ABSTRACT
Human T-Lymphotropic Virus type-1 (HTLV-1) is a unique retrovirus associated with both leukemogenesis and a specific neuroinflammatory condition known as HTLV-1-Associated Myelopathy (HAM). Currently, most proposed HAM biomarkers require invasive CSF sampling, which is not suitable for large cohorts or repeated prospective screening. To identify non-invasive biomarkers for incident HAM in a large Brazilian cohort of PLwHTLV-1 (n=615 with 6,673 person-years of clinical follow-up), we selected all plasma samples available at the time of entry in the cohort (between 1997-2019), in which up to 43 cytokines/chemokines and immune mediators were measured. Thus, we selected 110 People Living with HTLV-1 (PLwHTLV-1), of which 68 were neurologically asymptomatic (AS) at baseline and 42 HAM patients. Nine incident HAM cases were identified among 68 AS during follow-up. Using multivariate logistic regression, we found that lower IL-10, IL-4 and female sex were independent predictors of clinical progression to definite HAM (AUROC 0.91), and outperformed previously suggested biomarkers age, sex and proviral load (AUROC 0.77). Moreover, baseline IL-10 significantly predicted proviral load dynamics at follow-up in all PLwHTLV-1. In an exploratory analysis, we identified additional plasma biomarkers which were able to discriminate iHAM from either AS (IL6Rα, IL-27) or HAM (IL-29/IFN-λ1, Osteopontin, and TNFR2). In conclusion, female sex and low anti-inflammatory IL-10 and IL-4 are independent risk factors for incident HAM in PLwHTLV-1,while proviral load is not, in agreement with IL-10 being upstream of proviral load dynamics. Additional candidate biomarkers IL-29/IL-6R/TNFR2 represent plausible therapeutic targets for future clinical trials in HAM patients.
Subject(s)
Biomarkers , Human T-lymphotropic virus 1 , Interleukin-10 , Viral Load , Humans , Female , Male , Brazil/epidemiology , Human T-lymphotropic virus 1/immunology , Interleukin-10/blood , Biomarkers/blood , Middle Aged , Adult , HTLV-I Infections/immunology , HTLV-I Infections/blood , HTLV-I Infections/diagnosis , Proviruses , Cohort Studies , Paraparesis, Tropical Spastic/blood , Paraparesis, Tropical Spastic/immunology , Paraparesis, Tropical Spastic/virology , IncidenceABSTRACT
Here, we report on the anti-SARS-CoV-2 activity of PRO-2000, a sulfonated polyanionic compound. In Vero cells infected with the Wuhan, alpha, beta, delta or omicron variant, PRO-2000 displayed EC50 values of 1.1 µM, 2.4 µM, 1.3 µM, 2.1 µM and 0.11 µM, respectively, and an average selectivity index (i.e. ratio of cytotoxic versus antiviral concentration) of 172. Its anti-SARS-CoV-2 activity was confirmed by virus yield assays in Vero cells, Caco2 cells and A549 cells overexpressing ACE2 and TMPRSS2 (A549-AT). Using pseudoviruses bearing the SARS-CoV-2 spike (S), PRO-2000 was shown to block the S-mediated pseudovirus entry in Vero cells and A549-AT cells, with EC50 values of 0.091 µM and 1.6 µM, respectively. This entry process is initiated by interaction of the S glycoprotein with angiotensin-converting enzyme 2 (ACE2) and heparan sulfate proteoglycans. Surface Plasmon Resonance (SPR) studies showed that PRO-2000 binds to the receptor-binding domain (RBD) of S with a KD of 1.6 nM. Similar KD values (range: 1.2 nM-2.1 nM) were obtained with the RBDs of the alpha, beta, delta and omicron variants. In an SPR neutralization assay, PRO-2000 had no effect on the interaction between the RBD and ACE2. Instead, PRO-2000 was proven to inhibit binding of the RBD to a heparin-coated sensor chip, yielding an IC50 of 1.1 nM. To conclude, PRO-2000 has the potential to inhibit a broad range of SARS-CoV-2 variants by blocking the heparin-binding site on the S protein.
Subject(s)
Antiviral Agents , COVID-19 , Chlorocebus aethiops , Animals , Humans , Antiviral Agents/pharmacology , Angiotensin-Converting Enzyme 2 , Caco-2 Cells , Vero Cells , SARS-CoV-2 , Protein Binding , Spike Glycoprotein, CoronavirusABSTRACT
BACKGROUND: HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) is an incapacitating neuroinflammatory disorder for which no disease-modifying therapy is available, but corticosteroids provide some clinical benefit. Although HAM/TSP pathogenesis is not fully elucidated, older age, female sex and higher proviral load are established risk factors. We investigated systemic cytokines and a novel chronic inflammatory marker, GlycA, as possible biomarkers of immunopathogenesis and therapeutic response in HAM/TSP, and examined their interaction with established risk factors. PATIENTS AND METHODS: We recruited 110 People living with HTLV-1 (PLHTLV-1, 67 asymptomatic individuals and 43 HAM/TSP patients) with a total of 946 person-years of clinical follow-up. Plasma cytokine levels (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, TNF) and GlycA were quantified by Cytometric Bead Array and 1NMR, respectively. Cytokine signaling and prednisolone response were validated in an independent cohort by nCounter digital transcriptomics. We used multivariable regression, machine learning algorithms and Bayesian network learning for biomarker identification. RESULTS: We found that systemic IL-6 was positively correlated with both age (r = 0.50, p < 0.001) and GlycA (r = 0.45, p = 0.00049) in asymptomatics, revealing an 'inflammaging" signature which was absent in HAM/TSP. GlycA levels were higher in women (p = 0.0069), but cytokine levels did not differ between the sexes. IFN-γ (p = 0.007) and IL-17A (p = 0.0001) levels were increased in untreated HAM/TSP Multivariable logistic regression identified IL-17A and proviral load as independent determinants of clinical status, resulting in modest accuracy of predicting HAM/TSP status (64.1%), while a machine learning-derived decision tree classified HAM/TSP patients with 90.7% accuracy. Pre-treatment GlycA and TNF levels significantly predicted clinical worsening (measured by Osame Motor Disability Scale), independent of proviral load. In addition, a poor prednisolone response was significantly correlated with higher post-treatment IFN-γ levels. Likewise, a transcriptomic IFN signaling score, significantly correlated with previously proposed HAM/TSP biomarkers (CASP5/CXCL10/FCGR1A/STAT1), was efficiently blunted by in vitro prednisolone treatment of PBMC from PLHTLV-1 and incident HAM/TSP. CONCLUSIONS: An age-related increase in systemic IL-6/GlycA levels reveals inflammaging in PLHTLV-1, in the absence of neurological disease. IFN-γ and IL-17A are biomarkers of untreated HAM/TSP, while pre-treatment GlycA and TNF predict therapeutic response to prednisolone pulse therapy, paving the way for a precision medicine approach in HAM/TSP.
Subject(s)
HTLV-I Infections , Motor Disorders , Neuroinflammatory Diseases , Female , Humans , Bayes Theorem , Cytokines , Human T-lymphotropic virus 1 , Interleukin-17 , Interleukin-6 , Leukocytes, Mononuclear , Motor Disorders/virology , Neuroinflammatory Diseases/virology , HTLV-I Infections/complicationsABSTRACT
In previous investigations, we identified a class of 1,3,4-thiadiazole derivatives with antiviral activity. N-{3-(Methylsulfanyl)-1-[5-(phenylamino)-1,3,4-thiadiazole-2-yl]propyl}benzamide emerged as a relevant lead compound for designing novel influenza A virus inhibitors. In the present study, we elaborated on this initial lead by performing chemical synthesis and antiviral evaluation of a series of structural analogues. During this research, thirteen novel 1,3,4-thiadiazole derivatives were synthesized by the cyclization of the corresponding thiosemicarbazides as synthetic precursors. The structures and the purities of the synthesized compounds were confirmed through chromatographic and spectral data. Four L-methionine-based 1,3,4-thiadiazole derivatives displayed activity against influenza A virus, the two best compounds being 24 carrying a 5-(4-chlorophenylamino)-1,3,4-thiadiazole moiety and 30 possessing a 5-(benzoylamino)-1,3,4-thiadiazole structure [antiviral EC50 against influenza A/H3N2 virus: 4.8 and 7.4 µM, respectively]. The 1,3,4-thiadiazole derivatives were inactive against influenza B virus and a wide panel of unrelated DNA and RNA viruses. Compound 24 represents a new class of selective influenza A virus inhibitors acting during the virus entry process, as evidenced by our findings in a time-of-addition assay. Molecular descriptors and in silico prediction of ADMET properties of the active compounds were calculated. According to in silico ADMET and drug similarity studies, active compounds have been estimated to be good candidates for oral administration with no apparent toxicity considerations.
Subject(s)
Antiviral Agents/chemical synthesis , Methionine/chemistry , Thiadiazoles/chemistry , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Drug Design , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H3N2 Subtype/drug effects , Influenza A Virus, H3N2 Subtype/physiology , Influenza B virus/drug effects , Influenza B virus/physiology , Structure-Activity Relationship , Thiadiazoles/chemical synthesis , Thiadiazoles/pharmacology , Virus Internalization/drug effectsABSTRACT
To suppress serious influenza infections in persons showing insufficient protection from the vaccines, antiviral drugs are of vital importance. There is a need for novel agents with broad activity against influenza A (IAV) and B (IBV) viruses and with targets that differ from those of the current antivirals. We here report a new small molecule influenza virus inhibitor referred to as CPD A (chemical name: N-(pyridin-3-yl)thiophene-2-carboxamide). In an influenza virus minigenome assay, this non-nucleoside compound inhibited RNA synthesis of IAV and IBV with EC50 values of 2.3 µM and 2.6 µM, respectively. Robust in vitro activity was noted against a broad panel of IAV (H1N1 and H3N2) and IBV strains, with a median EC50 value of 0.20 µM, which is 185-fold below the 50% cytotoxic concentration. The action point in the viral replication cycle was located between 1 and 5 h p.i., showing a similar profile as ribavirin. Like this nucleoside analogue, CPD A was shown to cause strong depletion of the cellular GTP pool and, accordingly, its antiviral activity was antagonized when this pool was restored with exogenous guanosine. This aligns with the observed inhibition in a cell-based IMP dehydrogenase (IMPDH) assay, which seems to require metabolic activation of CPD A since no direct inhibition was seen in an enzymatic IMPDH assay. The combination of CPD A with ribavirin, another IMPDH inhibitor, proved strongly synergistic. To conclude, we established CPD A as a new inhibitor of influenza A and B virus replication and RNA synthesis, and support the potential of IMPDH inhibitors for influenza therapy with acceptable safety profile.
Subject(s)
Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , IMP Dehydrogenase/antagonists & inhibitors , Influenza A virus/drug effects , Influenza B virus/drug effects , Ribavirin/pharmacology , Cell Line , Drug Synergism , Humans , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H3N2 Subtype/drug effects , Influenza A virus/classification , Influenza, Human/drug therapyABSTRACT
In this study, a series of 10 quinoline analogues was evaluated for their in vitro antiviral activity against a panel of alpha- and beta-coronaviruses, including the severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2), as well as the human coronaviruses (HCoV) 229E and OC43. Chloroquine and hydroxychloroquine were the most potent with antiviral EC50 values in the range of 0.12-12 µM. Chloroquine displayed the most favorable selectivity index (i.e. ratio cytotoxic versus antiviral concentration), being 165 for HCoV-OC43 in HEL cells. Potent anti-coronavirus activity was also observed with amodiaquine, ferroquine and mefloquine, although this was associated with substantial cytotoxicity for mefloquine. Primaquine, quinidine, quinine and tafenoquine only blocked coronavirus replication at higher concentrations, while piperaquine completely lacked antiviral and cytotoxic effects. A time-of-addition experiment in HCoV-229E-infected HEL cells revealed that chloroquine interferes with viral entry at a post-attachment stage. Using confocal microscopy, no viral RNA synthesis could be detected upon treatment of SARS-CoV-2-infected cells with chloroquine. The inhibition of SARS-CoV-2 replication by chloroquine and hydroxychloroquine coincided with an inhibitory effect on the autophagy pathway as visualized by a dose-dependent increase in LC3-positive puncta. The latter effect was less pronounced or even absent with the other quinolines. In summary, we showed that several quinoline analogues, including chloroquine, hydroxychloroquine, amodiaquine, ferroquine and mefloquine, exhibit broad anti-coronavirus activity in vitro.
Subject(s)
Antimalarials/pharmacology , Antiviral Agents/pharmacology , Coronavirus Infections/drug therapy , Coronavirus/drug effects , Quinolines/pharmacology , Animals , Chlorocebus aethiops , Chloroquine/pharmacology , Coronavirus 229E, Human/drug effects , Coronavirus OC43, Human/drug effects , Humans , Hydroxychloroquine/pharmacology , SARS-CoV-2/drug effects , Vero Cells , Virus Internalization/drug effects , Virus Replication/drug effects , COVID-19 Drug TreatmentABSTRACT
Favipiravir (T-705) is a broad-spectrum antiviral drug that inhibits RNA viruses after intracellular conversion into its active form, T-705 ribofuranosyl 5'-triphosphate. We previously showed that T-705 is able to significantly inhibit the replication of chikungunya virus (CHIKV), an arbovirus transmitted by Aedes mosquitoes, in mammalian cells and in mouse models. In contrast, the effect of T-705 on CHIKV infection and replication in the mosquito vector is unknown. Since the antiviral activity of T-705 has been shown to be cell line-dependent, we studied here its antiviral efficacy in Aedes-derived mosquito cells and in Aedes aegypti mosquitoes. Interestingly, T-705 was devoid of anti-CHIKV activity in mosquito cells, despite being effective against CHIKV in Vero cells. By investigating the metabolic activation profile, we showed that, unlike Vero cells, mosquito cells were not able to convert T-705 into its active form. To explore whether alternative metabolization pathways might exist in vivo, Aedes aegypti mosquitoes were infected with CHIKV and administered T-705 via an artificial blood meal. Virus titrations of whole mosquitoes showed that T-705 was not able to reduce CHIKV infection in mosquitoes. Combined, these in vitro and in vivo data indicate that T-705 lacks antiviral activity in mosquitoes due to inadequate metabolic activation in this animal species.
ABSTRACT
Here, we report on the anti-influenza virus activity of the mannose-binding agents Hippeastrum hybrid agglutinin (HHA) and Galanthus nivalis agglutinin (GNA) and the (N-acetylglucosamine) n -specific Urtica dioica agglutinin (UDA). These carbohydrate-binding agents (CBA) strongly inhibited various influenza A(H1N1), A(H3N2), and B viruses in vitro, with 50% effective concentration values ranging from 0.016 to 83 nM, generating selectivity indexes up to 125,000. Somewhat less activity was observed against A/Puerto Rico/8/34 and an A(H1N1)pdm09 strain. In time-of-addition experiments, these CBA lost their inhibitory activity when added 30 min postinfection (p.i.). Interference with virus entry processes was also evident from strong inhibition of virus-induced hemolysis at low pH. However, a direct effect on acid-induced refolding of the viral hemagglutinin (HA) was excluded by the tryptic digestion assay. Instead, HHA treatment of HA-expressing cells led to a significant reduction of plasma membrane mobility. Crosslinking of membrane glycoproteins, through interaction with HA, could also explain the inhibitory effect on the release of newly formed virions when HHA was added at 6 h p.i. These CBA presumably interact with one or more N-glycans on the globular head of HA, since their absence led to reduced activity against mutant influenza B viruses and HHA-resistant A(H1N1) viruses. The latter condition emerged only after 33 cell culture passages in the continuous presence of HHA, and the A(H3N2) virus retained full sensitivity even after 50 passages. Thus, these CBA qualify as potent inhibitors of influenza A and B viruses in vitro with a pleiotropic mechanism of action and a high barrier for viral resistance.
Subject(s)
Amaryllidaceae , Herpesvirus 1, Cercopithecine , Influenza A Virus, H1N1 Subtype , Influenza, Human , Agglutinins , Antiviral Agents/pharmacology , Hemagglutinin Glycoproteins, Influenza Virus , Humans , Influenza A Virus, H3N2 Subtype , Influenza B virus , Mannose , Mannose-Binding Lectins , Plant Lectins , Virus ReplicationABSTRACT
The influenza virus hemagglutinin (HA) is an attractive target for antiviral therapy due to its essential role in mediating virus entry into the host cell. We here report the identification of a class of N-benzyl-4,4,-disubstituted piperidines as influenza A virus fusion inhibitors with specific activity against the H1N1 subtype. Using the highly efficient one-step Ugi four-component reaction, diverse library of piperidine-based analogues was synthesized and evaluated to explore the structure-activity relationships (SAR). Mechanistic studies, including resistance selection with the most active compound (2) demonstrated that it acts as an inhibitor of the low pH-induced HA-mediated membrane fusion process. Computational studies identified an as yet unrecognized fusion inhibitor binding site, which is located at the bottom of the HA2 stem in close proximity to the fusion peptide. A direct π-stacking interaction between the N-benzylpiperidine moiety of 2 and F9HA2 of the fusion peptide, reinforced with an additional π-stacking interaction with Y119HA2, and a salt bridge of the protonated piperidine nitrogen with E120HA2, were identified as important interactions to mediate ligand binding. This site rationalized the observed SAR and provided a structural explanation for the H1N1-specific activity of our inhibitors. Furthermore, the HA1-S326V mutation resulting in resistance to 2 is close to the proposed new binding pocket. Our findings point to the N-benzyl-4,4,-disubstituted piperidines as an interesting class of influenza virus inhibitors, representing the first example of fusion peptide binders with great potential for anti-influenza drug development.
Subject(s)
Antiviral Agents/pharmacology , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Influenza A Virus, H1N1 Subtype/drug effects , Piperidines/pharmacology , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Dogs , Dose-Response Relationship, Drug , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Humans , Influenza A Virus, H1N1 Subtype/metabolism , Madin Darby Canine Kidney Cells/drug effects , Madin Darby Canine Kidney Cells/virology , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Piperidines/chemical synthesis , Piperidines/chemistry , Structure-Activity RelationshipABSTRACT
The influenza virus hemagglutinin (HA) mediates membrane fusion after viral entry by endocytosis. The fusion process requires drastic low pH-induced HA refolding and is prevented by arbidol and tert-butylhydroquinone (TBHQ). We here report a class of superior inhibitors with indole-substituted spirothiazolidinone structure. The most active analogue 5f has an EC50 value against influenza A/H3N2 virus of 1â¯nM and selectivity index of almost 2000. Resistance data and in silico modeling indicate that 5f combines optimized fitting in the TBHQ/arbidol HA binding pocket with a capability for endosomal accumulation. Both criteria appear relevant to achieve superior inhibitors of HA-mediated fusion.
Subject(s)
Antiviral Agents/pharmacology , Hemagglutinin Glycoproteins, Influenza Virus/drug effects , Indoles/pharmacology , Influenza A Virus, H3N2 Subtype/drug effects , Influenza, Human/drug therapy , Spiro Compounds/pharmacology , Thiazolidines/pharmacology , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Dogs , Dose-Response Relationship, Drug , Humans , Hydrogen-Ion Concentration , Indoles/chemistry , Madin Darby Canine Kidney Cells/drug effects , Madin Darby Canine Kidney Cells/virology , Microbial Sensitivity Tests , Molecular Docking Simulation , Molecular Structure , Protein Refolding/drug effects , Spiro Compounds/chemistry , Structure-Activity Relationship , Thiazolidines/chemistryABSTRACT
Hemagglutinin is the surface protein of the influenza virus that mediates both binding and penetration of the virus into host cells. We here report on the synthesis and structure-activity relationship of some novel N-(1-thia-4-azaspiro[4.5]decan-4-yl)-carboxamide compounds carrying the 5-chloro-2-methoxybenzamide structure, designed as influenza virus fusion inhibitors. The carboxamides (1a-h, 2a-h) have a similar backbone structure as the fusion inhibitors that we reported on previously. Compounds 2b and 2d displayed inhibitory activity against influenza A/H3N2 virus replication (average antiviral EC50 : 2.1 µM for 2b and 3.4 µM for 2d). Data obtained in the hemolysis inhibition assay supported that these compounds act as inhibitors of the influenza virus hemagglutinin-mediated fusion process.
Subject(s)
Antiviral Agents/pharmacology , Aza Compounds/pharmacology , Influenza A Virus, H3N2 Subtype/drug effects , Spiro Compounds/pharmacology , Virus Replication/drug effects , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Aza Compounds/chemical synthesis , Aza Compounds/chemistry , Dose-Response Relationship, Drug , Microbial Sensitivity Tests , Molecular Docking Simulation , Molecular Structure , Spiro Compounds/chemical synthesis , Spiro Compounds/chemistry , Structure-Activity RelationshipABSTRACT
The antiviral drug T-705 (favipiravir) and its non-fluorinated analogue T-1105 inhibit the polymerases of RNA viruses after being converted to their ribonucleoside triphosphate (RTP) metabolite. We here compared the activation efficiency of T-705 and T-1105 in four cell lines that are commonly used for their antiviral evaluation. In MDCK cells, the levels of T-705-RTP were markedly lower than those of T-1105-RTP, while the opposite was seen in A549, Vero and HEK293T cells. In the latter three cell lines, T-1105 activation was hindered by inefficient conversion of the ribonucleoside monophosphate to the ribonucleoside diphosphate en route to forming the active triphosphate. Accordingly, T-1105 had better anti-RNA virus activity in MDCK cells, while T-705 was more potent in the other three cell lines. Additionally, we identified a fourth metabolite, the NAD analogue of T-705/T-1105, and showed that it can be formed by nicotinamide mononucleotide adenylyltransferase.
Subject(s)
Amides/pharmacology , Antiviral Agents/pharmacology , Cell Line , Pyrazines/pharmacology , RNA Viruses/drug effects , Animals , Cell Line/drug effects , Cell Line/metabolism , Cell Line/virology , Chlorocebus aethiops , Dogs , HEK293 Cells/drug effects , HEK293 Cells/metabolism , HEK293 Cells/virology , Humans , Madin Darby Canine Kidney Cells/drug effects , Madin Darby Canine Kidney Cells/metabolism , Madin Darby Canine Kidney Cells/virology , Ribonucleosides/metabolism , Vero Cells/drug effects , Vero Cells/metabolism , Vero Cells/virologyABSTRACT
The possible resistance of influenza virus against existing antiviral drugs calls for new therapeutic concepts. One appealing strategy is to inhibit virus entry, in particular at the stage of internalization. This requires a better understanding of virus-host interactions during the entry process, including the role of receptor tyrosine kinases (RTKs). To search for cellular targets, we evaluated a panel of 276 protein kinase inhibitors in a multicycle antiviral assay in Madin-Darby canine kidney cells. The RTK inhibitor Ki8751 displayed robust anti-influenza A and B virus activity and was selected for mechanistic investigations. Ki8751 efficiently disrupted the endocytic process of influenza virus in different cell lines carrying platelet-derived growth factor receptor ß (PDGFRß), an RTK that is known to act at GM3 ganglioside-positive lipid rafts. The more efficient virus entry in CHO-K1 cells compared to the wild-type ancestor (CHO-wt) cells indicated a positive effect of GM3, which is abundant in CHO-K1 but not in CHO-wt cells. Entering virus localized to GM3-positive lipid rafts and the PDGFRß-containing endosomal compartment. PDGFRß/GM3-dependent virus internalization involved PDGFRß phosphorylation, which was potently inhibited by Ki8751, and desialylation of activated PDGFRß by the viral neuraminidase. Virus uptake coincided with strong activation of the Raf/MEK/Erk cascade, but not of PI3K/Akt or phospholipase C-γ. We conclude that influenza virus efficiently hijacks the GM3-enhanced PDGFRß signalling pathway for cell penetration, providing an opportunity for host cell-targeting antiviral intervention.
Subject(s)
G(M3) Ganglioside/metabolism , Influenza, Human/metabolism , Influenza, Human/virology , Orthomyxoviridae Infections/metabolism , Orthomyxoviridae/pathogenicity , Receptor, Platelet-Derived Growth Factor beta/metabolism , Signal Transduction/physiology , Animals , CHO Cells , Cell Line , Cricetulus , Dogs , HEK293 Cells , Humans , Influenza, Human/drug therapy , Madin Darby Canine Kidney Cells , Orthomyxoviridae/drug effects , Orthomyxoviridae Infections/drug therapy , Phenylurea Compounds/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/physiology , Protein Kinase Inhibitors/pharmacology , Quinolines/pharmacology , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effects , Virus Internalization/drug effectsABSTRACT
Six series of semisynthetic lipophilic glycopeptide antibiotic derivatives were evaluated for in vitro activity against influenza A and B viruses. The new teicoplanin pseudoaglycon-derived lipoglycopeptides were prepared by coupling one or two side chains to the N-terminus of the glycopeptide core, using various conjugation methods. Three series of derivatives bearing two lipophilic groups were synthesized by attaching bis-alkylthio maleimides directly or through linkers of different lengths to the glycopeptide. Access to the fourth and fifth series of compounds was achieved by click chemistry, introducing single alkyl/aryl chains directly or through a tetraethylene glycol linker to the same position. A sixth group of semisynthetic derivatives was obtained by sulfonylation of the N-terminus. Of the 42 lipophilic teicoplanin pseudoaglycon derivatives tested, about half showed broad activity against influenza A and B viruses, with some of them having reasonable or no cytotoxicity. Minor differences in the side chain length as well as lipophilicity appeared to have significant impact on antiviral activity and cytotoxicity. Several lipoglycopeptides were also found to be active against human coronavirus.
Subject(s)
Antiviral Agents/pharmacology , Influenza A virus/drug effects , Influenza B virus/drug effects , Teicoplanin/pharmacology , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Microbial Sensitivity Tests , Molecular Structure , Structure-Activity Relationship , Teicoplanin/analogs & derivatives , Teicoplanin/chemistryABSTRACT
We here disclose chemical synthesis of ribonucleoside 5'-monophosphate (RMP), -diphosphate (RDP), and -triphosphate (RTP) and cycloSal-, Di PPro-, and Tri PPPro nucleotide prodrugs of the antiviral pseudobase T-1105. Moreover, we include one nucleoside diphosphate prodrug of the chemically less stable T-705. We demonstrate efficient T-1105-RDP and -RTP release from the Di PPro and Tri PPPro compounds by esterase activation. Using crude enzyme extracts, we saw rapid phosphorylation of T-1105-RDP into T-1105-RTP. In sharp contrast, phosphorylation of T-1105-RMP was not seen, indicating a yet unrecognized bottleneck in T-1105's metabolic activation. Accordingly, Di PPro and Tri PPPro compounds displayed improved cell culture activity against influenza A and B virus, which they retained in a mutant cell line incapable of activating the nucleobase parent. T-1105-RTP had a strong inhibitory effect against isolated influenza polymerase, and Di PPro-T-1105-RDP showed 4-fold higher potency in suppressing one-cycle viral RNA synthesis versus T-1105. Hence, our T-1105-RDP and -RTP prodrugs improve antiviral potency and achieve efficient metabolic bypass.
Subject(s)
Amides/chemistry , Amides/pharmacology , Orthomyxoviridae/drug effects , Prodrugs/metabolism , Pyrazines/chemistry , Pyrazines/pharmacology , Ribonucleotides/chemistry , Amides/metabolism , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Dogs , Madin Darby Canine Kidney Cells , Pyrazines/metabolismABSTRACT
Two series of easily accessible anilines were identified as inhibitors of influenza A virus subtype H1N1, and extensive chemical synthesis and analysis of the structure-activity relationship were performed. The compounds were shown to interfere with low pH-induced membrane fusion mediated by the H1 and H5 (group 1) hemagglutinin (HA) subtypes. A combination of virus resistance, HA interaction, and molecular dynamics simulation studies elucidated the binding site of these aniline-based influenza fusion inhibitors, which significantly overlaps with the pocket occupied by some H3 HA-specific inhibitors, indicating the high relevance of this cavity for drug design.
Subject(s)
Aniline Compounds/chemistry , Aniline Compounds/pharmacology , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H1N1 Subtype/physiology , Virus Internalization/drug effects , Aniline Compounds/metabolism , Binding Sites , HeLa Cells , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Humans , Influenza A Virus, H1N1 Subtype/metabolism , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Protein ConformationABSTRACT
A series of lipophilic teicoplanin pseudoaglycon derivatives, including alkyl-, aryl-, calixarene- and protected sugar-containing conjugates, were prepared using azide-alkyne click chemistry. Out of the conditions applied, the CuSO4-ascorbate reagent system proved to be more efficient than the Cu(I)I-Et3N-mediated reaction. Some of the new compounds have high in vitro activity against glycopeptide-resistant Gram-positive bacteria, including vanA-positive Enterococcus faecalis. A few of them also display promising in vitro anti-influenza activity.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Teicoplanin/analogs & derivatives , Triazoles/chemistry , Drug Resistance, Bacterial , Microbial Sensitivity Tests , Molecular Structure , Teicoplanin/chemical synthesis , Teicoplanin/pharmacologyABSTRACT
Adequate response to severe influenza infections or pandemic outbreaks requires two complementary strategies: preventive vaccination and antiviral therapy. The existing influenza drugs, M2 blockers and neuraminidase inhibitors, show modest clinical efficacy and established or potential resistance. In the past three years, several new agents have entered the clinical pipeline and already yielded some promising data from Phase 2 trials. For two main categories, that is, the broadly neutralizing anti-hemagglutinin antibodies and small-molecule inhibitors of the viral polymerase complex, crystallography was instrumental to guide drug development. These structural insights also aid to expand the activity spectrum towards influenza A plus B viruses, or conceive nucleoprotein or polymerase assembly inhibitors. The practice of influenza therapy should radically change in the next decade.
Subject(s)
Antiviral Agents/therapeutic use , Influenza Vaccines/administration & dosage , Influenza, Human/drug therapy , Antiviral Agents/pharmacology , Crystallography , Disease Outbreaks , Drug Design , Drug Resistance, Viral , Hemagglutinins, Viral/immunology , Humans , Influenza, Human/prevention & control , Influenza, Human/virology , PandemicsABSTRACT
T-705 (favipiravir) is a new antiviral agent in advanced clinical development for influenza therapy. It is supposed to act as an alternative substrate for the viral polymerase, causing inhibition of viral RNA synthesis or virus mutagenesis. These mechanisms were also proposed for ribavirin, an established and broad antiviral drug that shares structural similarity with T-705. We here performed a comparative analysis of the effects of T-705 and ribavirin on influenza virus and host cell functions. Influenza virus-infected cell cultures were exposed to T-705 or ribavirin during single or serial virus passaging. The effects on viral RNA synthesis and infectious virus yield were determined and mutations appearing in the viral genome were detected by whole-genome virus sequencing. In addition, the cellular nucleotide pools as well as direct inhibition of the viral polymerase enzyme were quantified. We demonstrate that the anti-influenza virus effect of ribavirin is based on IMP dehydrogenase inhibition, which results in fast and profound GTP depletion and an imbalance in the nucleotide pools. In contrast, T-705 acts as a potent and GTP-competitive inhibitor of the viral polymerase. In infected cells, viral RNA synthesis is completely inhibited by T-705 or ribavirin at ≥50 µM, whereas exposure to lower drug concentrations induces formation of noninfectious particles and accumulation of random point mutations in the viral genome. This mutagenic effect is 2-fold higher for T-705 than for ribavirin. Hence, T-705 and ribavirin both act as purine pseudobases but profoundly differ with regard to the mechanism behind their antiviral and mutagenic effects on influenza virus.
Subject(s)
Amides/pharmacology , Antiviral Agents/pharmacology , Gene Expression Regulation, Viral , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H3N2 Subtype/drug effects , Pyrazines/pharmacology , Reassortant Viruses/drug effects , Ribavirin/pharmacology , A549 Cells , Amides/chemistry , Animals , Antiviral Agents/chemistry , Chick Embryo , DNA-Directed RNA Polymerases/antagonists & inhibitors , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Dogs , Humans , IMP Dehydrogenase/antagonists & inhibitors , IMP Dehydrogenase/genetics , IMP Dehydrogenase/metabolism , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/growth & development , Influenza A Virus, H1N1 Subtype/metabolism , Influenza A Virus, H3N2 Subtype/genetics , Influenza A Virus, H3N2 Subtype/growth & development , Influenza A Virus, H3N2 Subtype/metabolism , Madin Darby Canine Kidney Cells , Mutation/drug effects , Pyrazines/chemistry , RNA, Viral/antagonists & inhibitors , RNA, Viral/biosynthesis , Reassortant Viruses/genetics , Reassortant Viruses/growth & development , Reassortant Viruses/metabolism , Ribavirin/chemistry , Sequence Analysis, RNA , Structure-Activity Relationship , Viral Proteins/antagonists & inhibitors , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Replication/drug effectsABSTRACT
Despite the close structural similarity between the heptapeptide cores of the glycopeptide antibiotics teicoplanin and ristocetin, synthetically modified derivatives of their aglycons show significantly different antibacterial and antiviral properties. The teicoplanin aglycon derivatives with one exception proved to be potent antibacterials but they did not exhibit anti-influenza virus activity. In contrast, the aglycoristocetin derivatives generally showed high anti-influenza virus activity and possessed moderate antibacterial activity. A systematic structure-activity relationship study has been carried out on ristocetin and teicoplanin aglycon derivatives, to explore which structural differences are responsible for these markedly different biological activities. According to electronic circular dichroism and in silico conformational studies, it was found that the differences in anti-influenza virus activity are mainly determined by the conformation of the heptapeptide core of the antibiotics controlled by the presence or absence of chloro substituents. Knowledge of the bioactive conformation will help to design new analogs with improved anti-influenza virus activity. For the teicoplanin derivatives, it was shown that derivatization to improve the antiviral efficacy was accompanied by a significant decrease in antibacterial activity.