RESUMO
The M2 proteins of influenza A and B viruses form acid-activated proton channels that are essential for the virus lifecycle. Proton selectivity is achieved by a transmembrane (TM) histidine whereas gating is achieved by a tryptophan residue. Although this functional apparatus is conserved between AM2 and BM2 channels, AM2 conducts protons exclusively inward whereas BM2 conducts protons in either direction depending on the pH gradient. Previous studies showed that in AM2, mutations of D44 abolished inward rectification of AM2, suggesting that the tryptophan gate is destabilized. To elucidate how charged residues C-terminal to the tryptophan regulates channel gating, here we investigate the structure and dynamics of H19 and W23 in a BM2 mutant, GDR-BM2, in which three BM2 residues are mutated to the corresponding AM2 residues, S16G, G26D and H27R. Whole-cell electrophysiological data show that GDR-BM2 conducts protons with inward rectification, identical to wild-type (WT) AM2 but different from WT-BM2. Solid-state NMR 15N and 13C spectra of H19 indicate that the mutant BM2 channel contains higher populations of cationic histidine and neutral τ tautomers compared to WT-BM2 at acidic pH. Moreover, 19F NMR spectra of 5-19F-labeled W23 resolve three peaks at acidic pH, suggesting three tryptophan sidechain conformations. Comparison of these spectra with the tryptophan spectra of other M2 peptides suggests that these indole sidechain conformations arise from interactions with the C-terminal charged residues and with the N-terminal cationic histidine. Taken together, these solid-state NMR data show that inward rectification in M2 proton channels is accomplished by tryptophan interactions with charged residues on both its C-terminal and N-terminal sides. Gating of these M2 proton channels is thus accomplished by a multi-residue complex with finely tuned electrostatic and aromatic interactions.
Assuntos
Histidina , Vírus da Influenza B , Prótons , Triptofano , Proteínas da Matriz Viral , Triptofano/química , Histidina/química , Histidina/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/genética , Vírus da Influenza B/química , Vírus da Influenza B/genética , Vírus da Influenza A/química , Vírus da Influenza A/metabolismo , Vírus da Influenza A/genética , Concentração de Íons de Hidrogênio , Canais Iônicos/química , Canais Iônicos/metabolismo , Canais Iônicos/genética , Mutação , Simulação de Dinâmica Molecular , Proteínas ViroporinasRESUMO
SARS-CoV-2 is still wreaking havoc all over the world with surging morbidity and high mortality. The main protease (Mpro ) is essential in the replication of SARS-CoV-2, enabling itself an active target for antiviral development. Herein, we reported the design and synthesis of a new class of peptidomimetics-constrained α, γ-AA peptides, based on which a series of aldehyde and ketoamide inhibitors of the Mpro of SARS-CoV-2 were prepared. The lead compounds showed excellent inhibitory activity in the FRET-based Mpro enzymatic assay not only for the Mpro of SARS-CoV-2 but also for SARS-CoV and MERS-CoV, along with HCoVs like HCoV-OC43, HCoV-229E, HCoV-NL63 and HKU1. The X-ray crystallographic results demonstrated that our compounds form a covalent bond with the catalytic Cys145. They also demonstrated effective antiviral activity against live SARS-CoV-2. Overall, the results suggest that α, γ-AA peptide could be a promising molecular scaffold in designing novel Mpro inhibitors of SARS-CoV-2 and other coronaviruses.
Assuntos
COVID-19 , Coronavirus Humano OC43 , Humanos , SARS-CoV-2 , Peptídeos/farmacologia , Antivirais/farmacologia , Inibidores de Proteases/químicaRESUMO
The COVID-19 pandemic spurred a broad interest in antiviral drug discovery. The SARS-CoV-2 main protease (Mpro) and papain-like protease (PLpro) are attractive antiviral drug targets given their vital roles in viral replication and modulation of host immune response. Structurally disparate compounds were reported as Mpro and PLpro inhibitors from either drug repurposing or rational design. Two polyphenols dieckol and 1,2,3,4,6-pentagalloylglucose (PGG) were recently reported as SARS-CoV-2 Mpro inhibitors. With our continuous interest in studying the mechanism of inhibition and resistance of Mpro inhibitors, we report herein our independent validation/invalidation of these two natural products. Our FRET-based enzymatic assay showed that neither dieckol nor PGG inhibited SARS-CoV-2 Mpro (IC50 > 20 µM), which is in contrary to previous reports. Serendipitously, PGG was found to inhibit the SARS-CoV-2 PLpro with an IC50 of 3.90 µM. The binding of PGG to PLpro was further confirmed in the thermal shift assay. However, PGG was cytotoxic in 293T-ACE2 cells (CC50 = 7.7 µM), so its intracellular PLpro inhibitory activity could not be quantified by the cell-based Flip-GFP PLpro assay. In addition, we also invalidated ebselen, disulfiram, carmofur, PX12, and tideglusib as SARS-CoV-2 PLpro inhibitors using the Flip-GFP assay. Overall, our results call for stringent hit validation, and the serendipitous discovery of PGG as a putative PLpro inhibitor might worth further pursuing. Graphical abstract.
RESUMO
The influenza A M2 wild-type (WT) proton channel is the target of the anti-influenza drug rimantadine. Rimantadine has two enantiomers, though most investigations into drug binding and inhibition have used a racemic mixture. Solid-state NMR experiments using the full length-M2 WT have shown significant spectral differences that were interpreted to indicate tighter binding for (R)- vs (S)-rimantadine. However, it was unclear if this correlates with a functional difference in drug binding and inhibition. Using X-ray crystallography, we have determined that both (R)- and (S)-rimantadine bind to the M2 WT pore with slight differences in the hydration of each enantiomer. However, this does not result in a difference in potency or binding kinetics, as shown by similar values for kon, koff, and Kd in electrophysiological assays and for EC50 values in cellular assays. We concluded that the slight differences in hydration for the (R)- and (S)-rimantadine enantiomers are not relevant to drug binding or channel inhibition. To further explore the effect of the hydration of the M2 pore on binding affinity, the water structure was evaluated by grand canonical ensemble molecular dynamics simulations as a function of the chemical potential of the water. Initially, the two layers of ordered water molecules between the bound drug and the channel's gating His37 residues mask the drug's chirality. As the chemical potential becomes more unfavorable, the drug translocates down to the lower water layer, and the interaction becomes more sensitive to chirality. These studies suggest the feasibility of displacing the upper water layer and specifically recognizing the lower water layers in novel drugs.
RESUMO
The main protease (Mpro) is a validated antiviral drug target of SARS-CoV-2. A number of Mpro inhibitors have now advanced to animal model study and human clinical trials. However, one issue yet to be addressed is the target selectivity over host proteases such as cathepsin L. In this study we describe the rational design of covalent SARS-CoV-2 Mpro inhibitors with novel cysteine reactive warheads including dichloroacetamide, dibromoacetamide, tribromoacetamide, 2-bromo-2,2-dichloroacetamide, and 2-chloro-2,2-dibromoacetamide. The promising lead candidates Jun9-62-2R (dichloroacetamide) and Jun9-88-6R (tribromoacetamide) had not only potent enzymatic inhibition and antiviral activity but also significantly improved target specificity over caplain and cathepsins. Compared to GC-376, these new compounds did not inhibit the host cysteine proteases including calpain I, cathepsin B, cathepsin K, cathepsin L, and caspase-3. To the best of our knowledge, they are among the most selective covalent Mpro inhibitors reported thus far. The cocrystal structures of SARS-CoV-2 Mpro with Jun9-62-2R and Jun9-57-3R reaffirmed our design hypothesis, showing that both compounds form a covalent adduct with the catalytic C145. Overall, these novel compounds represent valuable chemical probes for target validation and drug candidates for further development as SARS-CoV-2 antivirals.
Assuntos
Acetamidas/farmacologia , Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/química , Catepsina L/antagonistas & inibidores , Desenho de Fármacos , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Enterovirus D68 (EV-D68) is a viral pathogen that leads to severe respiratory illness and has been linked with the development of acute flaccid myelitis (AFM) in children. No vaccines or antivirals are currently available for EV-D68 infection, and treatment options for hospitalized patients are limited to supportive care. Here, we report the expression of the EV-D68 2A protease (2Apro) and characterization of its enzymatic activity. Furthermore, we discovered that telaprevir, an FDA-approved drug used for the treatment of hepatitis C virus (HCV) infections, is a potent antiviral against EV-D68 by targeting the 2Apro enzyme. Using a fluorescence resonance energy transfer-based substrate cleavage assay, we showed that the purified EV-D68 2Apro has proteolytic activity selective against a peptide sequence corresponding to the viral VP1-2A polyprotein junction. Telaprevir inhibits EV-D68 2Apro through a nearly irreversible, biphasic binding mechanism. In cell culture, telaprevir showed submicromolar-to-low-micromolar potency against several recently circulating neurotropic strains of EV-D68 in different human cell lines. To further confirm the antiviral drug target, serial viral passage experiments were performed to select for resistance against telaprevir. An N84T mutation near the active site of 2Apro was identified in resistant viruses, and this mutation reduced the potency of telaprevir in both the enzymatic and cellular antiviral assays. Collectively, we report for the first time the in vitro enzymatic activity of EV-D68 2Apro and the identification of telaprevir as a potent EV-D68 2Apro inhibitor. These findings implicate EV-D68 2Apro as an antiviral drug target and highlight the repurposing potential of telaprevir to treat EV-D68 infection.IMPORTANCE A 2014 EV-D68 outbreak in the United States has been linked to the development of acute flaccid myelitis in children. Unfortunately, no treatment options against EV-D68 are currently available, and the development of effective therapeutics is urgently needed. Here, we characterize and validate a new EV-D68 drug target, the 2Apro, and identify telaprevir-an FDA-approved drug used to treat hepatitis C virus (HCV) infections-as a potent antiviral with a novel mechanism of action toward 2Apro 2Apro functions as a viral protease that cleaves a peptide sequence corresponding to the VP1-2A polyprotein junction. The binding of telaprevir potently inhibits its enzymatic activity, and using drug resistance selection, we show that the potent antiviral activity of telaprevir was due to 2Apro inhibition. This is the first inhibitor to selectively target the 2Apro from EV-D68 and can be used as a starting point for the development of therapeutics with selective activity against EV-D68.
Assuntos
Antivirais/farmacologia , Enterovirus Humano D/efeitos dos fármacos , Infecções por Enterovirus/tratamento farmacológico , Oligopeptídeos/farmacologia , Células A549 , Linhagem Celular , Células HEK293 , Células HeLa , HumanosRESUMO
The Food and Drug Administration-approved influenza A antiviral amantadine inhibits the wild-type (WT) AM2 channel but not the S31N mutant predominantly found in circulating strains. In this study, serial viral passages were applied to select resistance against a newly developed isoxazole-conjugated adamantane inhibitor that targets the AM2 S31N channel. This led to the identification of the novel drug-resistant mutation L46P located outside the drug-binding site, which suggests an allosteric resistance mechanism. Intriguingly, when the L46P mutant was introduced to AM2 WT, the channel remained sensitive toward amantadine inhibition. To elucidate the molecular mechanism, molecular dynamics simulations and binding free energy molecular mechanics-generalized born surface area (MM-GBSA) calculations were performed on WT and mutant channels. It was found that the L46P mutation caused a conformational change in the N terminus of transmembrane residues 22-31 that ultimately broadened the drug-binding site of AM2 S31N inhibitor 4, which spans residues 26-34, but not of AM2 WT inhibitor amantadine, which spans residues 31-34. The MM-GBSA calculations showed stronger binding stability for 4 in complex with AM2 S31N compared with 4 in complex with AM2 S31N/L46P, and equal binding free energies of amantadine in complex with AM2 WT and AM2 L46P. Overall, these results demonstrate a unique allosteric resistance mechanism toward AM2 S31N channel blockers, and the L46P mutant represents the first experimentally confirmed drug-resistant AM2 mutant that is located outside of the pore where drug binds. SIGNIFICANCE STATEMENT: AM2 S31N is a high-profile antiviral drug target, as more than 95% of currently circulating influenza A viruses carry this mutation. Understanding the mechanism of drug resistance is critical in designing the next generation of AM2 S31N channel blockers. Using a previously developed AM2 S31N channel blocker as a chemical probe, this study was the first to identify a novel resistant mutant, L46P. The L46P mutant is located outside of the drug-binding site. Molecular dynamics simulations showed that L46P causes a dilation of drug-binding site between residues 22 and 31, which affects the binding of AM2 S31N channel blockers, but not the AM2 WT inhibitor amantadine.
Assuntos
Amantadina/farmacologia , Antivirais/farmacologia , Vírus da Influenza A/metabolismo , Mutação , Proteínas da Matriz Viral/genética , Regulação Alostérica/efeitos dos fármacos , Motivos de Aminoácidos , Animais , Antivirais/química , Sítios de Ligação , Cães , Farmacorresistência Viral , Feminino , Humanos , Vírus da Influenza A/efeitos dos fármacos , Células Madin Darby de Rim Canino , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Inoculações Seriadas , Relação Estrutura-Atividade , Proteínas da Matriz Viral/química , Xenopus laevisRESUMO
AM2 and BM2 proton channels are attractive antiviral drug targets due to their essential roles during influenza virus replication. Although both AM2 and BM2 are proton-selective ion channels, they share little sequence similarity except for the HXXXW sequence, which suggests that their proton conductance properties might differ. To test this hypothesis, we applied two-electrode voltage clamp electrophysiological assays to study the specific conductance, leakage current, channel activation, and inhibition of AM2 and BM2 proton channels. It was found that BM2 channel has a higher specific conductance than AM2 channel at pH5.5. Unlike AM2 channel, whose proton conductance is asymmetric (from viral exterior to interior), BM2 channel is capable of conducting proton in both directions. Moreover, BM2 requires a more acidic pH for channel activation than AM2, as revealed by its lower pKa values. Finally, both AM2 and BM2 can be inhibited by Cu(II) and Cu(I). Overall, the results from this side-by-side comparison of AM2 and BM2 channels reveal the structure-function relationships of these two viroporins, and such information might be important for the designing of novel ion channels.
Assuntos
Vírus da Influenza A/metabolismo , Canais Iônicos/metabolismo , Prótons , Proteínas da Matriz Viral/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Feminino , Concentração de Íons de Hidrogênio , Vírus da Influenza A/genética , Canais Iônicos/química , Canais Iônicos/genética , Modelos Moleculares , Mutação , Oócitos/metabolismo , Oócitos/fisiologia , Conformação Proteica , Homologia de Sequência do Ácido Nucleico , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Xenopus laevisRESUMO
Influenza viruses are respiratory pathogens that are responsible for annual influenza epidemics and sporadic influenza pandemics. Oseltamivir (Tamiflu®) is currently the only FDA-approved oral drug that is available for the prevention and treatment of influenza virus infection. However, its narrow therapeutic window, coupled with the increasing incidence of drug resistance, calls for the next generation of influenza antivirals. In this study, we discovered hesperadin, an aurora B kinase inhibitor, as a broad-spectrum influenza antiviral through forward chemical genomics screening. Hesperadin inhibits multiple human clinical isolates of influenza A and B viruses with single to submicromolar efficacy, including oseltamivir-resistant strains. Mechanistic studies revealed that hesperadin inhibits the early stage of viral replication by delaying the nuclear entry of viral ribonucleoprotein complex, thereby inhibiting viral RNA transcription and translation as well as viral protein synthesis. Moreover, a combination of hesperadin with oseltamivir shows synergistic antiviral activity, therefore hesperadin can be used either alone to treat infections by oseltamivir-resistant influenza viruses or used in combination with oseltamivir to delay resistance evolution among oseltamivir-sensitive strains. In summary, the discovery of hesperadin as a broad-spectrum influenza antiviral offers an alternative to combat future influenza epidemics and pandemics.
Assuntos
Antivirais/farmacologia , Indóis/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza B/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/farmacologia , Animais , Antivirais/química , Aurora Quinase B/antagonistas & inibidores , Células Cultivadas , Cães , Relação Dose-Resposta a Droga , Farmacorresistência Viral , Sinergismo Farmacológico , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/química , Células Madin Darby de Rim Canino , Oseltamivir/farmacologia , Inibidores de Proteínas Quinases/química , Sulfonamidas/química , Ensaio de Placa Viral , Replicação Viral/efeitos dos fármacosRESUMO
Adamantanes (amantadine and rimantadine) are one of the two classes of Food and Drug Administration-approved antiviral drugs used for the prevention and treatment of influenza A virus infections. They inhibit viral replication by blocking the wild-type (WT) M2 proton channel, thus preventing viral uncoating. However, their use was discontinued due to widespread drug resistance. Among a handful of drug-resistant mutants, M2-S31N is the predominant mutation and persists in more than 95% of currently circulating influenza A strains. We recently designed two classes of M2-S31N inhibitors, S31N-specific inhibitors and S31N/WT dual inhibitors, which are represented by N-[(5-cyclopropyl-1,2-oxazol-3-yl)methyl]adamantan-1-amine (WJ379) and N-[(5-bromothiophen-2-yl)methyl]adamantan-1-amine (BC035), respectively. However, their antiviral activities against currently circulating influenza A viruses and their genetic barrier to drug resistance are unknown. In this report, we evaluated the therapeutic potential of these two classes of M2-S31N inhibitors (WJ379 and BC035) by profiling their antiviral efficacy against multidrug-resistant influenza A viruses, in vitro drug resistance barrier, and synergistic effect with oseltamivir. We found that M2-S31N inhibitors were active against several influenza A viruses that are resistant to one or both classes of Food and Drug Administration-approved anti-influenza drugs. In addition, M2-S31N inhibitors display a higher in vitro genetic barrier to drug resistance than amantadine. The antiviral effect of WJ379 was also synergistic with oseltamivir carboxylate. Overall, these results reaffirm that M2-S31N inhibitors are promising antiviral drug candidates that warrant further development.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Moduladores de Transporte de Membrana/farmacologia , Proteínas Mutantes/metabolismo , Proteínas da Matriz Viral/metabolismo , Amantadina/farmacologia , Animais , Sequência de Bases , Cães , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/genética , Genes Virais , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Concentração Inibidora 50 , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Células Madin Darby de Rim Canino , Oseltamivir/farmacologia , Análise de Sequência de DNA , Inoculações SeriadasRESUMO
The influenza A virus M2 proton channel (A/M2) is the target of the antiviral drugs amantadine and rimantadine, whose use has been discontinued due to widespread drug resistance. Among the handful of drug-resistant mutants, S31N is found in more than 95% of the currently circulating viruses and shows greatly decreased inhibition by amantadine. The discovery of inhibitors of S31N has been hampered by the limited size, polarity, and dynamic nature of its amantadine-binding site. Nevertheless, we have discovered small-molecule drugs that inhibit S31N with potencies greater than amantadine's potency against WT M2. Drug binding locks the protein into a well-defined conformation, and the NMR structure of the complex shows the drug bound in the homotetrameric channel, threaded between the side chains of Asn31. Unrestrained molecular dynamics simulations predicted the same binding site. This S31N inhibitor, like other potent M2 inhibitors, contains a charged ammonium group. The ammonium binds as a hydrate to one of three sites aligned along the central cavity that appear to be hotspots for inhibition. These sites might stabilize hydronium-like species formed as protons diffuse through the outer channel to the proton-shuttling residue His37 near the cytoplasmic end of the channel.
Assuntos
Antivirais/química , Antivirais/farmacologia , Genes Fúngicos , Vírus da Influenza A/química , Vírus da Influenza A/genética , Mutação , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Amantadina/análogos & derivados , Amantadina/síntese química , Amantadina/química , Amantadina/farmacologia , Antivirais/síntese química , Sítios de Ligação , Desenho de Fármacos , Farmacorresistência Viral/genética , Humanos , Vírus da Influenza A/efeitos dos fármacos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relação Estrutura-Atividade , Proteínas da Matriz Viral/antagonistas & inibidoresRESUMO
Influenza viruses are the causative agents for seasonal influenza, which results in thousands of deaths and millions of hospitalizations each year. Moreover, sporadic transmission of avian or swan influenza viruses to humans often leads to an influenza pandemic, as there is no preimmunity in the human body to fight against such novel strains. The metastable genome of the influenza viruses, coupled with the reassortment of different strains from a wide range of host origins, leads to the continuous evolution of the influenza virus diversity. Such characteristics of influenza viruses present a grand challenge in devising therapeutic strategies to combat influenza virus infection. This review summarizes recent progress in designing small molecule inhibitors that target the drug-resistant influenza A virus M2 proton channels and highlights the contribution of mechanistic studies of proton conductance to drug discovery. The lessons learned throughout the course of M2 drug discovery might provide insights for designing inhibitors that target other therapeutically important ion channels.
Assuntos
Antivirais , Descoberta de Drogas/métodos , Vírus da Influenza A , Influenza Humana/tratamento farmacológico , Proteínas da Matriz Viral/antagonistas & inibidores , Antivirais/síntese química , Antivirais/química , Antivirais/uso terapêutico , Humanos , Influenza Humana/metabolismoRESUMO
Influenza virus infections lead to numerous deaths and millions of hospitalizations each year. One challenge facing anti-influenza drug development is the heterogeneity of the circulating influenza viruses, which comprise several strains with variable susceptibility to antiviral drugs. For example, the wild-type (WT) influenza A viruses, such as the seasonal H1N1, tend to be sensitive to antiviral drugs, amantadine and rimantadine, while the S31N mutant viruses, such as the pandemic 2009 H1N1 (H1N1pdm09) and seasonal H3N2, are resistant to this class of drugs. Thus, drugs targeting both WT and the S31N mutant are highly desired. We report our design of a novel class of dual inhibitors along with their ion channel blockage and antiviral activities. The potency of the most active compound 11 in inhibiting WT and the S31N mutant influenza viruses is comparable with that of amantadine in inhibiting WT influenza virus. Solution NMR studies and molecular dynamics (MD) simulations of drug-M2 interactions supported our design hypothesis: namely, the dual inhibitor binds in the WT M2 channel with an aromatic group facing down toward the C-terminus, while the same drug binds in the S31N M2 channel with its aromatic group facing up toward the N-terminus. The flip-flop mode of drug binding correlates with the structure-activity relationship (SAR) and has paved the way for the next round of rational design of broad-spectrum antiviral drugs.
Assuntos
Amantadina/farmacologia , Descoberta de Drogas , Farmacorresistência Viral/genética , Vírus da Influenza A/efeitos dos fármacos , Mutação , Inibidores da Bomba de Prótons/farmacologia , Bombas de Próton/metabolismo , Animais , Cães , Farmacorresistência Viral/efeitos dos fármacos , Vírus da Influenza A/genética , Células Madin Darby de Rim Canino , Simulação de Dinâmica Molecular , Porosidade , Ligação Proteica , Conformação Proteica , Inibidores da Bomba de Prótons/química , Inibidores da Bomba de Prótons/metabolismo , Bombas de Próton/química , Bombas de Próton/genética , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/metabolismo , Tiofenos/farmacologiaRESUMO
Influenza virus assembles and buds at the infected-cell plasma membrane. This involves extrusion of the plasma membrane followed by scission of the bud, resulting in severing the nascent virion from its former host. The influenza virus M2 ion channel protein contains in its cytoplasmic tail a membrane-proximal amphipathic helix that facilitates the scission process and is also required for filamentous particle formation. Mutation of five conserved hydrophobic residues to alanines within the amphipathic helix (M2 five-point mutant, or 5PM) reduced scission and also filament formation, whereas single mutations had no apparent phenotype. Here, we show that any two of these five residues mutated together to alanines result in virus debilitated for growth and filament formation in a manner similar to 5PM. Growth kinetics of the M2 mutants are approximately 2 logs lower than the wild-type level, and plaque diameter was significantly reduced. When the 5PM and a representative double mutant (I51A-Y52A) were introduced into A/WSN/33 M2, a strain that produces spherical particles, similar debilitation in viral growth occurred. Electron microscopy showed that with the 5PM and the I51A-Y52A A/Udorn/72 and WSN viruses, scission failed, and emerging virus particles exhibited a "beads-on-a-string" morphology. The major spike glycoprotein hemagglutinin is localized within lipid rafts in virus-infected cells, whereas M2 is associated at the periphery of rafts. Mutant M2s were more widely dispersed, and their abundance at the raft periphery was reduced, suggesting that the M2 amphipathic helix is required for proper localization in the host membrane and that this has implications for budding and scission.
Assuntos
Vírus da Influenza A/fisiologia , Proteínas da Matriz Viral/metabolismo , Montagem de Vírus , Liberação de Vírus , Substituição de Aminoácidos , Análise Mutacional de DNA , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/ultraestrutura , Microscopia Eletrônica , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas da Matriz Viral/genética , Ensaio de Placa ViralRESUMO
We compared the anti-influenza potencies of 57 adamantyl amines and analogs against influenza A virus with serine-31â M2 proton channel, usually termed as WT M2 channel, which is amantadine sensitive. We also tested a subset of these compounds against viruses with the amantadine-resistant L26F, V27A, A30T, G34E M2 mutant channels. Four compounds inhibited WT M2 virus inâ vitro with mid-nanomolar potency, with 27 compounds showing sub-micromolar to low micromolar potency. Several compounds inhibited L26F M2 virus inâ vitro with sub-micromolar to low micromolar potency, but only three compounds blocked L26F M2-mediated proton current as determined by electrophysiology (EP). One compound was found to be a triple blocker of WT, L26F, V27A M2 channels by EP assays, but did not inhibit V27A M2 virus inâ vitro, and one compound inhibited WT, L26F, V27A M2 inâ vitro without blocking V27A M2 channel. One compound blocked only L26F M2 channel by EP, but did not inhibit virus replication. The triple blocker compound is as long as rimantadine, but could bind and block V27A M2 channel due to its larger girth as revealed by molecular dynamics simulations, while MAS NMR informed on the interaction of the compound with M2(18-60) WT or L26F or V27A.
Assuntos
Influenza Humana , Simulação de Dinâmica Molecular , Humanos , Antivirais/química , Aminas/farmacologia , Prótons , Mutação , Influenza Humana/tratamento farmacológico , Amantadina/farmacologia , Amantadina/uso terapêutico , Proteínas da Matriz Viral/química , Farmacorresistência ViralRESUMO
The synthesis of several 6,7,8,9,10,11-hexahydro-9-methyl-5,7:9,11-dimethano-5H-benzocyclononen-7-amines is reported. Several of them display low micromolar NMDA receptor antagonist and/or trypanocidal activities. Two compounds are endowed with micromolar anti vesicular stomatitis virus activity, while only one compound shows micromolar anti-influenza activity. The anti-influenza activity of this compound does not seem to be mediated by blocking of the M2 protein.
Assuntos
Aminas/síntese química , Aminas/farmacologia , Vírus de DNA/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Trypanosoma brucei brucei/efeitos dos fármacos , Aminas/química , Antivirais/síntese química , Antivirais/química , Antivirais/farmacologia , Humanos , Receptores de N-Metil-D-Aspartato/metabolismo , Tripanossomicidas/síntese química , Tripanossomicidas/química , Tripanossomicidas/farmacologiaRESUMO
Influenza A virus M2 (A/M2) and the influenza B virus BM2 are both small integral membrane proteins that form proton-selective ion channels. Influenza A virus A/M2 channel is the target of the antiviral drug amantadine (and its methyl derivative rimantadine), whereas BM2 channel activity is not affected by the drug. The atomic structure of the pore-transmembrane (TM) domain peptide has been determined by x-ray crystallography [Stouffer et al. (2008) Nature 451:596-599] and of a larger M2 peptide by NMR methods [Schnell and Chou (2008) Nature 451:591-595]. The crystallographic data show electron density (at 3.5 A resolution) in the channel pore, consistent with amantadine blocking the pore of the channel. In contrast, the NMR data show 4 rimantadine molecules bound on the outside of the helices toward the cytoplasmic side of the membrane. Drug binding includes interactions with residues 40-45 and a polar hydrogen bond between rimantadine and aspartic acid residue 44 (D44). These 2 distinct drug-binding sites led to 2 incompatible drug inhibition mechanisms. We have generated chimeric channels between amantadine-sensitive A/M2 and amantadine-insensitive BM2 designed to define the drug-binding site. Two chimeras containing 5 residues of the A/M2 ectodomain and residues 24-36 of the A/M2 TM domain show 85% amantadine/rimantadine sensitivity and specific activity comparable to that of WT BM2. These functional data suggest that the amantadine/rimantadine binding site identified on the outside of the 4 helices is not the primary site associated with the pharmacologic inhibition of the A/M2 ion channel.
Assuntos
Amantadina/farmacologia , Vírus da Influenza B/efeitos dos fármacos , Canais Iônicos/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Proteínas Virais/antagonistas & inibidores , Sequência de Aminoácidos , Substituição de Aminoácidos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/química , Canais Iônicos/metabolismo , Dados de Sequência Molecular , Mutação/genética , Proteínas Recombinantes/química , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
The influenza A virus M2 protein (A/M2) is a homotetrameric pH-activated proton transporter/channel that mediates acidification of the interior of endosomally encapsulated virus. This 97-residue protein has a single transmembrane (TM) helix, which associates to form homotetramers that bind the anti-influenza drug amantadine. However, the minimal fragment required for assembly and proton transport in cellular membranes has not been defined. Therefore, the conductance properties of truncation mutants expressed in Xenopus oocytes were examined. A short fragment spanning residues 21-61, M2(21-61), was inserted into the cytoplasmic membrane and had specific, amantadine-sensitive proton transport activity indistinguishable from that of full-length A/M2; an epitope-tagged version of an even shorter fragment, M2(21-51)-FLAG, had specific activity within a factor of 2 of the full-length protein. Furthermore, synthetic fragments including a peptide spanning residues 22-46 were found to transport protons into liposomes in an amantadine-sensitive manner. In addition, the functionally important His-37 residue pK(a) values are highly perturbed in the tetrameric form of the protein, a property conserved in the TM peptide and full-length A/M2 in both micelles and bilayers. These data demonstrate that the determinants for folding, drug binding, and proton translocation are packaged in a remarkably small peptide that can now be studied with confidence.
Assuntos
Canais Iônicos/fisiologia , Fragmentos de Peptídeos/fisiologia , Proteínas da Matriz Viral/fisiologia , Amantadina/farmacologia , Sequência de Aminoácidos , Animais , Transporte Biológico/efeitos dos fármacos , Dopaminérgicos/farmacologia , Feminino , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Canais Iônicos/química , Canais Iônicos/genética , Potenciais da Membrana/efeitos dos fármacos , Microinjeções , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Multimerização Proteica , Prótons , Homologia de Sequência de Aminoácidos , Termodinâmica , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , XenopusRESUMO
SARS-CoV-2 encodes two viral cysteine proteases, the main protease (Mpro) and the papain-like protease (PLpro), both of which are validated antiviral drug targets. PLpro is involved in the cleavage of viral polyproteins as well as immune modulation by removing ubiquitin and interferon-stimulated gene product 15 (ISG15) from host proteins. Therefore, targeting PLpro might be a two-pronged approach. Several compounds including YM155, cryptotanshinone, tanshinone I, dihydrotanshinone I, tanshinone IIA, SJB2-043, 6-thioguanine, and 6-mercaptopurine were recently identified as SARS-CoV-2 PLpro inhibitors through high-throughput screenings. In this study, we aim to validate/invalidate the reported PLpro inhibitors using a combination of PLpro target-specific assays including enzymatic FRET assay, thermal shift binding assay (TSA), and cell-based FlipGFP assay. Collectively, our results showed that all compounds tested either did not show binding or led to denaturation of PLpro in the TSA binding assay, which might explain their weak enzymatic inhibition in the FRET assay. In addition, none of the compounds showed cellular PLpro inhibition as revealed by the FlipGFP assay. Therefore, more efforts are needed to search for potent and specific SARS-CoV-2 PLpro inhibitors.