RESUMO
Covalent target modulation with small molecules has been emerging as a promising strategy for drug discovery. However, covalent inhibitory antibody remains unexplored due to the lack of efficient strategies to engineer antibody with desired bioactivity. Herein, we developed an intracellular selection method to generate covalent inhibitory antibody against human rhinovirus 14 (HRV14) 3C protease through unnatural amino acid mutagenesis along the heavy chain complementarity-determining region 3 (CDR-H3). A library of antibody mutants was thus constructed and screened in vivo through co-expression with the target protease. Using this screening strategy, six covalent antibodies with proximity-enabled bioactivity were identified, which were shown to covalently target HRV14-3C protease with high inhibitory potency and exquisite selectivity. Compared to structure-based rational design, this library-based screening method provides a simple and efficient way for the discovery and engineering of covalent antibody for enzyme inhibition.
Assuntos
Proteases Virais 3C/antagonistas & inibidores , Anticorpos/farmacologia , Regiões Determinantes de Complementaridade/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Rhinovirus/enzimologia , Proteases Virais 3C/metabolismo , Anticorpos/química , Inibidores de Cisteína Proteinase/química , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Coronaviruses cause diseases in humans and livestock. The SARS-CoV-2 is infecting millions of human beings, with high morbidity and mortality worldwide. The main protease (Mpro) of coronavirus plays a pivotal role in viral replication and transcription, which, in theory, is an attractive drug target for antiviral drug development. It has been extensively discussed whether Xanthohumol is able to help COVID-19 patients. Here, we report that Xanthohumol, a small molecule in clinical trials from hops (Humulus lupulus), was a potent pan-inhibitor for various coronaviruses by targeting Mpro, for example, betacoronavirus SARS-CoV-2 (IC50 value of 1.53 µM), and alphacoronavirus PEDV (IC50 value of 7.51 µM). Xanthohumol inhibited Mpro activities in the enzymatical assays, while pretreatment with Xanthohumol restricted the SARS-CoV-2 and PEDV replication in Vero-E6 cells. Therefore, Xanthohumol is a potent pan-inhibitor of coronaviruses and an excellent lead compound for further drug development.
Assuntos
Proteases Virais 3C/antagonistas & inibidores , Flavonoides/química , Propiofenonas/química , Inibidores de Proteases/química , SARS-CoV-2/enzimologia , Proteases Virais 3C/química , Proteases Virais 3C/metabolismo , Alphacoronavirus/enzimologia , Alphacoronavirus/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , COVID-19/virologia , Domínio Catalítico , Chlorocebus aethiops , Coronavirus/enzimologia , Coronavirus/fisiologia , Flavonoides/metabolismo , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Humanos , Simulação de Acoplamento Molecular , Propiofenonas/metabolismo , Propiofenonas/farmacologia , Propiofenonas/uso terapêutico , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , SARS-CoV-2/isolamento & purificação , Alinhamento de Sequência , Células Vero , Replicação Viral/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
The generation of bioactive molecules from inactive precursors is a crucial step in the chemical evolution of life, however, mechanistic insights into this aspect of abiogenesis are scarce. Here, we investigate the protein-catalyzed formation of antivirals by the 3C-protease of enterovirus D68. The enzyme induces aldol condensations yielding inhibitors with antiviral activity in cells. Kinetic and thermodynamic analyses reveal that the bioactivity emerges from a dynamic reaction system including inhibitor formation, alkylation of the protein target by the inhibitors, and competitive addition of non-protein nucleophiles to the inhibitors. The most active antivirals are slowly reversible inhibitors with elongated target residence times. The study reveals first examples for the chemical evolution of bio-actives through protein-catalyzed, non-enzymatic C-C couplings. The discovered mechanism works under physiological conditions and might constitute a native process of drug development.
Assuntos
Proteases Virais 3C/antagonistas & inibidores , Antivirais/química , Enterovirus Humano D/enzimologia , Evolução Química , Proteases Virais 3C/metabolismo , Antivirais/metabolismo , Antivirais/farmacologia , Biocatálise , Carbono/química , Enterovirus Humano D/efeitos dos fármacos , Humanos , Cinética , TermodinâmicaRESUMO
BACKGROUND: Human rhinovirus 3C protease (HRV-3Cpro) plays a crucial role in viral proliferation, establishing it as a prime target for antiviral therapy. However, research on identifying HRV-3Cpro inhibitors is still limited. OBJECTIVE: This study had two primary objectives: first, to validate the efficacy of an end-point colorimetric assay, previously developed by our team, for identifying potential inhibitors of HRV-3Cpro; and second, to discover phytochemicals in medicinal plants that inhibit the enzyme's activity. METHODS: Rupintrivir, a well-known inhibitor of HRV-3Cpro, was used to validate the colorimetric assay. Following this, we conducted a two-step in silico screening of 2532 phytochemicals, which led to the identification of eight active compounds: apigenin, carnosol, chlorogenic acid, kaempferol, luteolin, quercetin, rosmarinic acid, and rutin. We subsequently evaluated these candidates in vitro. To further investigate the inhibitory potential of the most promising candidates, namely, carnosol and rosmarinic acid, molecular docking studies were performed to analyze their binding interactions with HRV-3Cpro. RESULTS: The colorimetric assay we previously developed is effective in identifying compounds that selectively inhibit HRV-3Cpro. Carnosol and rosmarinic acid emerged as potent inhibitors, inhibiting HRV-3Cpro activity in vitro by over 55%. Our analysis indicated that carnosol and rosmarinic acid exert their inhibitory effects through a competitive mechanism. Molecular docking confirmed their competitive binding to the enzyme's active site. CONCLUSION: Carnosol and rosmarinic acid warrant additional investigation for their potential in the development of common cold treatment. By highlighting these compounds as effective HRV-3Cpro inhibitors, our study presents a promising approach for discovering phytochemical inhibitors against proteases from similar pathogens.
Assuntos
Proteases Virais 3C , Antivirais , Resfriado Comum , Simulação de Acoplamento Molecular , Compostos Fitoquímicos , Rhinovirus , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/química , Compostos Fitoquímicos/isolamento & purificação , Proteases Virais 3C/antagonistas & inibidores , Rhinovirus/enzimologia , Rhinovirus/efeitos dos fármacos , Humanos , Antivirais/farmacologia , Antivirais/química , Resfriado Comum/tratamento farmacológico , Resfriado Comum/virologia , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade , Inibidores de Proteases/farmacologia , Inibidores de Proteases/químicaRESUMO
It is widely accepted that Hepatitis A virus (HAV) is responsible for liver failure and even death in older people and in people with other serious health issues; so, proposing new compounds with inhibitory activity can help to treated of these disease's. In current study, a new class of quinolines is proposed with inhibitor activity of the HAV proteinase. So, in the first step, fused quinoline derivatives has been synthesized in short reaction time (12.0 min) and high efficiency yields (94%) in presence of 1-carboxymethyl-2,3-dimethylimidazolium iodide ([cmdmim]I) ionic liquid catalyst using a new method. In the following, chemical reactivity and inhibitory activity of synthesized quinolines were evaluated in density functional theory (DFT) framework and molecular docking methodologies. High global softness (0.67 eV), low HOMOSWBNNT-LUMO4a gap (4.78 eV), and more negative adsorption energy (- 87.9 kJ mol-1) in these quinolines reveal that the 4a and 4b compounds have better delivery than other quinolines using SWBNNT as suitable carrier to target cells. Molecular docking shows that the best cavity of the HAV has - 134.2 kJ mol-1 interaction energy involving bonding and non-bonding interactions. In fact, these interactions are between fused quinolines with especial geometries and sidechain flexibility amino acids residues inside the best binding site of the HAV, as hydrogen bonding, steric, and electrostatic interactions. So, these interactions imply that proposed fused quinolines have good inhibitor activity for the HAV.
Assuntos
Proteases Virais 3C/antagonistas & inibidores , Antivirais/farmacologia , Hepatite A/tratamento farmacológico , Quinolinas/farmacologia , Antivirais/química , Antivirais/uso terapêutico , Hepatite A/virologia , Vírus da Hepatite A/efeitos dos fármacos , Vírus da Hepatite A/enzimologia , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Quinolinas/química , Quinolinas/uso terapêutico , Relação Estrutura-AtividadeRESUMO
Foot-and-mouth-disease virus (FMDV) is a picornavirus that causes a highly contagious disease of cloven-hoofed animals resulting in economic losses worldwide. The 3C protease (3Cpro) is the main protease essential in the picornavirus life cycle, which is an attractive antiviral target. Here, we used computer-aided virtual screening to filter potential anti-FMDV agents from the natural phytochemical compound libraries. The top 23 filtered compounds were examined for anti-FMDV activities by a cell-based assay, two of which possessed antiviral effects. In the viral and post-viral entry experiments, luteolin and isoginkgetin could significantly block FMDV growth with low 50% effective concentrations (EC50). Moreover, these flavonoids could reduce the viral load as determined by RT-qPCR. However, their prophylactic activities were less effective. Both the cell-based and the fluorescence resonance energy transfer (FRET)-based protease assays confirmed that isoginkgetin was a potent FMDV 3Cpro inhibitor with a 50% inhibition concentration (IC50) of 39.03 ± 0.05 and 65.3 ± 1.7 µM, respectively, whereas luteolin was less effective. Analyses of the protein-ligand interactions revealed that both compounds fit in the substrate-binding pocket and reacted to the key enzymatic residues of the 3Cpro. Our findings suggested that luteolin and isoginkgetin are promising antiviral agents for FMDV and other picornaviruses.
Assuntos
Proteases Virais 3C/antagonistas & inibidores , Antivirais/farmacologia , Biflavonoides/farmacologia , Inibidores Enzimáticos/farmacologia , Vírus da Febre Aftosa/efeitos dos fármacos , Vírus da Febre Aftosa/enzimologia , Febre Aftosa/virologia , Luteolina/farmacologia , Proteases Virais 3C/química , Proteases Virais 3C/genética , Proteases Virais 3C/metabolismo , Animais , Antivirais/química , Biflavonoides/química , Simulação por Computador , Inibidores Enzimáticos/química , Vírus da Febre Aftosa/química , Vírus da Febre Aftosa/genética , Humanos , Luteolina/química , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologiaRESUMO
The fatal pathogen enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease (HFMD), which leads to serious neurological syndromes. While there are no effective clinical agents available for EV71 treatment thus far, EV71 3C protease (3Cpro), a cysteine protease encoded by the virus, has become a promising drug target for discovery of antiviral drugs, given that it plays a crucial role in virus proliferation and interferes with host cell function. Here, we report two inhibitors of EV71 3Cpro, FOPMC and FIOMC, that were developed from previously reported cyanohydrin derivative (R)-1 by replacing the acyl cyanohydrin group with 4-iminooxazolidin-2-one. FOPMC and FIOMC have potent antiviral activity and dramatically improved metabolic stability. These two inhibitors demonstrated broad anti-EV effects on various cell lines and five epidemic viral strains. We further illuminated the binding models between 3Cpro and FOPMC/FIOMC through molecular docking and molecular dynamics simulations. The substitution of an acyl cyanohydrin group with 4-iminooxazolidin-2-one does make FOPMC and FIOMC potent anti-EV71 drug candidates as universal nonclassical bioisosteres with a cyanohydrin moiety. IMPORTANCE EV71 is one of the most epidemic agents of HFMD. Thus far, there are no antiviral drugs available for clinical usage. The conserved EV71 3Cpro plays pivotal roles in virus proliferation and defense host immunity, as well as having no homology in host cells, making it a most promising antiviral target. In this work, we identified that propyl- and isopropyl-substituted 4-iminooxazolidin-2-one moieties (FOPMC and FIOMC) effectively inhibited five epidemic viral strains in rhabdomyosarcoma (RD), HEK-293T, and VeroE6 cell lines. The inhibition mechanism was also illustrated with molecular docking and molecular dynamics (MD) simulations. The successful replacement of the labile cyanohydrin greatly improved the stability and pharmacokinetic properties of (R)-1, making 4-iminooxazolidin-2-one a nonclassical bioisosteric moiety of cyanohydrin. This discovery addressed a critical issue of the primitive structural scaffold of these promising anti-EV71 inhibitors and could lead to their development as broad-spectrum anti-EV agents.
Assuntos
Proteases Virais 3C , Antivirais , Enterovirus Humano A , Replicação Viral , Animais , Humanos , Proteases Virais 3C/antagonistas & inibidores , Antivirais/química , Antivirais/farmacologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Enterovirus Humano A/efeitos dos fármacos , Enterovirus Humano A/crescimento & desenvolvimento , Doença de Mão, Pé e Boca/tratamento farmacológico , Doença de Mão, Pé e Boca/prevenção & controle , Doença de Mão, Pé e Boca/virologia , Células HEK293 , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Nitrilas/química , Nitrilas/farmacologia , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
As one of the principal etiological agents of hand, foot, and mouth disease (HFMD), enterovirus 71 (EV71) is associated with severe neurological complications or fatal diseases, while without effective medications thus far. Here we applied dually activated Michael acceptor to develop a series of reversible covalent compounds for EV71 3C protease (3Cpro), a promising antiviral drug target that plays an essential role during viral replication by cleaving the precursor polyprotein, inhibiting host protein synthesis, and evading innate immunity. Among them, cyanoacrylate and Boc-protected cyanoarylamide derivatives (SLQ-4 and SLQ-5) showed effective antiviral activity against EV71. The two inhibitors exhibited broad antiviral effects, acting on RD, 293T, and Vero cell lines, as well as on EV71 A, B, C, CVA16, and CVB3 viral strains. We further determined the binding pockets between the two inhibitors and 3Cpro based on docking studies. These results, together with our previous studies, provide evidence to elucidate the mechanism of action of these two reversible covalent inhibitors and contribute to the development of clinically effective medicines to treat EV71 infections.
Assuntos
Proteases Virais 3C/antagonistas & inibidores , Antivirais/farmacologia , Enterovirus Humano A/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Proteases Virais 3C/química , Acrilamidas/química , Acrilamidas/farmacologia , Animais , Antivirais/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cianoacrilatos/química , Cianoacrilatos/farmacologia , Enterovirus/classificação , Enterovirus/efeitos dos fármacos , Infecções por Enterovirus/virologia , Humanos , Simulação de Acoplamento Molecular , Inibidores de Proteases/química , Replicação Viral/efeitos dos fármacosRESUMO
Human rhinoviruses (HRVs) are the predominant infectious agents for the common cold worldwide. The HRV-C species cause severe illnesses in children and are closely related to acute exacerbations of asthma. 3C protease, a highly conserved enzyme, cleaves the viral polyprotein during replication and assists the virus in escaping the host immune system. These key roles make 3C protease an important drug target. A few structures of 3Cs complexed with an irreversible inhibitor rupintrivir have been determined. These structures shed light on the determinants of drug specificity. Here we describe the structures of HRV-C15 3C in free and inhibitor-bound forms. The volume-decreased S1' subsite and half-closed S2 subsite, which were thought to be unique features of enterovirus A 3C proteases, appear in the HRV-C 3C protease. Rupintrivir assumes an "intermediate" conformation in the complex, which might open up additional avenues for the design of potent antiviral inhibitors. Analysis of the features of the three-dimensional structures and the amino acid sequences of 3C proteases suggest new applications for existing drugs.