RESUMO
Influenza viruses contribute significantly to the global health burden, necessitating the development of strategies against transmission as well as effective antiviral treatments. The present study reports a biomimetic strategy inspired by the natural antiviral properties of mucins. A bovine serum albumin (BSA) conjugate decorated with the multivalent neuraminidase inhibitor Zanamivir (ZA-BSA) was synthesized using copper-free click chemistry. This synthetic pseudo-mucin exhibited potent neuraminidase inhibitory activity against several influenza strains. Virus capture and growth inhibition assays demonstrated its effective absorption of virion particles and ability to prevent viral infection in nanomolar concentrations. Investigation of the underlying antiviral mechanism of ZA-BSA revealed a dual mode of action, involving disruption of the initial stages of host-cell binding and fusion by inducing viral aggregation, followed by blocking the release of newly assembled virions by targeting neuraminidase activity. Notably, the conjugate also exhibited potent inhibitory activity against Oseltamivir-resistant neuraminidase variant comparable to the monomeric Zanamivir. These findings highlight the application of multivalent drug presentation on protein scaffold to mimic mucin adsorption of viruses, together with counteracting drug resistance. This innovative approach has potential for the creation of antiviral agents against influenza and other viral infections.
Assuntos
Antivirais , Mucinas , Neuraminidase , Vírion , Zanamivir , Neuraminidase/antagonistas & inibidores , Neuraminidase/metabolismo , Zanamivir/farmacologia , Zanamivir/química , Antivirais/farmacologia , Antivirais/química , Mucinas/metabolismo , Mucinas/química , Humanos , Vírion/efeitos dos fármacos , Animais , Soroalbumina Bovina/química , Cães , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Células Madin Darby de Rim Canino , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/enzimologiaRESUMO
Sialosides containing C8-modified sialic acids are challenging synthetic targets but potentially useful probes for diagnostic substrate profiling of sialidases and elucidating the binding specificity of sialic acid-interacting proteins. Here, we demonstrate efficient chemoenzymatic methods for synthesizing para-nitrophenol-tagged α2-3- and α2-6-linked sialyl galactosides containing C8-acetamido, C8-azido, or C8-amino derivatized N-acetylneuraminic acid (Neu5Ac). High-throughput substrate specificity studies showed that the C8-modification of sialic acid significantly changes its recognition by sialidases from humans, various bacteria, and different influenza A and B viruses. Sialosides carrying Neu5Ac with a C8-azido modification were generally well tolerated by all the sialidases we tested, whereas sialosides containing C8-acetamido-modified Neu5Ac were only cleaved by selective bacterial sialidases. In contrast, sialosides with C8-amino-modified Neu5Ac were cleaved by a combination of selective bacterial and influenza A virus sialidases. These results indicate that sialosides terminated with a C8-amino or C8-acetamido-modified sialic acid can be used with other sialosides for diagnostic profiling of disease-causing sialidase-producing pathogens.
Assuntos
Neuraminidase , Ácidos Siálicos , Neuraminidase/metabolismo , Especificidade por Substrato , Humanos , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Bactérias/enzimologia , Orthomyxoviridae/enzimologia , Vírus da Influenza A/enzimologiaRESUMO
Influenza virus neuraminidase (NA) is a major antiviral drug target and has recently reemerged as a key target of antibody-mediated protective immunity. Here we show that recombinant NAs across non-bat subtypes adopt various tetrameric conformations, including an "open" state that may help explain poorly understood variations in NA stability across viral strains and subtypes. We use homology-directed protein design to uncover the structural principles underlying these distinct tetrameric conformations and stabilize multiple recombinant NAs in the "closed" state, yielding two near-atomic resolution structures of NA by cryo-EM. In addition to enhancing thermal stability, conformational stabilization improves affinity to protective antibodies elicited by viral infection, including antibodies targeting a quaternary epitope and the broadly conserved catalytic site. Stabilized NAs can also be integrated into viruses without affecting fitness. Our findings provide a deeper understanding of NA structure, stability, and antigenicity, and establish design strategies for reinforcing the conformational integrity of recombinant NA proteins.
Assuntos
Neuraminidase , Orthomyxoviridae/enzimologia , Proteínas Virais , Anticorpos Antivirais , Epitopos , Neuraminidase/química , Proteínas Recombinantes/química , Proteínas Virais/químicaRESUMO
Influenza viruses are responsible for contagious respiratory illnesses in humans and cause seasonal epidemics and occasional pandemics worldwide. Previously, we identified a quinolinone derivative PA-49, which inhibited the influenza virus RNA-dependent RNA polymerase (RdRp) by targeting PA-PB1 interaction. This paper reports the structure optimization of PA-49, which resulted in the identification of 3-((dibenzylamino)methyl)quinolinone derivatives with more potent anti-influenza virus activity. During the optimization, the hit compound 89, which was more active than PA-49, was identified. Further optimization and scaffold hopping of 89 led to the most potent compounds 100 and a 1,8-naphthyridinone derivative 118, respectively. We conclusively determined that compounds 100 and 118 suppressed the replication of influenza virus and exhibited anti-influenza virus activity against both influenza virus types A and B in the range of 50% effective concentration (EC50) = 0.061-0.226 µM with low toxicity (50% cytotoxic concentration (CC50) >10 µM).
Assuntos
Antivirais/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/enzimologia , Animais , Antivirais/química , Antivirais/toxicidade , Linhagem Celular , Cães , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/toxicidade , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza B/efeitos dos fármacos , Células Madin Darby de Rim Canino , Modelos Moleculares , Simulação de Acoplamento Molecular , Relação Estrutura-AtividadeRESUMO
The growing resistance of the influenza virus to widely used competitive neuraminidase inhibitors occupying the active site of the enzyme requires the development of bifunctional compounds that can simultaneously interact with other regulatory sites on the protein surface. When developing such an inhibitor and combining structural fragments that could be located in the sialic acid cavity of the active site and the adjacent 430-cavity, it is necessary to select a suitable linker not only for connecting the fragments, but also to ensure effective interactions with the unique arginine triad Arg118-Arg292-Arg371 of neuraminidase. Using molecular modeling, we have demonstrated the usefulness of the sulfonamide group in the linker design and the potential advantage of this functional group over other isosteric analogues.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Neuraminidase/metabolismo , Orthomyxoviridae/enzimologia , Sulfonamidas/química , Antivirais/síntese química , Antivirais/química , Domínio Catalítico , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Modelos Moleculares , Simulação de Acoplamento Molecular , Neuraminidase/antagonistas & inibidores , Neuraminidase/química , Orthomyxoviridae/efeitos dos fármacos , Relação Estrutura-Atividade , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Influenza is an acute respiratory infection caused by the influenza virus, but few drugs are available for its treatment. Consequently, researchers have been engaged in efforts to discover new antiviral mechanisms that can lay the foundation for novel anti-influenza drugs. The viral RNA-dependent RNA polymerase (RdRp) is an enzyme that plays an indispensable role in the viral infection process, which is directly linked to the survival of the virus. Methods of inhibiting PB1-PB2 (basic polymerase 1-basic polymerase 2) interactions, which are a key part of RdRp enzyme activity, are integral in the design of novel antiviral drugs, a specific PB1-PB2 interactions inhibitor has not been reported. We have screened Enamine's database and conducted a parallel screening of multiple docking schemes, followed by simulations of molecular dynamics to determine the structure of a stable ligand-PB1 complex. We also calculated the free energy of binding between the screened compounds and PB1 protein. Ultimately, we screened and identified a potential PB1-PB2 inhibitor using the ADMET prediction model.
Assuntos
Antivirais/farmacologia , Orthomyxoviridae/efeitos dos fármacos , Antivirais/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Orthomyxoviridae/química , Orthomyxoviridae/enzimologia , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Proteínas Virais/químicaRESUMO
Seasonal influenza A and B viruses represent a global concern. Antiviral drugs are crucial to treat severe influenza in high-risk patients and prevent virus spread in case of a pandemic. The emergence of viruses showing drug resistance, in particular for the recently licensed polymerase inhibitor baloxavir marboxil, drives the need for developing alternative antivirals. The endonuclease activity residing in the N-terminal domain of the polymerase acidic protein (PAN) is crucial for viral RNA synthesis and a validated target for drug design. Its function can be impaired by molecules bearing a metal-binding pharmacophore (MBP) able to coordinate the two divalent metal ions in the active site. In the present work, the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold is explored for the inhibition of influenza virus PA endonuclease. The structure-activity relationship was analysed by modifying the substituents on the lipophilic moiety linked to the MBP. The new compounds exhibited nanomolar inhibitory activity in a FRET-based enzymatic assay, and a few compounds (15-17, 21) offered inhibition in the micromolar range, in a cell-based influenza virus polymerase assay. When investigated against a panel of PA-mutant forms, compound 17 was shown to retain full activity against the baloxavir-resistant I38T mutant. This was corroborated by docking studies providing insight into the binding mode of this novel class of PA inhibitors.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Isoindóis/farmacologia , Orthomyxoviridae/efeitos dos fármacos , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HEK293 , Humanos , Isoindóis/síntese química , Isoindóis/química , Simulação de Acoplamento Molecular , Estrutura Molecular , Orthomyxoviridae/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Relação Estrutura-Atividade , Proteínas Virais/metabolismoRESUMO
Visualization of virus-infected cells is usually performed by immunostaining with an antiviral antibody. On the other hand, we established an easy method for fluorescence (FL) imaging of cells infected with influenza A and B viruses and some paramyxoviruses without the need for cell fixation and an antiviral antibody. These viruses and the cells they have infected express the viral surface enzyme "neuraminidase" or "hemagglutinin-neuraminidase" that shows sialidase activity. Sialidase activity is fluorescently visualized by using a sialidase fluorogenic probe developed in our previous study. The probe enables histochemical FL imaging of the virus-infected cells and is applicable to virus isolation and detection of an influenza virus resistant to antiinfluenza drugs of sialidase inhibitors.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Fluorescência , Neuraminidase/metabolismo , Imagem Óptica/métodos , Infecções por Orthomyxoviridae/metabolismo , Orthomyxoviridae/enzimologia , Animais , Células COS , Chlorocebus aethiops , Cães , Células Madin Darby de Rim Canino , Neuraminidase/genética , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/isolamento & purificação , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Células VeroRESUMO
Hemagglutinin and neuraminidase, which constitute the glycoprotein spikes expressed on the surface of influenza A and B viruses, are the most exposed parts of the virus and play critical roles in the viral lifecycle. As such, they make prominent targets for the immune response and antiviral drugs. Neuraminidase inhibitors, particularly oseltamivir, constitute the most commonly used antivirals against influenza viruses, and they have proved their clinical utility against seasonal and emerging influenza viruses. However, the emergence of resistant strains remains a constant threat and consideration. Antivirals targeting the hemagglutinin protein are relatively new and have yet to gain global use but are proving to be effective additions to the antiviral repertoire, with a relatively high threshold for the emergence of resistance. Here we review antiviral drugs, both approved for clinical use and under investigation, that target the influenza virus hemagglutinin and neuraminidase proteins, focusing on their mechanisms of action and the emergence of resistance to them.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Orthomyxoviridae/efeitos dos fármacos , Proteínas do Envelope Viral/antagonistas & inibidores , Animais , Antivirais/classificação , Antivirais/metabolismo , Ensaios Clínicos como Assunto , Inibidores Enzimáticos/farmacologia , Hemaglutininas Virais/metabolismo , Humanos , Influenza Humana/tratamento farmacológico , Camundongos , Neuraminidase/antagonistas & inibidores , Orthomyxoviridae/química , Orthomyxoviridae/classificação , Orthomyxoviridae/enzimologia , Infecções por Orthomyxoviridae/tratamento farmacológico , Oseltamivir/farmacologiaRESUMO
Influenza virus RNA-dependent RNA polymerase (FluPol) transcribes the viral RNA genome in the infected cell nucleus. In the 1970s, researchers showed that viral transcription depends on host RNA polymerase II (RNAP II) activity and subsequently that FluPol snatches capped oligomers from nascent RNAP II transcripts to prime its own transcription. Exactly how this occurs remains elusive. Here, we review recent advances in the mechanistic understanding of FluPol transcription and early events in RNAP II transcription that are relevant to cap-snatching. We describe the known direct interactions between FluPol and the RNAP II C-terminal domain and summarize the transcription-related host factors that have been found to interact with FluPol. We also discuss open questions regarding how FluPol may be targeted to actively transcribing RNAP II and the exact context and timing of cap-snatching, which is presumed to occur after cap completion but before the cap is sequestered by the nuclear cap-binding complex.
Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Orthomyxoviridae/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Transcrição Gênica , Proteínas Virais/metabolismo , Humanos , Orthomyxoviridae/patogenicidade , Proteínas de Ligação ao Cap de RNA/genética , Proteínas de Ligação ao Cap de RNA/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genéticaRESUMO
Altered sialylation patterns play a role in chronic autoimmune diseases such as rheumatoid arthritis (RA). Recent studies have shown the pro-inflammatory activities of immunoglobulins (Igs) with desialylated sugar moieties. The role of neuraminidases (NEUs), enzymes which are responsible for the cleavage of terminal sialic acids (SA) from sialoglycoconjugates, is not fully understood in RA. We investigated the impact of zanamivir, an inhibitor of the influenza virus neuraminidase, and mammalian NEU2/3 on clinical outcomes in experimental arthritides studies. The severity of arthritis was monitored and IgG titers were measured by ELISA. (2,6)-linked SA was determined on IgG by ELISA and on cell surfaces by flow cytometry. Zanamivir at a dose of 100 mg/kg (zana-100) significantly ameliorated collagen-induced arthritis (CIA), whereas zana-100 was ineffective in serum transfer-induced arthritis. Systemic zana-100 treatment reduced the number of splenic CD138+/TACI+ plasma cells and CD19+ B cells, which was associated with lower IgG levels and an increased sialylation status of IgG compared to controls. Our data reveal the contribution of NEU2/3 in CIA. Zanamivir down-modulated the T and B cell-dependent humoral immune response and induced an anti-inflammatory milieu by inhibiting sialic acid degradation. We suggest that neuraminidases might represent a promising therapeutic target for RA and possibly also for other antibody-mediated autoimmune diseases.
Assuntos
Anti-Inflamatórios/administração & dosagem , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Inibidores Enzimáticos/administração & dosagem , Neuraminidase/antagonistas & inibidores , Zanamivir/administração & dosagem , Animais , Artrite Experimental/induzido quimicamente , Colágeno/efeitos adversos , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos C57BL , Orthomyxoviridae/enzimologia , Ácidos Siálicos/metabolismoRESUMO
Encouraged by our earlier discovery of N1-selective inhibitors, the 150-cavity of influenza virus neuraminidases (NAs) could be further exploited to yield more potent oseltamivir derivatives. Herein, we report the design, synthesis and biological evaluation of a series of novel oseltamivir derivatives via the structural modifications at C5-NH2 of oseltamivir targeting 150-cavity. Among them, compound 5c bearing 4-(3-methoxybenzyloxy)benzyl group exhibited the most potent activity, which was lower or modestly improved activities than oseltamivir carboxylate (OSC) against N1 (H1N1), N1 (H5N1) and N1 (H5N1-H274Y). Specifically, there was 30-fold loss of activity against the wild-type strain H1N1. However, 5c displayed 4.85-fold more potent activity than OSC against H5N1-H274Y NA. Also, 5c demonstrated low cytotoxicity in vitro and no acute toxicity in mice. Molecular docking studies provided insights into the high potency of 5c against N1 and N1-H274Y mutant NAs. Besides, the in silico prediction of physicochemical properties and CYP enzymatic inhibitory ability of representative compounds were conducted to evaluate their drug-like properties.
Assuntos
Antivirais/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Neuraminidase/antagonistas & inibidores , Orthomyxoviridae/efeitos dos fármacos , Oseltamivir/farmacologia , Animais , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Neuraminidase/genética , Neuraminidase/metabolismo , Orthomyxoviridae/enzimologia , Oseltamivir/análogos & derivados , Oseltamivir/química , Relação Estrutura-AtividadeRESUMO
Neuraminidase (NA) inhibitors are one of the two major classes of antivirals available for the treatment and prevention of influenza. X-ray crystal structure determination of NA head domains and their complexes with various inhibitors are of importance for the design and optimization of anti-influenza drugs. However, the globular tetrameric properties of NA head domains may produce crystals with pathological imperfections, lattice-translocation defects, making structure determination no longer straightforward. In this report, using a crystal of the NA head domain from the Wuhan Asiatic toad influenza virus as an example, the identification and solution of this type of crystal pathology are presented. Furthermore, its underlying mechanism of formation is explored.
Assuntos
Anuros/virologia , Neuraminidase/química , Infecções por Orthomyxoviridae/virologia , Orthomyxoviridae/enzimologia , Proteínas Virais/química , Animais , Domínio Catalítico , Modelos MolecularesRESUMO
This work describes a set of discovery research studies of an influenza cap-dependent endonuclease (CEN) inhibitor with a carbamoyl pyridone bicycle (CAB) scaffold. Using influenza CEN inhibitory activity, antiviral activity and pharmacokinetic (PK) parameters as indices, structure activity relationships (SAR) studies were performed at the N-1 and N-3 positions on the CAB scaffold, which is a unique template to bind two metals. The hydrophobic substituent at the N-1 position is extremely important for CEN inhibitory activity and antiviral activity, and dihydrodibenzothiepine is the most promising pharmacophore. The compound (S)-13i showed potent virus titer reduction over oseltamivir phosphate in an in vivo mouse model. The CAB compound described herein served as the lead compound of baloxavir marboxil with a tricyclic scaffold, which was approved in Japan and the USA in 2018.
Assuntos
Antivirais/farmacologia , Endonucleases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Orthomyxoviridae/efeitos dos fármacos , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Endonucleases/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Estrutura Molecular , Orthomyxoviridae/enzimologia , Relação Estrutura-AtividadeRESUMO
The recently discovered 340-cavity in influenza neuraminidase (NA) N6 and N7 subtypes has introduced new possibilities for rational structure-based drug design. However, the plasticity of the 340-loop (residues 342-347) and the role of the 340-loop in NA activity and substrate binding have not been deeply exploited. Here, we investigate the mechanism of 340-cavity formation and demonstrate for the first time that seven of nine NA subtypes are able to adopt an open 340-cavity over 1.8 µs total molecular dynamics simulation time. The finding that the 340-loop plays a role in the sialic acid binding pathway suggests that the 340-cavity can function as a druggable pocket. Comparing the open and closed conformations of the 340-loop, the side chain orientation of residue 344 was found to govern the formation of the 340-cavity. Additionally, the conserved calcium ion was found to substantially influence the stability of the 340-loop. Our study provides dynamical evidence supporting the 340-cavity as a druggable hotspot at the atomic level and offers new structural insight in designing antiviral drugs.
Assuntos
Antivirais/farmacologia , Desenvolvimento de Medicamentos , Neuraminidase/química , Orthomyxoviridae/enzimologia , Sítios de Ligação , Cálcio/química , Íons , Modelos Moleculares , Simulação de Dinâmica Molecular , Ácido N-Acetilneuramínico/química , Análise de Componente Principal , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Neuraminidase (NA) is one of the targets for the development of new antivirals against the influenza virus. The recombinant Escherichia coli cells, namely the strains BL21(DE3)pLysS and ArcticExpress(DE3) were used to produce the influenza virus neuraminidase. Although the different conditions of induction were tested, the accumulation of over-expressed NA in insoluble fraction occurred independently of these conditions. The level of over-expressed protein represents 26.15 % of the total cellular proteins. Therefore, the aim of these study was to design the procedure for isolation of recombinant neuraminidase from IBs and subsequently its solubilization and refolding to its native and active form. The highest purity of IBs (86 %) was achieved after repeatedly washing for at least five times with 2 M urea. The best solubilizing agent for releasing NA from IBs was the solution of 8 M urea at pH 8.0 with 94.8 ± 0.4 mg/L released proteins. The most appropriate buffer for refolding of solubilized NA was found to be 50 mM Tris-HCl at pH 7.5 (102 ± 24.2 mg proteins) and the addition of glycerol or arginine had no stimulating effect on protein recovery. The determination of non-glycosylated activity of refolded NA monomer (Km = 0.51 g/L; Vmax = 9.73 U/mg; kcat = 8.76 s-1) using fetuin as a substrate in the coupled enzyme reaction system was the highlight of this work. This procedure provides a way to produce active form of NA monomer by recombinant E. coli cells.
Assuntos
Escherichia coli/metabolismo , Corpos de Inclusão/metabolismo , Neuraminidase/isolamento & purificação , Orthomyxoviridae/enzimologia , Proteínas Virais/isolamento & purificação , Escherichia coli/genética , Neuraminidase/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Virais/metabolismoRESUMO
Influenza viruses harboring treatment-emergent I38F/M/N/T substitutions in the polymerase acidic (PA) endonuclease exhibited reduced susceptibility to baloxavir and were associated with virus rebound and variable clinical response in clinical trials. US regulatory review of registrational trial data also identified treatment-emergent PA substitutions E23K in A/H1N1 viruses and E23G/K, A37T, and E199G in A/H3N2 viruses, which conferred reduced susceptibility to baloxavir, although to a lesser degree than I38F/M/N/T substitutions, and were associated with virus rebound. Although these non-I38 substitutions emerged less frequently than substitutions at I38, they represent alternate pathways to baloxavir virologic resistance and should be monitored accordingly.
Assuntos
Substituição de Aminoácidos , Farmacorresistência Viral , Influenza Humana/virologia , Mutação , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/genética , RNA Polimerase Dependente de RNA/genética , Antivirais/farmacologia , Antivirais/uso terapêutico , Dibenzotiepinas/farmacologia , Dibenzotiepinas/uso terapêutico , Humanos , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Vírus da Influenza B/classificação , Vírus da Influenza B/genética , Influenza Humana/tratamento farmacológico , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Orthomyxoviridae/classificação , Orthomyxoviridae/enzimologia , Piridonas/farmacologia , Piridonas/uso terapêutico , Resultado do Tratamento , Triazinas/farmacologia , Triazinas/uso terapêutico , Carga ViralRESUMO
BACKGROUND: The novel cap-dependent endonuclease inhibitor baloxavir marboxil was approved in February 2018 for the treatment of influenza virus infection in Japan. In vitro studies have revealed that an I38T substitution in the polymerase acidic subunit (PA) is associated with reduced susceptibility of influenza viruses to baloxavir. OBJECTIVES: Development of a rapid and simple method for monitoring influenza A(H1N1)pdm09, A(H3N2), and B viruses possessing the I38T substitution in PA. METHODS: Three assays were developed based on RNase H2-dependent PCR (rhPCR) and named A/H1pdm PA_I38T rhPCR, A/H3 PA_I38T rhPCR, and B PA_I38T rhPCR. The assays were evaluated using cDNAs synthesized from in vitro-transcribed PA gene RNA controls, RNAs purified from viruses isolated in the 2017/2018 and 2018/2019 influenza seasons, and RNAs purified from clinical specimens collected in the 2018/2019 influenza season. RESULTS: The assays developed in this study accurately discriminated PA I38 and PA T38 with high sensitivity. CONCLUSIONS: Our assays should be considered a powerful tool for monitoring the emergence of baloxavir-resistant influenza viruses.
Assuntos
Substituição de Aminoácidos/efeitos dos fármacos , Antivirais/farmacologia , Dibenzotiepinas/farmacologia , Farmacorresistência Viral/genética , Técnicas de Diagnóstico Molecular/métodos , Morfolinas/farmacologia , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/genética , Piridonas/farmacologia , Triazinas/farmacologia , Substituição de Aminoácidos/genética , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/enzimologia , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/enzimologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza B/efeitos dos fármacos , Vírus da Influenza B/enzimologia , Vírus da Influenza B/genética , Influenza Humana/diagnóstico , Influenza Humana/virologia , Japão , Limite de Detecção , Técnicas de Diagnóstico Molecular/normas , Orthomyxoviridae/enzimologia , Ribonuclease H/genética , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Neuraminidase inhibitors (NAIs) are the only class of antivirals in clinical use against influenza virus approved worldwide. However, approximately 1-3% of circulating strains present resistance mutations to oseltamivir (OST), the most used NAI. Therefore, it is important to catalogue new molecules to inhibit influenza virus, especially OST-resistant strains. Natural products from tropical plants used for human consumption represent a worthy class of substances. Their use could be stimulated in resource-limited setting where the access to expensive antiviral therapies is restricted. METHODS: We evaluated the anti-influenza virus activity of agathisflavone derived from Anacardium occidentale L. RESULTS: The neuraminidase (NA) activity of wild-type and OST-resistant influenza virus was inhibited by agathisflavone, with IC50 values ranging from 20 to 2.0 µM, respectively. Agathisflavone inhibited influenza virus replication with EC50 of 1.3 µM. Sequential passages of the virus in the presence of agathisflavone revealed the emergence of mutation R249S, A250S and R253Q in the NA gene. These changes are outside the OST binding region, meaning that agathisflavone targets this viral enzyme at a region different than conventional NAIs. CONCLUSION: Altogether our data suggest that agathisflavone has a promising chemical structure for the development of anti-influenza drugs.
Assuntos
Anacardium/química , Biflavonoides/farmacologia , Inibidores Enzimáticos/farmacologia , Neuraminidase/antagonistas & inibidores , Orthomyxoviridae/efeitos dos fármacos , Compostos Fitoquímicos/farmacologia , Animais , Biflavonoides/química , Biflavonoides/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/virologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Neuraminidase/metabolismo , Orthomyxoviridae/enzimologia , Compostos Fitoquímicos/química , Compostos Fitoquímicos/isolamento & purificação , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
The limited therapeutic options and increasing drug-resistance call for next-generation influenza antivirals. Due to the essential function in viral replication and high sequence conservation among influenza viruses, influenza polymerase PA-PB1 protein-protein interaction becomes an attractive drug target. Here, we developed an in vitro split luciferase complementation-based assay to speed up screening of PA-PB1 interaction inhibitors. By screening 10,000 compounds, we identified two PA-PB1 interaction inhibitors, R160792 and R151785, with potent and broad-spectrum antiviral activity against a panel of influenza A and B viruses, including amantadine-, oseltamivir-, or dual resistant strains. Further mechanistic study reveals that R151785 inhibits PA nuclear localization, reduces the levels of viral RNAs and proteins, and inhibits viral replication at the intermediate stage, all of which are in line with its antiviral mechanism of action. Overall, we developed a robust high throughput-screening assay for screening broad-spectrum influenza antivirals targeting PA-PB1 interaction and identified R151785 as a promising antiviral drug candidate.