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
The emergence of SARS-CoV-2 variants and drug-resistant mutants calls for additional oral antivirals. The SARS-CoV-2 papain-like protease (PLpro) is a promising but challenging drug target. We designed and synthesized 85 noncovalent PLpro inhibitors that bind to a recently discovered ubiquitin binding site and the known BL2 groove pocket near the S4 subsite. Leads inhibited PLpro with the inhibitory constant Ki values from 13.2 to 88.2 nanomolar. The co-crystal structures of PLpro with eight leads revealed their interaction modes. The in vivo lead Jun12682 inhibited SARS-CoV-2 and its variants, including nirmatrelvir-resistant strains with EC50 from 0.44 to 2.02 micromolar. Oral treatment with Jun12682 improved survival and reduced lung viral loads and lesions in a SARS-CoV-2 infection mouse model, suggesting that PLpro inhibitors are promising oral SARS-CoV-2 antiviral candidates.
Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19 , Proteases Semelhantes à Papaína de Coronavírus , Inibidores de Protease de Coronavírus , Desenho de Fármacos , SARS-CoV-2 , Animais , Camundongos , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/química , Modelos Animais de Doenças , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Inibidores de Protease de Coronavírus/administração & dosagem , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologia , Administração Oral , Cristalografia por Raios X , Relação Estrutura-Atividade , Carga Viral/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos BALB CRESUMO
The virally encoded 3C-like protease (3CLpro) is a well-validated drug target for the inhibition of coronaviruses including Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Most inhibitors of 3CLpro are peptidomimetic, with a γ-lactam in place of Gln at the P1 position of the pseudopeptide chain. An effort was pursued to identify a viable alternative to the γ-lactam P1 mimetic which would improve physicochemical properties while retaining affinity for the target. Discovery of a 2-tetrahydrofuran as a suitable P1 replacement that is a potent enzymatic inhibitor of 3CLpro in SARS-CoV-2 virus is described herein.
Assuntos
Antivirais , Inibidores de Protease de Coronavírus , Furanos , Antivirais/química , Antivirais/farmacologia , Lactamas , Peptídeo Hidrolases , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , SARS-CoV-2 , Furanos/química , Inibidores de Protease de Coronavírus/químicaRESUMO
BACKGROUND: Simnotrelvir is an oral 3-chymotrypsin-like protease inhibitor that has been found to have in vitro activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and potential efficacy in a phase 1B trial. METHODS: In this phase 2-3, double-blind, randomized, placebo-controlled trial, we assigned patients who had mild-to-moderate coronavirus disease 2019 (Covid-19) and onset of symptoms within the past 3 days in a 1:1 ratio to receive 750 mg of simnotrelvir plus 100 mg of ritonavir or placebo twice daily for 5 days. The primary efficacy end point was the time to sustained resolution of symptoms, defined as the absence of 11 Covid-19-related symptoms for 2 consecutive days. Safety and changes in viral load were also assessed. RESULTS: A total of 1208 patients were enrolled at 35 sites in China; 603 were assigned to receive simnotrelvir and 605 to receive placebo. Among patients in the modified intention-to-treat population who received the first dose of trial drug or placebo within 72 hours after symptom onset, the time to sustained resolution of Covid-19 symptoms was significantly shorter in the simnotrelvir group than in the placebo group (180.1 hours [95% confidence interval {CI}, 162.1 to 201.6] vs. 216.0 hours [95% CI, 203.4 to 228.1]; median difference, -35.8 hours [95% CI, -60.1 to -12.4]; P = 0.006 by Peto-Prentice test). On day 5, the decrease in viral load from baseline was greater in the simnotrelvir group than in the placebo group (mean difference [±SE], -1.51±0.14 log10 copies per milliliter; 95% CI, -1.79 to -1.24). The incidence of adverse events during treatment was higher in the simnotrelvir group than in the placebo group (29.0% vs. 21.6%). Most adverse events were mild or moderate. CONCLUSIONS: Early administration of simnotrelvir plus ritonavir shortened the time to the resolution of symptoms among adult patients with Covid-19, without evident safety concerns. (Funded by Jiangsu Simcere Pharmaceutical; ClinicalTrials.gov number, NCT05506176.).
Assuntos
COVID-19 , Inibidores de Protease de Coronavírus , Adulto , Humanos , Administração Oral , Antivirais/administração & dosagem , Antivirais/efeitos adversos , Antivirais/farmacologia , Antivirais/uso terapêutico , China , Proteínas M de Coronavírus/antagonistas & inibidores , Proteínas M de Coronavírus/metabolismo , Inibidores de Protease de Coronavírus/administração & dosagem , Inibidores de Protease de Coronavírus/efeitos adversos , Inibidores de Protease de Coronavírus/farmacologia , Inibidores de Protease de Coronavírus/uso terapêutico , COVID-19/metabolismo , COVID-19/terapia , Tratamento Farmacológico da COVID-19/métodos , Método Duplo-Cego , Ritonavir/administração & dosagem , Ritonavir/efeitos adversos , Ritonavir/farmacologia , Ritonavir/uso terapêutico , SARS-CoV-2/efeitos dos fármacos , Fatores de Tempo , Combinação de MedicamentosRESUMO
BACKGROUND: Oral antiviral drugs with improved antiviral potency and safety are needed to address current challenges in clinical practice for treatment of COVID-19, including the risks of rebound, drug-drug interactions, and emerging resistance. METHODS: Olgotrelvir (STI-1558) is designed as a next-generation antiviral targeting the SARS-CoV-2 main protease (Mpro), an essential enzyme for SARS-CoV-2 replication, and human cathepsin L (CTSL), a key enzyme for SARS-CoV-2 entry into host cells. FINDINGS: Olgotrelvir is a highly bioavailable oral prodrug that is converted in plasma to its active form, AC1115. The dual mechanism of action of olgotrelvir and AC1115 was confirmed by enzyme activity inhibition assays and co-crystal structures of AC1115 with SARS-CoV-2 Mpro and human CTSL. AC1115 displayed antiviral activity by inhibiting replication of all tested SARS-CoV-2 variants in cell culture systems. Olgotrelvir also inhibited viral entry into cells using SARS-CoV-2 Spike-mediated pseudotypes by inhibition of host CTSL. In the K18-hACE2 transgenic mouse model of SARS-CoV-2-mediated disease, olgotrelvir significantly reduced the virus load in the lungs, prevented body weight loss, and reduced cytokine release and lung pathologies. Olgotrelvir demonstrated potent activity against the nirmatrelvir-resistant Mpro E166 mutants. Olgotrelvir showed enhanced oral bioavailability in animal models and in humans with significant plasma exposure without ritonavir. In phase I studies (ClinicalTrials.gov: NCT05364840 and NCT05523739), olgotrelvir demonstrated a favorable safety profile and antiviral activity. CONCLUSIONS: Olgotrelvir is an oral inhibitor targeting Mpro and CTSL with high antiviral activity and plasma exposure and is a standalone treatment candidate for COVID-19. FUNDING: Funded by Sorrento Therapeutics.
Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19 , Inibidores de Protease de Coronavírus , SARS-CoV-2 , Animais , Humanos , Camundongos , Antivirais/farmacologia , Antivirais/uso terapêutico , Catepsina L/antagonistas & inibidores , COVID-19/prevenção & controle , Modelos Animais de Doenças , Camundongos Transgênicos , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Tratamento Farmacológico da COVID-19/métodosRESUMO
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
New lead drugs to treat COVID-19 are beginning to emerge.
Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19 , Proteases 3C de Coronavírus , Inibidores de Protease de Coronavírus , Pandemias , Humanos , COVID-19/prevenção & controle , Pandemias/prevenção & controle , Descoberta de Drogas , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidoresRESUMO
We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.
Assuntos
Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus , Inibidores de Protease de Coronavírus , Descoberta de Drogas , SARS-CoV-2 , Humanos , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Simulação de Acoplamento Molecular , Inibidores de Protease de Coronavírus/síntese química , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologia , Relação Estrutura-Atividade , Cristalografia por Raios XRESUMO
Global coronavirus disease 2019 (COVID-19) pandemic still threatens human health and public safety, and the development of effective antiviral agent is urgently needed. The SARS-CoV-2 main protease (Mpro) and papain-like protease (PLpro) are vital proteins in viral replication and promising therapeutic targets. Additionally, PLpro also modulates host immune response by cleaving ubiquitin and interferon-stimulated gene product 15 (ISG15) from ISGylated host proteins. In this report, we identified [1,2]selenazolo[5,4-c]pyridin-3(2H)-one and benzo[d]isothiazol-3(2H)-one as attractive scaffolds of PLpro and Mpro inhibitors. The representative compounds 6c and 7e exhibited excellent PLpro inhibition with percent inhibition of 42.9% and 44.9% at 50 nM, respectively. The preliminary enzyme kinetics experiment and fluorescent labelling experiment results determined that 6c was identified as a covalent PLpro inhibitor, while 7e was a non-covalent inhibitor. Molecular docking and dynamics simulations revealed that 6c and 7e bound to Zn-finger domain of PLpro. Compounds 6c and 7e were also identified to potent Mpro inhibitors, and they exhibited potent antiviral activities in SARS-CoV-2 infected Vero E6 cells, with EC50 value of 3.9 µM and 7.4 µM, respectively. In addition, the rat liver homogenate half-life of 6c and 7e exceeded 24 h. These findings suggest that 6c and 7e are promising led compounds for further development of PLpro/Mpro dual-target antiviral drugs.
Assuntos
COVID-19 , Proteases Semelhantes à Papaína de Coronavírus , Inibidores de Protease de Coronavírus , Animais , Humanos , Ratos , Antivirais/farmacologia , Corantes , Endopeptidases , Simulação de Acoplamento Molecular , Peptídeo Hidrolases , SARS-CoV-2 , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologia , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidoresRESUMO
The effect of mutations of the catalytic dyad residues of SARS-CoV-2 main protease (MProWT) on the thermodynamics of binding of covalent inhibitors comprising nitrile [nirmatrelvir (NMV), NBH2], aldehyde (GC373), and ketone (BBH1) warheads to MPro is examined together with room temperature X-ray crystallography. When lacking the nucleophilic C145, NMV binding is â¼400-fold weaker corresponding to 3.5 kcal/mol and 13.3 °C decrease in free energy (ΔG) and thermal stability (Tm), respectively, relative to MProWT. The H41A mutation results in a 20-fold increase in the dissociation constant (Kd), and 1.7 kcal/mol and 1.4 °C decreases in ΔG and Tm, respectively. Increasing the pH from 7.2 to 8.2 enhances NMV binding to MProH41A, whereas no significant change is observed in binding to MProWT. Structures of the four inhibitor complexes with MPro1-304/C145A show that the active site geometries of the complexes are nearly identical to that of MProWT with the nucleophilic sulfur of C145 positioned to react with the nitrile or the carbonyl carbon. These results support a two-step mechanism for the formation of the covalent complex involving an initial non-covalent binding followed by a nucleophilic attack by the thiolate anion of C145 on the warhead carbon. Noncovalent inhibitor ensitrelvir (ESV) exhibits a binding affinity to MProWT that is similar to NMV but differs in its thermodynamic signature from NMV. The binding of ESV to MProC145A also results in a significant, but smaller, increase in Kd and decrease in ΔG and Tm, relative to NMV.
Assuntos
COVID-19 , Inibidores de Protease de Coronavírus , SARS-CoV-2 , Humanos , Carbono , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologia , Lactamas , Leucina , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Nitrilas , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologiaRESUMO
CONTEXT: In the replication of SARS-CoV-2, the main protease (Mpro/3CLpro) is significant. It is conserved in a number of novel coronavirus variations, and no known human proteases share its cleavage sites. Therefore, 3CLpro is an ideal target. In the report, we screened five potential inhibitors (1543, 2308, 3717, 5606, and 9000) of SARS-CoV-2 Mpro through a workflow. The calculation of MM-GBSA binding free energy showed that three of the five potential inhibitors (1543, 2308, 5606) had similar inhibitor effects to X77 against Mpro of SARS-CoV-2. In conclusion, the manuscript lays the groundwork for the design of Mpro inhibitors. METHODS: In the virtual screening phase, we used structure-based virtual screening (Qvina2.1) and ligand-based virtual screening (AncPhore). In the molecular dynamic simulation part, we used the Amber14SB + GAFF force field to perform molecular dynamic simulation of the complex for 100 ns (Gromacs2021.5) and performed MM-GBSA binding free energy calculation according to the simulation trajectory.
Assuntos
Proteases 3C de Coronavírus , Inibidores de Protease de Coronavírus , Simulação de Dinâmica Molecular , SARS-CoV-2 , Humanos , Endopeptidases , Simulação de Acoplamento Molecular , Farmacóforo , SARS-CoV-2/enzimologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologiaRESUMO
Ligand-based drug design methods are thought to require large experimental datasets to become useful for virtual screening. In this work, we propose a computational strategy to design novel inhibitors of coronavirus main protease, Mpro. The pipeline integrates publicly available screening and binding affinity data in a two-stage machine-learning model using the recent MACAW embeddings. Once trained, the model can be deployed to rapidly screen large libraries of molecules in silico. Several hundred thousand compounds were virtually screened and 10 of them were selected for experimental testing. From these 10 compounds, 8 showed a clear inhibitory effect on recombinant Mpro, with half-maximal inhibitory concentration values (IC50) in the range 0.18-18.82 µM. Cellular assays were also conducted to evaluate cytotoxic, haemolytic, and antiviral properties. A promising lead compound against coronavirus Mpro was identified with dose-dependent inhibition of virus infectivity and minimal toxicity on human MRC-5 cells.
Assuntos
COVID-19 , Proteases 3C de Coronavírus , Humanos , SARS-CoV-2 , Inibidores de Protease de Coronavírus , Ligantes , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Proteínas não Estruturais Virais/metabolismo , Cisteína Endopeptidases/metabolismo , Antivirais/farmacologia , Antivirais/química , Simulação de Acoplamento MolecularRESUMO
The new pandemic caused by the coronavirus (SARS-CoV-2) has become the biggest challenge that the world is facing today. It has been creating a devastating global crisis, causing countless deaths and great panic. The search for an effective treatment remains a global challenge owing to controversies related to available vaccines. A great research effort (clinical, experimental, and computational) has emerged in response to this pandemic, and more than 125000 research reports have been published in relation to COVID-19. The majority of them focused on the discovery of novel drug candidates or repurposing of existing drugs through computational approaches that significantly speed up drug discovery. Among the different used targets, the SARS-CoV-2 main protease (Mpro), which plays an essential role in coronavirus replication, has become the preferred target for computational studies. In this review, we examine a representative set of computational studies that use the Mpro as a target for the discovery of small-molecule inhibitors of COVID-19. They will be divided into two main groups, structure-based and ligand-based methods, and each one will be subdivided according to the strategies used in the research. From our point of view, the use of combined strategies could enhance the possibilities of success in the future, permitting to development of more rigorous computational studies in future efforts to combat current and future pandemics.
Assuntos
Antivirais , COVID-19 , Proteases 3C de Coronavírus , Inibidores de Protease de Coronavírus , Descoberta de Drogas , Humanos , Antivirais/farmacologia , Simulação de Acoplamento Molecular , SARS-CoV-2 , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Protease de Coronavírus/farmacologiaRESUMO
The recent global health emergency caused by the coronavirus disease 2019 (COVID-19) pandemic has taken a heavy toll, both in terms of lives and economies. Vaccines against the disease have been developed, but the efficiency of vaccination campaigns worldwide has been variable due to challenges regarding production, logistics, distribution and vaccine hesitancy. Furthermore, vaccines are less effective against new variants of the SARS-CoV-2 virus and vaccination-induced immunity fades over time. These challenges and the vaccines' ineffectiveness for the infected population necessitate improved treatment options, including the inhibition of the SARS-CoV-2 main protease (Mpro). Drug repurposing to achieve inhibition could provide an immediate solution for disease management. Here, we used structure-based virtual screening (SBVS) to identify natural products (from NP-lib) and FDA-approved drugs (from e-Drug3D-lib and Drugs-lib) which bind to the Mpro active site with high-affinity and therefore could be designated as potential inhibitors. We prioritized nine candidate inhibitors (e-Drug3D-lib: Ciclesonide, Losartan and Telmisartan; Drugs-lib: Flezelastine, Hesperidin and Niceverine; NP-lib: three natural products) and predicted their half maximum inhibitory concentration using DeepPurpose, a deep learning tool for drug-target interactions. Finally, we experimentally validated Losartan and two of the natural products as in vitro Mpro inhibitors, using a bioluminescence resonance energy transfer (BRET)-based Mpro sensor. Our study suggests that existing drugs and natural products could be explored for the treatment of COVID-19.
Assuntos
Antivirais , Produtos Biológicos , COVID-19 , Proteases 3C de Coronavírus , Inibidores de Protease de Coronavírus , SARS-CoV-2 , Humanos , Antivirais/química , Antivirais/farmacologia , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Losartan/química , Losartan/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidoresRESUMO
We recently demonstrated that inhibitor binding reorganizes the oxyanion loop of a monomeric catalytic domain of SARS CoV-2 main protease (MPro) from an unwound (E) to a wound (active, E*) conformation, independent of dimerization. Here we assess the effect of the flanking N-terminal residues, to imitate the MPro precursor prior to its autoprocessing, on conformational equilibria rendering stability and inhibitor binding. Thermal denaturation (Tm) of C145A mutant, unlike H41A, increases by 6.8 °C, relative to wild-type mature dimer. An inactivating H41A mutation to maintain a miniprecursor containing TSAVL[Q or E] of the flanking nsp4 sequence in an intact form [(-6)MProH41A and (-6*)MProH41A, respectively], and its corresponding mature MProH41A were systematically examined. While the H41A mutation exerts negligible effect on Tm and dimer dissociation constant (Kdimer) of MProH41A, relative to the wild type MPro, both miniprecursors show a 4-5 °C decrease in Tm and > 85-fold increase in Kdimer as compared to MProH41A. The Kd for the binding of the covalent inhibitor GC373 to (-6*)MProH41A increases â¼12-fold, relative to MProH41A, concomitant with its dimerization. While the inhibitor-free dimer exhibits a state in transit from E to E* with a conformational asymmetry of the protomers' oxyanion loops and helical domains, inhibitor binding restores the asymmetry to mature-like oxyanion loop conformations (E*) but not of the helical domains. Disorder of the terminal residues 1-2 and 302-306 observed in both structures suggest that N-terminal autoprocessing is tightly coupled to the E-E* equilibrium and stable dimer formation.
Assuntos
Proteases 3C de Coronavírus , Inibidores de Protease de Coronavírus , SARS-CoV-2 , Humanos , Domínio Catalítico , Cristalografia por Raios X , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/genética , Estabilidade Proteica , Mutação , Inibidores de Protease de Coronavírus/químicaRESUMO
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
New variants of the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) emerged and spread rapidly all over the world, which strongly supports the need for pharmacological options to complement vaccine strategies. Main protease (Mpro or 3CLpro) is a critical enzyme in the life cycle of SARS-CoV-2 and appears to be highly conserved among different genera of coronaviruses, making it an ideal target for the development of drugs with broad-spectrum property. PF-07304814 developed by Pfizer is an intravenously administered inhibitor targeting SARS-CoV-2 Mpro. Here we showed that PF-07304814 displays broad-spectrum inhibitory activity against Mpros from multiple coronaviruses. Crystal structures of Mpros of SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV-NL63 bound to the inhibitor PF-07304814 revealed a conserved ligand-binding site, providing new insights into the mechanism of inhibition of viral replication. A detailed analysis of these crystal structures complemented by comprehensive comparison defined the key structural determinants essential for inhibition and illustrated the binding mode of action of Mpros from different coronaviruses. In view of the importance of Mpro for the medications of SARS-CoV-2 infection, insights derived from the present study should accelerate the design of pan-coronaviral main protease inhibitors that are safer and more effective.
Assuntos
Proteases 3C de Coronavírus , Inibidores de Protease de Coronavírus , Indóis , Leucina , Pirrolidinonas , SARS-CoV-2 , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologia , Desenho de Fármacos , Humanos , Indóis/química , Indóis/farmacologia , Leucina/química , Leucina/farmacologia , Ligantes , Ligação Proteica , Pirrolidinonas/química , Pirrolidinonas/farmacologia , 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
In response to the current pandemic caused by the novel SARS-CoV-2, we design new compounds based on Lopinavir structure as an FDA-approved antiviral agent which is currently under more evaluation in clinical trials for COVID-19 patients. This is the first example of the preparation of Lopinavir isosteres from the main core of Lopinavir conducted to various heterocyclic fragments. It is proposed that main protease inhibitors play an important role in the cycle life of coronavirus. Thus, the protease inhibition effect of synthesized compounds was studied by molecular docking method. All of these 10 molecules, showing a good docking score compared. Molecular dynamics (MD) simulations also confirmed the stability of the best-designed compound in Mpro active site.Communicated by Ramaswamy H. Sarma.