ABSTRACT
With CA16, enterovirus-71 is the causative agent of hand foot and mouth disease (HFMD) which occurs mostly in children under 5 years-old and responsible of several outbreaks since a decade. Most of the time, HFMD is a mild disease but can progress to severe complications such as meningitis, brain stem encephalitis, acute flaccid paralysis (AFP) and even death; EV71 has been identified in all severe cases. Therefore, it is actually one of the most public health issues that threatens children's life. [Formula: see text] is a protease which plays important functions in EV71 infection. To date, a lot of [Formula: see text] inhibitors have been tested but none of them has been approved yet. Therefore, a drug screening is still an utmost importance in order to treat and/or prevent EV71 infections. This work highlights the EV71 life cycle, [Formula: see text] functions and [Formula: see text] inhibitors recently screened. It permits to well understand all mechanisms about [Formula: see text] and consequently allow further development of drugs targeting [Formula: see text]. Thus, this review is helpful for screening of more new [Formula: see text] inhibitors or for designing analogues of well known [Formula: see text] inhibitors in order to improve its antiviral activity.
Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical/methods , Enterovirus A, Human/drug effects , Enzyme Inhibitors/pharmacology , Hand, Foot and Mouth Disease/drug therapy , RNA, Viral/antagonists & inhibitors , Animals , Antiviral Agents/isolation & purification , Child , Drug Evaluation, Preclinical/trends , Enterovirus A, Human/enzymology , Enzyme Inhibitors/isolation & purification , Hand, Foot and Mouth Disease/complications , Hand, Foot and Mouth Disease/virology , Humans , Mice , Nucleic Acid Synthesis Inhibitors/pharmacology , PhylogenyABSTRACT
Enterovirus A71 (EV-A71) is a human pathogen causing hand, foot and mouth disease (HFMD) which seriously threatened the safety and lives of infants and young children. However, there are no licensed direct antiviral agents to cure the HFMD. In this study, a series of quinoline formamide analogues as effective enterovirus inhibitors were developed, subsequent systematic structure-activity relationship (SAR) studies demonstrated that these quinoline formamide analogues exhibited good potency to treat EV-A71 infection. As described, the most efficient EV-A71 inhibitor 6i showed good anti-EV-A71 activity (EC50 = 1.238 µM) in RD cells. Furthermore, compound 6i could effectively prevent death of virus infected mice at dose of 6 mg/kg. When combined with emetine (0.1 mg/kg), this treatment could completely prevent the clinical symptoms and death of virus infected mice. Mechanism study indicated that compound 6i inhibited EV-A71 via targeting 2C helicase, thus impeding RNA remodeling and metabolism. Taken together, these data indicated that 6i is a promising EV-A71 inhibitor and worth extensive preclinical investigation as a lead compound.
Subject(s)
Antiviral Agents/pharmacology , Dibucaine/pharmacology , Enterovirus A, Human/drug effects , Enterovirus Infections/drug therapy , Enzyme Inhibitors/pharmacology , RNA Helicases/antagonists & inhibitors , Viral Proteins/antagonists & inhibitors , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Dibucaine/chemical synthesis , Dibucaine/chemistry , Dose-Response Relationship, Drug , Drug Therapy, Combination , Enterovirus A, Human/enzymology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Mice , Mice, Inbred Strains , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , RNA Helicases/metabolism , Structure-Activity Relationship , Viral Proteins/metabolismABSTRACT
Hand, foot and mouth disease (HFMD) is a serious, highly contagious disease. HFMD caused by Enterovirus 71 (EV71), results in severe complications and even death. The pivotal role of EV71 3Cpro in the viral life cycle makes it an attractive target for drug discovery and development to treat HFMD. In this study, we identified novel EV71 3Cpro inhibitors by docking-based virtual screening. Totally 50 compounds were selected to test their inhibitory activity against EV71 3Cpro. The best inhibitor DC07090 exhibited the inhibition potency with an IC50 value of 21.72 ± 0.95 µM without apparent toxicity (CC50 > 200 µM). To explore structure-activity relationship of DC07090, 15 new derivatives were designed, synthesized and evaluated in vitro enzyme assay accordingly. Interestingly, four compounds showed inhibitory activities against EV71 3Cpro and only DC07090 inhibited EV71 replication with an EC50 value of 22.09 ± 1.07 µM. Enzyme inhibition kinetic experiments showed that the compound was a reversible and competitive inhibitor. The Ki value was determined to be 23.29 ± 12.08 µM. Further molecular docking, MD simulation and mutagenesis studies confirmed the binding mode of DC07090 and EV71 3Cpro. Besides, DC07090 could also inhibit coxsackievirus A16 (CVA16) replication with an EC50 value of 27.76 ± 0.88 µM. Therefore, DC07090 represents a new non-peptidyl small molecule inhibitor for further development of antiviral therapy against EV71 or other picornaviruses.
Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Enterovirus A, Human/enzymology , Oxazoles/chemistry , Oxazoles/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Viral Proteins/antagonists & inhibitors , 3C Viral Proteases , Antiviral Agents/metabolism , Binding Sites , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Drug Evaluation, Preclinical , Enterovirus A, Human/drug effects , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Oxazoles/metabolism , Protein Conformation , Pyridines/metabolism , Structure-Activity Relationship , User-Computer Interface , Viral Proteins/chemistry , Viral Proteins/metabolismABSTRACT
Hand, Foot and Mouth Disease is a highly contagious disease caused by a range of human enteroviruses. Outbreaks occur regularly, especially in the Asia-Pacific region, putting a burden on public healthcare systems. Currently, there is no antiviral for treating this infectious disease and the only vaccines are limited to circulation in China, presenting an unmet medical need that needs to be filled urgently. The human enterovirus 3 C protease has been deemed a plausible drug target due to its essential roles in viral replication. In this study, we designed and synthesized 10 analogues of the Rhinovirus 3 C protease inhibitor, Rupintrivir, and tested their 3 C protease inhibitory activities followed by a cellular assay using human enterovirus 71 (EV71)-infected human RD cells. Our results revealed that a peptide-based compound containing a trifluoromethyl moiety to be the most potent analogue, with an EC50 of 65 nM, suggesting its potential as a lead for antiviral drug discovery.
Subject(s)
Antiviral Agents/pharmacology , Enterovirus A, Human/drug effects , Enterovirus A, Human/enzymology , Peptides/pharmacology , Protease Inhibitors/pharmacology , Viral Proteins/antagonists & inhibitors , 3C Viral Proteases , Antiviral Agents/chemistry , Cell Line , Cysteine Endopeptidases , Drug Evaluation, Preclinical , Drug Synergism , Enterovirus/drug effects , Humans , Inhibitory Concentration 50 , Peptides/chemistry , Protease Inhibitors/chemistry , Virus Replication/drug effectsABSTRACT
Luteoloside is a member of the flavonoids family that exhibits several bioactivities including anti-microbial and anti-cancer activities. However, the antiviral activity of luteoloside against enterovirus 71 (EV71) and the potential mechanism(s) responsible for this effect remain unknown. In this study, the antiviral potency of luteoloside against EV71 and its inhibitory effects on 3C protease activity were evaluated. First, we investigated the cytotoxicity of luteoloside against rhabdomyosarcoma (RD) cells, which was the cell line selected for an in vitro infection model. In a subsequent antiviral assay, the cytopathic effect of EV71 was significantly and dose-dependently relieved by the administration of luteoloside (EC50 = 0.43 mM, selection index = 5.3). Using a plaque reduction assay, we administered luteoloside at various time points and found that the compound reduced EV71 viability in RD cells rather than increasing defensive mobilization or viral absorption. Moreover, biochemical studies focused on VP1 (a key structural protein of EV71) mRNA transcript and protein levels also revealed the inhibitory effects of luteoloside on the EV71 viral yield. Finally, we performed inhibition assays using luteoloside to evaluate its effect on recombinant 3C protease activity. Our results demonstrated that luteoloside blocked 3C protease enzymatic activity in a dose-dependent manner (IC50 = 0.36 mM) that was similar to the effect of rutin, which is a well-known C3 protease inhibitor. Collectively, the results from this study indicate that luteoloside can block 3C protease activity and subsequently inhibit EV71 production in vitro.
Subject(s)
Antiviral Agents/pharmacology , Cysteine Endopeptidases/chemistry , Cysteine Proteinase Inhibitors/pharmacology , Enterovirus A, Human/drug effects , Enterovirus Infections/drug therapy , Glucosides/pharmacology , Luteolin/pharmacology , Viral Proteins/chemistry , 3C Viral Proteases , Antiviral Agents/chemistry , Cell Line, Tumor , Cysteine Proteinase Inhibitors/chemistry , Drug Evaluation, Preclinical , Enterovirus A, Human/enzymology , Enterovirus A, Human/physiology , Enterovirus Infections/virology , Humans , Inhibitory Concentration 50 , Medicine, Chinese Traditional , Viral Proteins/antagonists & inhibitorsABSTRACT
Enterovirus A71 (EV-A71) causes severe complications: encephalitis, pulmonary edema, and death. No effective drug has been approved for clinical use. This study investigated the antiviral effects of flavonoids against EV-A71. An in vitro inhibitor screening assay using recombinant EV-A71 3C protease (3Cpro) demonstrated fisetin and rutin inhibiting 3Cpro enzymatic activity in a dose-dependent manner. Cell-based fluorescence resonance energy transfer (FRET) assay with an EV-A71 3Cpro cleavage motif probe also confirmed that fisetin and rutin inhibited the replication of EV-A71 in cells. A virus replication assay indicated that fisetin and rutin reduced significantly the EV-A71-induced cytopathic effect and viral plaque titers in RD cells culture. The IC(50) values of plaque reduction against EV-A71 were 85 µM for fisetin and 110 µM for rutin. Therapeutic indices (CC50/IC50 of plaque reduction assays) of fisetin and rutin exceeded 10. The study suggests that fisetin and rutin inhibit the replication of EV-A71.