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1.
Acta Virol ; 65(1): 82-88, 2021.
Article in English | MEDLINE | ID: mdl-33827225

ABSTRACT

Hepatitis B virus (HBV) is a partially double-stranded DNA virus that specifically targets hepatocytes. It is considered a major health issue due to its high prevalence and the life-threatening consequences of chronic infection, including liver cirrhosis and hepatocellular carcinoma. Despite widespread vaccination against HBV, millions of people live with chronic HBV infection. Existing antiviral therapies fail to achieve full HBV elimination, so most patients with the disease require lifelong treatment. The search for new antiviral therapy strategies is hindered by the limited availability of in vitro HBV infection models that are able to support the full HBV life cycle. Therefore, the development and optimization of cellular models are crucial to the search for drugs effective against HBV. In this study, we optimized an in vitro HBV infection model consisting of two cell lines: HepAD38 cells, which are able to produce infectious HBV; and HepG2-NTCP cells, which are susceptible to HBV infection. We showed that prolonged production of HBV in the "donor" cells and HBV inoculation of the "acceptor" cells simultaneously with seeding improves the established procedure. This modified protocol was proven effective in experiments involving compounds with known activity against HBV, suggesting its utility for future high-throughput screening. Keywords: HBV; HBV in vitro models; HepG2-NTCP; HepAD38.


Subject(s)
Hepatitis B , Symporters , Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , Hepatitis B/drug therapy , Hepatitis B virus/genetics , Hepatocytes , High-Throughput Screening Assays , Humans , Organic Anion Transporters, Sodium-Dependent/pharmacology , Symporters/pharmacology , Virus Replication
2.
J Antimicrob Chemother ; 76(4): 1010-1018, 2021 03 12.
Article in English | MEDLINE | ID: mdl-33367751

ABSTRACT

BACKGROUND: The development and clinical implementation of the cap-dependent endonuclease (CEN) inhibitor baloxavir marboxil was a breakthrough in influenza therapy, but it was associated with the emergence of drug-resistant variants. OBJECTIVES: To design and synthesize structural analogues of CEN inhibitors and evaluate their safety, pharmacokinetics and antiviral potency in vitro and in vivo. METHODS: The drug candidate AV5124 and its active metabolite AV5116 were synthesized based on pharmacophore modelling. Stability in plasma and microsomes, plasma protein binding, cytotoxicity and antiviral activities were assessed in vitro. Pharmacokinetics after IV or oral administration were analysed in CD-1 mice. Acute toxicity and protective efficacy against lethal A(H1N1)pdm09 influenza virus challenge were examined in BALB/c mice. RESULTS: Pharmacophore model-assisted, 3D molecular docking predicted key supramolecular interactions of the metal-binding group and bulky hydrophobic group of AV5116 with the CEN binding site (Protein Data Bank code: 6FS6) that are essential for high antiviral activity. AV5116 inhibited influenza virus polymerase complexes in cell-free assays and replication of oseltamivir-susceptible and -resistant influenza A and B viruses at nanomolar concentrations. Notably, AV5116 was equipotent or more potent than baloxavir acid (BXA) against WT (I38-WT) viruses and viruses with reduced BXA susceptibility carrying an I38T polymerase acidic (PA) substitution. AV5116 exhibited low cytotoxicity in Madin-Darby canine kidney cells and lacked mitochondrial toxicity, resulting in favourable selective indices. Treatment with 20 or 50 mg/kg AV5124 prevented death in 60% and 100% of animals, respectively. CONCLUSIONS: Overall, AV5124 and A5116 are promising inhibitors of the influenza virus CEN and warrant further development as potent anti-influenza agents.


Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza, Human , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Dibenzothiepins , Dogs , Endonucleases , Humans , Influenza, Human/drug therapy , Mice , Mice, Inbred BALB C , Molecular Docking Simulation , Morpholines , Pyridones , Triazines
3.
J Med Chem ; 63(17): 9403-9420, 2020 09 10.
Article in English | MEDLINE | ID: mdl-32787099

ABSTRACT

4-Substituted 2,4-dioxobutanoic acids inhibit influenza virus cap-dependent endonuclease (CEN) activity. Baloxavir marboxil, 4, is approved for treating influenza virus infections. We describe here the synthesis and biological evaluation of active compounds, 5a-5g, and their precursors (6a, 6b, 6d, and 6e) with flexible bulky hydrophobic groups instead of the rigid polyheterocyclic moieties. In silico docking confirmed the ability of 5a-5g to bind to the active site of influenza A CEN (PDB code: 6FS6) like baloxavir acid, 3. These novel compounds inhibited polymerase complex activity, inhibited virus replication in cells, prevented death in a lethal influenza A virus mouse challenge model, and dramatically lowered viral lung titers. 5a and 5e potently inhibited different influenza genera in vitro. Precursors 6a and 6d demonstrated impressive mouse oral bioavailability with 6a, providing effective in vivo protection. Thus, these novel compounds are potent CEN inhibitors with in vitro and in vivo activity comparable to baloxavir.


Subject(s)
Dibenzothiepins/chemistry , Dibenzothiepins/pharmacology , Endonucleases/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Influenza A Virus, H1N1 Subtype/enzymology , Morpholines/chemistry , Morpholines/pharmacology , Pyridones/chemistry , Pyridones/pharmacology , Triazines/chemistry , Triazines/pharmacology , Animals , Dibenzothiepins/adverse effects , Dibenzothiepins/pharmacokinetics , Endonucleases/chemistry , Enzyme Inhibitors/adverse effects , Enzyme Inhibitors/pharmacokinetics , Female , HEK293 Cells , Humans , Influenza A Virus, H1N1 Subtype/drug effects , Mice , Models, Molecular , Morpholines/adverse effects , Morpholines/pharmacokinetics , Protein Conformation , Pyridones/adverse effects , Pyridones/pharmacokinetics , Tissue Distribution , Triazines/adverse effects , Triazines/pharmacokinetics
4.
Mar Drugs ; 18(4)2020 Apr 22.
Article in English | MEDLINE | ID: mdl-32331442

ABSTRACT

The enzymatic depolymerization of fucoidans from brown algae allowed the production of their standardized derivatives with different biological activities. This work aimed to compare the antiviral activities of native (FeF) and modified with enzyme (FeHMP) fucoidans from F. evanescens. The cytotoxicity and antiviral activities of the FeF and FeHMP against herpes viruses (HSV-1, HSV-2), enterovirus (ECHO-1), and human immunodeficiency virus (HIV-1) in Vero and human MT-4 cell lines were examined by methylthiazolyltetrazolium bromide (MTT) and cytopathic effect (CPE) reduction assays, respectively. The efficacy of fucoidans in vivo was evaluated in the outbred mice model of vaginitis caused by HSV-2. We have shown that both FeF and FeHMP significantly inhibited virus-induced CPE in vitro and were more effective against HSV. FeF exhibited antiviral activity against HSV-2 with a selective index (SI) > 40, and FeHMP with SI ˃ 20, when they were added before virus infection or at the early stages of the HSV-2 lifecycle. Furthermore, in vivo studies showed that after intraperitoneal administration (10 mg/kg), both FeF and FeHMP protected mice from lethal intravaginal HSV-2 infection to approximately the same degree (44-56%). Thus, FeF and FeHMP have comparable potency against several DNA and RNA viruses, allowing us to consider the studied fucoidans as promising broad-spectrum antivirals.


Subject(s)
Antiviral Agents/pharmacology , Fucus/chemistry , Polysaccharides/pharmacology , Viruses/drug effects , Animals , Antiviral Agents/isolation & purification , Chlorocebus aethiops , DNA Viruses/drug effects , Disease Models, Animal , Female , Humans , Mice , Polysaccharides/isolation & purification , RNA Viruses/drug effects , Vaginitis/drug therapy , Vaginitis/virology , Vero Cells
5.
Molecules ; 25(7)2020 Mar 30.
Article in English | MEDLINE | ID: mdl-32235534

ABSTRACT

Free radical reactions play an important role in biological functions of living systems. The balance between oxidants and antioxidants is necessary for the normal homeostasis of cells and organisms. Experimental works demonstrate the role of oxidative stress that is caused by influenza virus as well as the toxic effects of some antiviral drugs. Therefore, antiviral drugs should be characterized by its pro- and antioxidant activity, because it can affect its therapeutic efficiency. The aim of the study was to quantify the antioxidant capacity and propose the mechanism of the antioxidant effect of the antiviral drug Umifenovir (Arbidol®). The kinetic chemiluminescence with the 2,2'-azobis (2-amidinopropane) dihydrochloride + luminol system was used to quantify the antioxidant capacity of Umifenovir relative to the standard compound Trolox. With computer simulation, the reaction scheme and rate constants were proposed. The antioxidant capacity of 0.9 µM Umifenovir (maximum concentration of Umifenovir in blood after oral administration of 200 mg) was as high as 1.65 ± 0.18 µM of Trolox. Thus, the total antioxidant capacity of Umifenovir is comparable to the antioxidant capacity of Trolox. Unlike Trolox, Umifenovir reacts with free radicals in two stages. For Trolox, the free radical scavenging rate constant was k = 2000 nM-1 min.-1, for Umifenovir k1 = 300 nM-1min.-1, k2 = 4 nM-1min.-1. Slower kinetics of Umifenovir provides the prolonged antioxidant effect when compared to Trolox. This phenomenon can make a serious contribution to the compensation of oxidative stress that is caused by a viral disease and the therapeutic effect of the drug.


Subject(s)
Antioxidants/chemistry , Antiviral Agents/chemistry , Computer Simulation , Indoles/chemistry , Models, Chemical , Kinetics
6.
Vaccine ; 38(4): 859-867, 2020 01 22.
Article in English | MEDLINE | ID: mdl-31718898

ABSTRACT

Clinical and historical data underscore the ability of influenza viruses to ally with Staphylococcus aureus and predispose the host for secondary bacterial pneumonia, which is a leading cause of influenza-associated mortality. This is fundamental because no vaccine for S. aureus is available and the number of antibiotic-resistant strains is alarmingly rising. Hence, this leaves influenza vaccination the only strategy to prevent postinfluenza staphylococcal infections. In the present work, we assessed the off-target effects of a Tnms42 insect cell-expressed BEI-treated Gag-VLP preparation expressing the HA of A/Puerto Rico/8/1934 (H1N1) in preventing S. aureus superinfection in mice pre-infected with a homologous or heterologous H1N1 viral challenge strain. Our results demonstrate that matched anti-hemagglutinin immunity elicited by a VLP preparation may suffice to prevent morbidity and mortality caused by lethal secondary bacterial infection. This effect was observed even when employing a single low antigen dose of 50 ng HA per animal. However, induction of anti-hemagglutinin immunity alone was not helpful in inhibiting heterologous viral replication and subsequent bacterial infection. Our results indicate the potential of the VLP vaccine approach in terms of immunogenicity but suggest that anti-HA immunity should not be considered as the sole preventive method for combatting influenza and postinfluenza bacterial infections.


Subject(s)
Influenza Vaccines/administration & dosage , Influenza, Human/prevention & control , Staphylococcal Infections/prevention & control , Vaccines, Virus-Like Particle/administration & dosage , Animals , Female , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Humans , Influenza A Virus, H1N1 Subtype/immunology , Influenza Vaccines/immunology , Influenza, Human/complications , Insecta , Mice , Mice, Inbred BALB C , Superinfection/prevention & control , Vaccination , Vaccines, Virus-Like Particle/immunology , Virus Replication/immunology
7.
Vaccines (Basel) ; 7(4)2019 Nov 17.
Article in English | MEDLINE | ID: mdl-31744208

ABSTRACT

: Influenza virus infections pre-dispose an individual to secondary pneumococcal infections, which represent a serious public health concern. Matching influenza vaccination was demonstrated helpful in preventing postinfluenza bacterial infections and associated illnesses in humans. Yet, the impact of influenza hemagglutinin (HA)-specific immunity alone in this dual-infection scenario remains elusive. In the present study, we assessed the protective effect of neutralizing and non-neutralizing anti-hemagglutinin immunity in a BALB/c influenza-pneumococcus superinfection model. Our immunogens were insect cell-expressed hemagglutinin-Gag virus-like particles that had been differentially-treated for the inactivation of bioprocess-related baculovirus impurities. We evaluated the potential of several formulations to restrain the primary infection with vaccine-matched or -mismatched influenza strains and secondary bacterial replication. In addition, we investigated the effect of anti-HA immunity on the interferon status in mouse lungs prior to bacterial challenge. In our experimental setup, neutralizing anti-HA immunity provided significant but incomplete protection from postinfluenza bacterial superinfection, despite effective control of viral replication. In view of this, it was surprising to observe a survival advantage with non-neutralizing adaptive immunity when using a heterologous viral challenge strain. Our findings suggest that both neutralizing and non-neutralizing anti-HA immunity can reduce disease and mortality caused by postinfluenza pneumococcal infections.

8.
J Med Virol ; 91(4): 588-597, 2019 04.
Article in English | MEDLINE | ID: mdl-30431664

ABSTRACT

Antiviral drugs can play a significant role in the control of influenza. Umifenovir (Arbidol) is licensed and widely used in Russia for the prophylaxis and/or treatment of influenza. We evaluated the susceptibility to umifenovir of reference influenza A and B viruses and influenza A viruses isolated from patients in the ARBITR clinical trial in 2012-2014 seasons. Using an MDCK cell-based enzyme-linked immunosorbent assay (ELISA), we showed that the replication of antigenically dominant human influenza A and B viruses was efficiently inhibited by umifenovir. The wild-type А/Perth/265/2009 (H1N1)pdm09, A/Fukui/45/2004 (H3N2), and B/Perth/211/2001 viruses and their oseltamivir-resistant counterparts were susceptible to umifenovir among in vitro laboratory assays. All 18 clinical isolates of influenza A viruses obtained before and during therapy were susceptible to umifenovir with 50% effective concentration (EC 50 ) ranging from 8.4 ± 1.1 to 17.4 ± 5.4 µM. No molecular markers of umifenovir resistance were identified in influenza viruses isolate d from patients at 3, 5, and 7 days after initiation of therapy. None of the viruses isolated before and during umifenovir therapy displayed reduced susceptibility to neuraminidase (NA) inhibitors. Thus, umifenovir is effective against influenza viruses circulating in 2012-2014 seasons, and therapy did not lead to the emergence of drug-resistant variants.


Subject(s)
Antiviral Agents/pharmacology , Indoles/pharmacology , Influenza, Human/virology , Orthomyxoviridae/drug effects , Adolescent , Adult , Aged , Animals , Clinical Trials as Topic , Dogs , Female , Humans , Madin Darby Canine Kidney Cells , Male , Microbial Sensitivity Tests , Middle Aged , Orthomyxoviridae/isolation & purification , Russia , Virus Replication/drug effects , Young Adult
9.
J Antimicrob Chemother ; 69(7): 1892-902, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24729605

ABSTRACT

OBJECTIVES: Development of a novel drug candidate with improved potency against influenza virus neuraminidase compared with currently available therapeutics, and high activity against oseltamivir-resistant viruses. METHODS: A number of synthetic compounds were evaluated for antiviral properties in vitro and in vivo. Three-dimensional molecular docking, assisted by a pharmacophore model, was applied to classify compounds within the series by their inhibitory potency. Compound stability in blood and in animal models was determined. Pharmacokinetic studies in dogs and rats after oral or intravenous administration were performed. RESULTS: A novel highly potent drug candidate [(3R,4R,5S)-5-[(diaminomethylene)amino]-3-(1-ethylpropoxy)-4-[(fluoroacetyl)amino]cyclohex-1-ene-1-carboxylic acid; AV5080] was synthesized and tested. AV5080 exhibited high activity against influenza virus neuraminidase in vitro, with IC(50) values of 0.03 nM and 0.07 nM against the neuraminidase of A/Duck/Minnesota/1525/1981/H5N1 and A/Perth/265/2009/H1N1 (wild-type), respectively. Notably, AV5080 was highly active against oseltamivir-resistant influenza viruses. CONCLUSIONS: Based on the results presented in this study, AV5080 is a promising novel oral drug candidate for the treatment of influenza, including oseltamivir-resistant types. Further pre-clinical development of AV5080 is warranted.


Subject(s)
Antiviral Agents/isolation & purification , Enzyme Inhibitors/isolation & purification , Neuraminidase/antagonists & inhibitors , Viral Proteins/antagonists & inhibitors , Administration, Oral , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Dogs , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Female , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H1N1 Subtype/enzymology , Influenza A Virus, H5N1 Subtype/drug effects , Influenza A Virus, H5N1 Subtype/enzymology , Inhibitory Concentration 50 , Molecular Docking Simulation , Rats
10.
J Antimicrob Chemother ; 69(5): 1311-24, 2014 May.
Article in English | MEDLINE | ID: mdl-24428978

ABSTRACT

OBJECTIVES: Development of a novel drug candidate with improved activity against influenza virus neuraminidase (NA) compared with currently available therapeutics. METHODS: Synthesized compounds were evaluated in vitro and in vivo. Three-dimensional molecular docking was successfully applied to classify compounds within the series by inhibitory potency. Stability was investigated in blood samples and in animal models. A pharmacokinetic study was performed in dogs and rats using peroral and intravenous administration. RESULTS: A novel highly potent drug candidate [(3R,4R,5S)-4-(2,2-difluoroacetylamino)-5-amino-3-(1-ethyl-propoxy)-cyclohex-1-enecarboxylic acid; AV5027] and its prodrug ethyl ester (AV5075S) were synthesized and tested. AV5027 and AV5075S exhibit picomolar activity against influenza virus NA. AV5075S inhibited NA in a model of pneumonia using mouse-adapted A/Aichi/2/68 (H3N2) virus significantly more strongly than oseltamivir phosphate. A general metabolic pathway was constructed for the parent compound based on experimental results and theoretical analyses. CONCLUSIONS: AV5075S can be reasonably regarded as a novel 'next in class' oral drug candidate for the treatment of influenza.


Subject(s)
Antiviral Agents/administration & dosage , Prodrugs/administration & dosage , Administration, Intravenous , Administration, Oral , Animals , Disease Models, Animal , Dogs , Female , Male , Mice , Orthomyxoviridae Infections/drug therapy , Orthomyxoviridae Infections/virology , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , Rats, Sprague-Dawley , Treatment Outcome
11.
Antiviral Res ; 100(3): 698-708, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24416774

ABSTRACT

A medium-sized focused library of novel Oseltamivir structural analogues with promising antiviral activity was successfully synthesized using a combinatorial approach. The synthesized compounds were then thoroughly evaluated in neuraminidase- and cell-based assays. As a result, (3R,4R,5S)-4-(2,2-difluoroacetylamino)-5-amino-3-(1-ethyl-propoxy)-cyclohex-1-enecarboxylic acid (AV5027) was identified as novel Hit-compound with picomolar potency. QSAR analysis was carried out based on the obtained biological data. Computational modeling was performed using a 3D-molecular docking approach and classical regression analysis. The developed integral model demonstrated a sufficient prediction accuracy and tolerance to evaluate compounds based on their potential activity against neuraminidase (NA) at least within the scaffold. Several compounds from the series can be reasonably regarded as promising anti-influenza drug-candidates.


Subject(s)
Antiviral Agents/pharmacology , Cyclohexanecarboxylic Acids/pharmacology , Fluoroacetates/pharmacology , Neuraminidase/antagonists & inhibitors , Viral Proteins/antagonists & inhibitors , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Antiviral Agents/toxicity , Combinatorial Chemistry Techniques , Cyclohexanecarboxylic Acids/chemical synthesis , Cyclohexanecarboxylic Acids/chemistry , Cyclohexanecarboxylic Acids/toxicity , Dogs , Drug Design , Fluoroacetates/chemical synthesis , Fluoroacetates/chemistry , Fluoroacetates/toxicity , Influenza A virus/drug effects , Influenza A virus/enzymology , Influenza B virus/drug effects , Influenza B virus/enzymology , Madin Darby Canine Kidney Cells , Models, Molecular , Molecular Docking Simulation , Molecular Structure , Quantitative Structure-Activity Relationship
12.
Antiviral Res ; 81(2): 132-40, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19028526

ABSTRACT

The antiviral drug arbidol (ARB), which is licensed in Russia for use against influenza, is known to inhibit early membrane fusion events in influenza A and B virus replication. To investigate in more detail the target and mechanism of ARB action we generated and studied the characteristics of ARB-resistant influenza virus mutants. Observations of the ARB susceptibility of reassortants between A/Singapore/1/57(H2N2) and A/chicken/Germany/27(H7N7, "Weybridge" strain) and of mutants of the latter virus identified the virus haemagglutinin (HA) as the major determinant of ARB sensitivity. ARB-resistant mutants, selected from the most sensitive reassortant, possessed single amino acid substitutions in the HA2 subunit which caused an increase in the pH of fusion and the associated conformational change in HA. ARB was shown to stabilize the HA by causing a 0.2 pH unit reduction in the pH of the transition to the low pH form, which was specifically abrogated by the resistance mutations. Some of the resistance mutations, which reduce acid stability and would disrupt ARB-HA interactions, are located in the vicinity of a potential ARB binding site identified using the docking programme Gold. Together, the results of these investigations indicate that ARB falls within a class of inhibitor which interacts with HA to stabilize it against the low pH transition to its fusogenic state and consequently inhibit HA-mediated membrane fusion during influenza virus infection.


Subject(s)
Antiviral Agents/pharmacology , Drug Resistance, Viral , Indoles/pharmacology , Influenza A Virus, H2N2 Subtype/drug effects , Influenza A Virus, H7N7 Subtype/drug effects , Reassortant Viruses/drug effects , Amino Acid Substitution/genetics , Animals , Cell Line , Dogs , Hemagglutinins, Viral/chemistry , Hemagglutinins, Viral/genetics , Microbial Sensitivity Tests , Models, Molecular , Mutation, Missense , Protein Conformation
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