tratamento Farmacológico da COVID-19 , Cannabis , Inibidores de Protease de Coronavírus , Peptídeos , SARS-CoV-2 , Humanos , Cannabis/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias/prevenção & controle , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/isolamento & purificaçãoRESUMO
The coronavirus pandemic has affected more than 150 million people, while over 3.25 million people have died from the coronavirus disease 2019 (COVID-19). As there are no established therapies for COVID-19 treatment, drugs that inhibit viral replication are a promising target; specifically, the main protease (Mpro) that process CoV-encoded polyproteins serves as an Achilles heel for assembly of replication-transcription machinery as well as down-stream viral replication. In the search for potential antiviral drugs that target Mpro, a series of cembranoid diterpenes from the biologically active soft-coral genus Sarcophyton have been examined as SARS-CoV-2 Mpro inhibitors. Over 360 metabolites from the genus were screened using molecular docking calculations. Promising diterpenes were further characterized by molecular dynamics (MD) simulations based on molecular mechanics-generalized Born surface area (MM-GBSA) binding energy calculations. According to in silico calculations, five cembranoid diterpenes manifested adequate binding affinities as Mpro inhibitors with ΔGbinding < -33.0 kcal/mol. Binding energy and structural analyses of the most potent Sarcophyton inhibitor, bislatumlide A (340), was compared to darunavir, an HIV protease inhibitor that has been recently subjected to clinical-trial as an anti-COVID-19 drug. In silico analysis indicates that 340 has a higher binding affinity against Mpro than darunavir with ΔGbinding values of -43.8 and -34.8 kcal/mol, respectively throughout 100 ns MD simulations. Drug-likeness calculations revealed robust bioavailability and protein-protein interactions were identified for 340; biochemical signaling genes included ACE, MAPK14 and ESR1 as identified based on a STRING database. Pathway enrichment analysis combined with reactome mining revealed that 340 has the capability to re-modulate the p38 MAPK pathway hijacked by SARS-CoV-2 and antagonize injurious effects. These findings justify further in vivo and in vitro testing of 340 as an antiviral agent against SARS-CoV-2.
Assuntos
Antozoários/química , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Protease de Coronavírus/farmacologia , Diterpenos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , COVID-19/virologia , Proteases 3C de Coronavírus/metabolismo , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/isolamento & purificação , Diterpenos/química , Diterpenos/isolamento & purificação , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , SARS-CoV-2/enzimologia , SARS-CoV-2/patogenicidade , Relação Estrutura-AtividadeRESUMO
The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses an unprecedented global health crisis. It is particularly urgent to develop clinically effective therapies to contain the pandemic. The main protease (Mpro) and the RNA-dependent RNA polymerase (RdRP), which are responsible for the viral polyprotein proteolytic process and viral genome replication and transcription, respectively, are two attractive drug targets for SARS-CoV-2. This review summarizes up-to-date progress in the structural and pharmacological aspects of those two key targets above. Different classes of inhibitors individually targeting Mpro and RdRP are discussed, which could promote drug development to treat SARS-CoV-2 infection.
Assuntos
Antivirais/química , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Inibidores de Protease de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , RNA-Polimerase RNA-Dependente de Coronavírus/química , Inibidores Enzimáticos/química , SARS-CoV-2/enzimologia , Antivirais/farmacologia , Inibidores de Protease de Coronavírus/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Conformação Proteica , SARS-CoV-2/efeitos dos fármacosRESUMO
SARS-CoV-2 papain-like protease is considered as an important potential target for anti-SARS-CoV-2 drug discovery due to its crucial roles in viral spread and innate immunity. Here, we have utilized an in silico molecular docking approach to identify the possible inhibitors of the SARS-CoV-2 papain-like protease, by screening 21 antiviral, antifungal and anticancer compounds. Among them, Neobavaisoflavone has the highest binding energy for SARS-CoV-2 papain-like protease. These molecules could bind near the SARS-CoV-2 papain-like protease crucial catalytic triad, ubiquitination and ISGylation residues: Trp106, Asn109, Cys111, Met208, Lys232, Pro247, Tyr268, Gln269, His272, Asp286 and Thr301. Because blocking the papain-like protease is an important strategy in fighting against viruses, these compounds might be promising candidates for therapeutic intervention against COVID-19.
Assuntos
Proteases Semelhantes à Papaína de Coronavírus/química , Inibidores de Protease de Coronavírus/química , Inibidores de Cisteína Proteinase/química , Descoberta de Drogas/métodos , Isoflavonas/química , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Inibidores de Protease de Coronavírus/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Isoflavonas/farmacologia , Ligantes , Simulação de Acoplamento Molecular , Ligação ProteicaRESUMO
Human ubiquitin carboxyl-terminal hydrolase-2 (USP2) inhibitors, such as thiopurine analogs, have been reported to inhibit SARS-CoV papain-like proteases (PLpro). The PLpro have significant functional implications in the innate immune response during SARS-CoV-2 infection and considered an important antiviral target. Both proteases share strikingly similar USP fold with right-handed thumb-palm-fingers structural scaffold and conserved catalytic triad Cys-His-Asp/Asn. In this urgency situation of COVID-19 outbreak, there is a lack of in-vitro facilities readily available to test SARS-CoV-2 inhibitors in whole-cell assays. Therefore, we adopted an alternate route to identify potential USP2 inhibitor through integrated in-silico efforts. After an extensive virtual screening protocol, the best compounds were selected and tested. The compound Z93 showed significant IC50 value against Jurkat (9.67 µM) and MOTL-4 cells (11.8 µM). The binding mode of Z93 was extensively analyzed through molecular docking, followed by MD simulations, and molecular interactions were compared with SARS-CoV-2. The relative binding poses of Z93 fitted well in the binding site of both proteases and showed consensus π-π stacking and H-bond interactions with histidine and aspartate/asparagine residues of the catalytic triad. These results led us to speculate that compound Z93 might be the first potential chemical lead against SARS-CoV-2 PLpro, which warrants in-vitro evaluations.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Protease de Coronavírus/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Antivirais/química , COVID-19/virologia , Linhagem Celular Tumoral , Proteases 3C de Coronavírus/metabolismo , Inibidores de Protease de Coronavírus/química , Avaliação Pré-Clínica de Medicamentos , Humanos , Células Jurkat , Modelos Moleculares , Estrutura Molecular , Ubiquitina Tiolesterase/metabolismoRESUMO
The SARS-CoV-2 main protease (Mpro ) cleaves along the two viral polypeptides to release non-structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease. Here, we used native mass spectrometry to characterize the functional unit of Mpro . Analysis of the monomer/dimer equilibria reveals a dissociation constant of Kd =0.14±0.03â µM, indicating MPro has a strong preference to dimerize in solution. We characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, and screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the dimer, slow the rate of substrate processing by ≈35 %. This information, together with analysis of the x-ray crystal structures, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity.