ABSTRACT
Zika virus (ZIKV) is a member of the Flaviviridae family that can cause neurological disorders and congenital malformations. The NS2B-NS3 viral serine protease is an attractive target for the development of new antiviral agents against ZIKV. We report here a SAR study on a series of substrate-like linear tripeptides that inhibit in a non-covalent manner the NS2B-NS3 protease. Optimization of the residues at positions P1, P2, P3 and of the N-terminal and C-terminal portions of the tripeptide allowed the identification of inhibitors with sub-micromolar potency with phenylglycine as arginine-mimicking group and benzylamide as C-terminal fragment. Further SAR exploration and application of these structural changes to a series of peptides having a 4-substituted phenylglycine residue at the P1 position led to potent compounds showing double digit nanomolar inhibition of the Zika protease (IC50 = 30 nM) with high selectivity against trypsin-like proteases and the proteases of other flavivirus, such as Dengue 2 virus (DEN2V) and West Nile virus (WNV).
Subject(s)
Antiviral Agents/pharmacology , Peptides/pharmacology , Protease Inhibitors/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Zika Virus/drug effects , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Dengue Virus/drug effects , Dose-Response Relationship, Drug , Humans , Microbial Sensitivity Tests , Molecular Structure , Peptides/chemical synthesis , Peptides/chemistry , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , RNA Helicases/antagonists & inhibitors , RNA Helicases/metabolism , Serine Endopeptidases/metabolism , Structure-Activity Relationship , Viral Nonstructural Proteins/metabolism , West Nile virus/drug effects , Zika Virus/enzymologyABSTRACT
The naturally occurring nucleotide 3'-deoxy-3',4'-didehydro-cytidine-5'-triphosphate (ddhCTP) was recently found to exert potent and broad-spectrum antiviral activity. However, nucleoside 5'-triphosphates in general are not cell-permeable, which precludes the direct use of ddhCTP as a therapeutic. To harness the therapeutic potential of this endogenous antiviral nucleotide, we synthesized phosphoramidate prodrug HLB-0532247 (1) and found it to result in dramatically elevated levels of ddhCTP in cells. We compared 1 and 3'-deoxy-3',4'-didehydro-cytidine (ddhC) and found that 1 more effectively reduces titers of Zika and West Nile viruses in cell culture with minimal nonspecific toxicity to host cells. We conclude that 1 is a promising antiviral agent based on a novel strategy of facilitating elevated levels of the endogenous ddhCTP antiviral nucleotide.
Subject(s)
Antiviral Agents/pharmacology , Cytidine Triphosphate/pharmacology , West Nile virus/drug effects , Zika Virus/drug effects , Animals , Antiviral Agents/chemistry , Cell Line , Chlorocebus aethiops , Cytidine Triphosphate/chemistry , Dose-Response Relationship, Drug , Humans , Microbial Sensitivity Tests , Molecular Structure , Structure-Activity RelationshipABSTRACT
Dengue virus (DENV) and West Nile virus (WNV) are arthropod-transmitted flaviviruses that cause systemic vascular leakage and encephalitis syndromes, respectively, in humans. However, the viral factors contributing to these specific clinical disorders are not completely understood. Flavivirus nonstructural protein 1 (NS1) is required for replication, expressed on the cell surface, and secreted as a soluble glycoprotein, reaching high levels in the blood of infected individuals. Extracellular DENV NS1 and WNV NS1 interact with host proteins and cells, have immune evasion functions, and promote endothelial dysfunction in a tissue-specific manner. To characterize how differences in DENV NS1 and WNV NS1 might function in pathogenesis, we generated WNV NS1 variants with substitutions corresponding to residues found in DENV NS1. We discovered that the substitution NS1-P101K led to reduced WNV infectivity in the brain and attenuated lethality in infected mice, although the virus replicated efficiently in cell culture and peripheral organs and bound at wild-type levels to brain endothelial cells and complement components. The P101K substitution resulted in reduced NS1 antigenemia in mice, and this was associated with reduced WNV spread to the brain. Because exogenous administration of NS1 protein rescued WNV brain infectivity in mice, we conclude that circulating WNV NS1 facilitates viral dissemination into the central nervous system and impacts disease outcomes. IMPORTANCE Flavivirus NS1 serves as an essential scaffolding molecule during virus replication but also is expressed on the cell surface and is secreted as a soluble glycoprotein that circulates in the blood of infected individuals. Although extracellular forms of NS1 are implicated in immune modulation and in promoting endothelial dysfunction at blood-tissue barriers, it has been challenging to study specific effects of NS1 on pathogenesis without disrupting its key role in virus replication. Here, we assessed WNV NS1 variants that do not affect virus replication and evaluated their effects on pathogenesis in mice. Our characterization of WNV NS1-P101K suggests that the levels of NS1 in the circulation facilitate WNV dissemination to the brain and affect disease outcomes. Our findings facilitate understanding of the role of NS1 during flavivirus infection and support antiviral strategies for targeting circulating forms of NS1.
Subject(s)
Viral Nonstructural Proteins/metabolism , West Nile virus/metabolism , Animals , Brain/metabolism , Brain/virology , Dengue Virus/drug effects , Dengue Virus/immunology , Dengue Virus/metabolism , Endothelial Cells , Female , Flavivirus/pathogenicity , Immune Evasion , Male , Mice , Mice, Inbred C57BL , Viral Nonstructural Proteins/analysis , Viral Nonstructural Proteins/blood , Viral Nonstructural Proteins/genetics , Virus Replication/genetics , Virus Replication/physiology , West Nile Fever/immunology , West Nile virus/drug effects , West Nile virus/immunologyABSTRACT
A series of 7-deazaadenine ribonucleosides bearing alkyl, alkenyl, alkynyl, aryl, or hetaryl groups at position 7 as well as their 5'-O-triphosphates and two types of monophosphate prodrugs (phosphoramidates and S-acylthioethanol esters) were prepared and tested for antiviral activity against selected RNA viruses (Dengue, Zika, tick-borne encephalitis, West Nile, and SARS-CoV-2). The modified triphosphates inhibited the viral RNA-dependent RNA polymerases at micromolar concentrations through the incorporation of the modified nucleotide and stopping a further extension of the RNA chain. 7-Deazaadenosine nucleosides bearing ethynyl or small hetaryl groups at position 7 showed (sub)micromolar antiviral activities but significant cytotoxicity, whereas the nucleosides bearing bulkier heterocycles were still active but less toxic. Unexpectedly, the monophosphate prodrugs were similarly or less active than the corresponding nucleosides in the in vitro antiviral assays, although the bis(S-acylthioethanol) prodrug 14h was transported to the Huh7 cells and efficiently released the nucleoside monophosphate.
Subject(s)
Antiviral Agents/pharmacology , Prodrugs/pharmacology , Purines/pharmacology , RNA Viruses/drug effects , Ribonucleosides/pharmacology , COVID-19/virology , Cell Line, Tumor , Dengue Virus/drug effects , Encephalitis Viruses, Tick-Borne/drug effects , Humans , Phosphates/pharmacology , Purine Nucleosides , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/drug effects , West Nile virus/drug effects , Zika Virus/drug effects , COVID-19 Drug TreatmentABSTRACT
Emerging viruses like dengue, West Nile, chikungunya, and Zika can cause widespread viral epidemics. Developing novel drugs or vaccines against specific targets for each virus is a difficult task. As obligate parasites, all viruses exploit common cellular pathways, providing the possibility to develop broad-spectrum antiviral agents targeting host factors. The human DEAD-box RNA helicase DDX3X is an essential cofactor for viral replication but dispensable for cell viability. Herein, we exploited the presence of a unique structural motif of DDX3X not shared by other cellular enzymes to develop a theoretical model to aid in the design of a novel class of highly selective inhibitors acting against such specific targets, thus limiting off-targeting effects. High-throughput virtual screening led us to identify hit compound 5, endowed with promising antienzymatic activity. To improve its aqueous solubility, 5 and its two enantiomers were synthesized and converted into their corresponding acetate salts (compounds 11, 12, and 13). In vitro mutagenesis and biochemical and cellular assays further confirmed that the developed molecules were selective for DDX3X and were able to suppress replication of West Nile and dengue viruses in infected cells in the micromolar range while showing no toxicity for uninfected cells. These results provide proof of principle for a novel strategy in developing highly selective and broad-spectrum antiviral molecules active against emerging and dangerous viral pathogens. This study paves the way for the development of larger focused libraries targeting such domain to expand SAR studies and fully characterize their mode of interaction.
Subject(s)
Antiviral Agents/pharmacology , DEAD-box RNA Helicases/antagonists & inhibitors , Dengue Virus/drug effects , Enzyme Inhibitors/pharmacology , West Nile virus/drug effects , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/toxicity , Arabidopsis/enzymology , Cell Line, Tumor , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/genetics , Drosophila/enzymology , Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/toxicity , Hepacivirus/enzymology , Humans , Microbial Sensitivity Tests , Molecular Docking Simulation , Mutation , Proof of Concept Study , Protein Domains , Virus Replication/drug effectsABSTRACT
Dengue and West Nile virus are rapidly spreading global pathogens for which no specific therapeutic treatments are available. One of the promising targets for drug discovery against dengue and other flaviviruses is the viral serine protease NS2B-NS3. We present the design, synthesis, and in vitro and cellular characterization of a novel chemotype of potent small-molecule non-peptidic dengue protease inhibitors derived from 4-benzyloxyphenylglycine. A newly developed luciferase-based DENV-2 protease reporter system in HeLa cells (DENV2proHeLa) was employed to determine the activity of the compounds in a cellular environment. Specificity and selectivity of the DENV2proHeLa system were confirmed by viral titer reduction assays. The compounds reach low micromolar to upper nanomolar inhibitory potency in cell-based assays, are selective against other serine proteases, and do not show relevant cytotoxicity. An extensive structure-activity relationship study provides a perspective for further drug development against flaviviral infections.
Subject(s)
Dengue Virus/drug effects , Genes, Reporter/drug effects , Protease Inhibitors/administration & dosage , Protease Inhibitors/chemistry , Virus Replication/drug effects , West Nile virus/drug effects , Animals , Chlorocebus aethiops , Dengue Virus/physiology , Dose-Response Relationship, Drug , Genes, Reporter/physiology , HeLa Cells , Humans , Vero Cells , Virus Replication/physiology , West Nile virus/physiologyABSTRACT
Nucleoside analogs are widely used for the treatment of viral diseases (Hepatitis B/C, herpes and human immunodeficiency virus, HIV) and various malignancies. ALS-8176, a prodrug of the 4'-chloromethyl-2'-deoxy-2'-fluoro nucleoside ALS-8112, was evaluated in hospitalized infants for the treatment of respiratory syncytial virus (RSV), but was abandoned for unclear reasons. Based on the structure of ALS-8112, a series of novel 4'-modified-2'-deoxy-2'-fluoro nucleosides were synthesized. Newly prepared compounds were evaluated against RSV, but also against a panel of RNA viruses, including Dengue, West Nile, Chikungunya, and Zika viruses. Unfortunately, none of the compounds showed marked antiviral activity against these viruses.
Subject(s)
Antiviral Agents/chemical synthesis , Deoxycytidine/analogs & derivatives , Deoxyribonucleosides/chemical synthesis , Prodrugs/chemical synthesis , Animals , Antiviral Agents/pharmacology , Cell Line, Tumor , Chikungunya virus/drug effects , Chikungunya virus/growth & development , Cricetulus , Dengue Virus/drug effects , Dengue Virus/growth & development , Deoxycytidine/chemical synthesis , Deoxycytidine/pharmacology , Deoxyribonucleosides/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/virology , Hepatocytes/drug effects , Hepatocytes/virology , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/virology , Microbial Sensitivity Tests , Primary Cell Culture , Prodrugs/pharmacology , Respiratory Syncytial Virus, Human/drug effects , Respiratory Syncytial Virus, Human/growth & development , T-Lymphocytes/drug effects , T-Lymphocytes/virology , Treatment Failure , Virus Replication/drug effects , West Nile virus/drug effects , West Nile virus/growth & development , Zika Virus/drug effects , Zika Virus/growth & developmentABSTRACT
Introduction: West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus, which is endemic in many countries, especially in Europe and in North America, where the virus has increased its activity in the recent years. No vaccines nor antiviral drugs are available for the prevention and treatment of WNV infection in humans.Areas covered: This review article describes viral and host targets that have been addressed by anti-WNV drug discovery studies and summarizes the most relevant anti-WNV candidate compounds identified so far, focusing on those showing antiviral efficacy in in vivo models and broad-spectrum anti-flavivirus activity.Expert opinion: The most promising anti-WNV drug candidates target conserved enzymatic motifs in viral NS3 protease and NS5 polymerase and are effective against different flaviviruses. Targeting host factors required for viral infection and replication and modulation of host innate antiviral response are also promising approaches, which may lead to the development of compounds with broad-spectrum antiviral activity, a desirable feature for an antiviral drug.
Subject(s)
Antiviral Agents/pharmacology , Drug Discovery/methods , West Nile Fever/drug therapy , Animals , Drug Development , Humans , Immunity, Innate , Mosquito Vectors , Virus Replication/drug effects , West Nile Fever/transmission , West Nile Fever/virology , West Nile virus/drug effectsABSTRACT
Sofosbuvir, a licensed nucleotide analog targeting hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), has been recently evaluated as a broad anti-Flavivirus lead candidate revealing activity against Zika and Dengue viruses both in vitro and in animal models. In this study, the in vitro antiviral activity of sofosbuvir against West Nile virus (WNV) was determined by plaque assay (PA) and Immunodetection Assay (IA) in human cell lines and by enzymatic RdRp assay. By PA, the sofosbuvir half-maximal inhibitory concentration (IC50) was 1.2 ± 0.3 µM in Huh-7, 5.3 ± 0.9 µM in U87, 7.8 ± 2.5 µM in LN-18 and 63.4 ± 14.1 µM in A549 cells. By IA, anti-WNV activity was confirmed in both hepatic (Huh-7, 1.7 ± 0.5 µM) and neuronal (U87, 7.3 ± 2.0 µM) cell types. Sofosbuvir was confirmed to inhibit the purified WNV RdRp (IC50 11.1 ± 4.6 µM). In vitro resistance selection experiments were performed by propagating WNV in the Huh-7 cell line with two-fold increasing concentrations of sofosbuvir. At 80 µM, a significantly longer time for viral breakthrough was observed compared with lower concentrations (18 vs. 7-9 days post infection; p = 0.029), along with the detection of the S604T mutation, corresponding to the well-known S282T substitution in the motif B of HCV NS5B, which confers resistance to sofosbuvir. Molecular docking experiments confirmed that the S604T mutation within the catalytic site of RdRp affected the binding mode of sofosbuvir. To our knowledge, this is the first report of the antiviral activity of sofosbuvir against WNV as well as of selection of mutants in vitro.
Subject(s)
Antiviral Agents/pharmacology , Drug Resistance, Viral/genetics , Sofosbuvir/pharmacology , Virus Replication/drug effects , West Nile virus/drug effects , A549 Cells , Cell Line , Hepatocytes/virology , Humans , Inhibitory Concentration 50 , Molecular Docking Simulation , Mutation , Neurons/virology , Ribavirin/pharmacology , West Nile virus/physiologyABSTRACT
The ß-lactam ring represents a valuable moiety that can induce covalent binding of an inhibitor to its target. In this study, we explored di- and tripeptides with ß-lactam electrophilic warheads as inhibitors of dengue and West Nile virus NS2B-NS3 protease. Tripeptides with a (3S)-ß-lactam moiety displayed the highest activity, with IC50 and EC50 values in the lower micromolar range in biochemical and cellular assays. The activity against dengue protease was in general higher than against West Nile virus protease. The compounds were inactive against the off-targets thrombin and trypsin. Liquid chromatography-mass spectrometry experiments revealed that tripeptide-ß-lactam inhibitors bind to the protease in two distinct binding modes. Only one binding mode leads to a covalent, but reversible, interaction of the ß-lactam ring with the catalytic serine, followed by release of the inhibitor with opened ß-lactam ring. The other binding mode leads to the cleavage of the peptide backbone. This observation provides the first experimental evidence that benzyloxyphenylglycine in flaviviral protease inhibitors is positioned in the prime site of the enzyme.
Subject(s)
Antiviral Agents/pharmacology , Oligopeptides/pharmacology , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , beta-Lactams/pharmacology , Antiviral Agents/chemical synthesis , Antiviral Agents/metabolism , Catalytic Domain , Cell Line, Tumor , Dengue Virus/chemistry , Dengue Virus/drug effects , Dipeptides/chemical synthesis , Dipeptides/metabolism , Dipeptides/pharmacology , Humans , Microbial Sensitivity Tests , Molecular Docking Simulation , Oligopeptides/chemical synthesis , Oligopeptides/metabolism , Protein Binding , RNA Helicases/antagonists & inhibitors , RNA Helicases/metabolism , Serine Endopeptidases/chemistry , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/metabolism , Viral Nonstructural Proteins/metabolism , West Nile virus/chemistry , West Nile virus/drug effects , beta-Lactams/chemical synthesis , beta-Lactams/metabolismABSTRACT
Imbalanced datasets, comprising of more inactive compounds relative to the active ones, are a common challenge in ligand-based model building workflows for drug discovery. This is particularly true for neglected tropical diseases since efforts to identify therapeutics for these diseases are often limited. In this report, we analyze the performance of several undersampling strategies in modeling the Dengue Virus 2 (DENV2) inhibitory activity, as well as the anti-flaviviral activities for the West Nile (WNV) and Zika (ZIKV) viruses. To this end, we build datasets comprising of 1218 (159 actives and 1059 inactives), 1044 (132 actives and 912 inactives) and 302 (75 actives and 227 inactives) molecules with known DENV2, WNV and ZIKV inhibitory activity profiles, respectively. We develop ensemble classifiers for these endpoints and compare the performance of the different undersampling algorithms on external sets. It is observed that data pruning algorithms yield superior performance relative to data selection algorithms. The best overall performance is provided by the one-sided selection algorithm with test set balanced accuracy (BACC) values of 0.84, 0.74 and 0.77 for the DENV2, WNV and ZIKV inhibitory activities, respectively. For the model building, we use the recently proposed GT-STAF information indices, and compare the predictivity of 3 molecular fragmentation approaches: connected subgraphs, substructure and alogp atom types, which are observed to show comparable performance. On the other hand, a combination of indices based on these fragmentation strategies enhances the predictivity of the built ensembles. The built models could be useful for screening new molecules with possible DENV, WNV and ZIKV inhibitory activities. ADMET modelers are encouraged to adopt undersampling algorithms in their workflows when dealing with imbalanced datasets.
Subject(s)
Antiviral Agents/pharmacology , Drug Discovery/methods , Flaviviridae/drug effects , Support Vector Machine , Antiviral Agents/chemistry , Dengue Virus/drug effects , Flaviviridae Infections/drug therapy , Humans , West Nile virus/drug effects , Zika Virus/drug effectsABSTRACT
We report the full polyprotein genomic sequence of a West Nile virus strain isolated from Eurasian magpies dying with neurologic signs in Greece. Our findings demonstrate the local genetic evolution of the West Nile virus strain responsible for a human disease outbreak in the country that began in 2010.
Subject(s)
Bird Diseases/virology , West Nile Fever/veterinary , West Nile virus/drug effects , West Nile virus/genetics , Animals , Bird Diseases/epidemiology , Birds/virology , Disease Outbreaks , Genome, Viral , Genomics/methods , Greece/epidemiology , Humans , Phylogeny , West Nile virus/isolation & purificationABSTRACT
West Nile virus (WNV) is a neurotropic flavivirus and the leading cause of mosquito-borne encephalitis in the United States. Recent studies in humans have found that dysfunctional T cell responses strongly correlate with development of severe WNV neuroinvasive disease. However, the contributions of human dendritic cells (DCs) in priming WNV-specific T cell immunity remains poorly understood. Here, we demonstrate that human monocyte derived DCs (moDCs) support productive viral replication following infection with a pathogenic strain of WNV. Antiviral effector gene transcription was strongly induced during the log phase of viral growth, while secretion of type I interferons (IFN) occurred with delayed kinetics. Activation of RIG-I like receptor (RLR) or type I IFN signaling prior to log phase viral growth significantly diminished viral replication, suggesting that early activation of antiviral programs can block WNV infection. In contrast to the induction of antiviral responses, WNV infection did not promote transcription or secretion of proinflammatory (interleukin-6 [IL-6], granulocyte-macrophage colony-stimulating factor [GM-CSF], CCL3, CCL5, and CXCL9) or T cell modulatory (IL-4, IL-12, and IL-15) cytokines. There was also minimal induction of molecules associated with antigen presentation and T cell priming, including the costimulatory molecules CD80, CD86, and CD40. Functionally, WNV-infected moDCs dampened allogenic CD4 and CD8 T cell activation and proliferation. Combining these observations, we propose a model whereby WNV subverts human DC activation to compromise priming of WNV-specific T cell immunity.IMPORTANCE West Nile virus (WNV) is an encephalitic flavivirus that remains endemic in the United States. Previous studies have found dysfunctional T cell responses correlate to severe disease outcomes during human WNV infection. Here, we sought to better understand the ability of WNV to program human dendritic cells (DCs) to prime WNV-specific T cell responses. While productive infection of monocyte-derived DCs activated antiviral and type I interferon responses, molecules associated with inflammation and programming of T cells were minimally induced. Functionally, WNV-infected DCs dampened T cell activation and proliferation during an allogeneic response. Combined, our data support a model whereby WNV infection of human DCs compromises WNV-specific T cell immunity.
Subject(s)
Dendritic Cells/immunology , Monocytes/immunology , T-Lymphocytes/immunology , West Nile Fever/immunology , West Nile virus/immunology , Animals , Antiviral Agents/pharmacology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Line , Cell Proliferation , Chlorocebus aethiops , Cytokines/metabolism , DEAD Box Protein 58 , Dendritic Cells/virology , Flavivirus , Humans , Immunity, Innate , Interferon Type I/metabolism , Lymphocyte Activation , Receptors, Immunologic , Vero Cells , Virus Replication , West Nile Fever/virology , West Nile virus/drug effectsABSTRACT
The innate response to a pathogen is critical in determining the outcome of the infection. However, the interplay of different cellular responses that are activated following viral infection and their contribution to innate antiviral signalling has not been clearly established. This work shows that flaviviruses, including Dengue, Zika, West Nile and Tick-borne encephalitis viruses, activate the unfolded protein response before transcription of interferon regulatory factor 3 induced genes. Infection in conditions of unfolded protein response priming leads to early activation of innate antiviral responses and cell intrinsic inhibition of viral replication, which is interferon regulatory factor 3 dependent. These results demonstrate that the unfolded protein response is not only a physiological reaction of the cell to viral infection, but also synergizes with pattern recognition sensing to mount a potent antiviral response.
Subject(s)
Antiviral Agents/pharmacology , Flavivirus Infections/immunology , Host-Pathogen Interactions/physiology , Immunity, Innate/drug effects , Signal Transduction/drug effects , Signal Transduction/immunology , Unfolded Protein Response/drug effects , Animals , Chlorocebus aethiops , DEAD Box Protein 58/metabolism , Dengue/immunology , Dengue Virus/drug effects , Encephalitis Viruses, Tick-Borne/drug effects , Encephalitis, Tick-Borne/immunology , Endoribonucleases/metabolism , Female , Humans , Interferon Regulatory Factor-3/metabolism , Mice , Protein Serine-Threonine Kinases/metabolism , Receptors, Immunologic , Transcriptome , Vero Cells , Virus Replication/drug effects , West Nile Fever/immunology , West Nile virus/drug effects , Zika Virus/drug effects , Zika Virus Infection/immunologyABSTRACT
Currently, more than 70 flaviviruses were identified and reported in the literature, whose Dengue (DENV), Zika (ZIKV), and West Nile (WNV) viruses have been responsible for millions of cases of infections worldwide, mainly in developing countries. These viruses are transmitted by the bite of mosquitoes from genus Aedes, or Culex and, in some cases, Stegomyia. Despite numerous efforts to identify a selective, safe, and effective antiviral agent, there is no currently approved drug for the treatment of flaviviral infections. Then, current pharmacological therapy has the objective to treat the clinical symptoms. Various peptidomimetics and peptide-derivatives have been synthesized and evaluated against several biological targets from flaviviruses with different applications, such as diagnosis, E protein inhibitors, entry inhibitors, virucidal inhibitors, and also viral replication inhibitors. Flaviviral replication depends on the NS3pro that is completely activated when it is complexed to its cofactor, NS2B; forming a viral enzymatic complex. The development of NS2B-NS3pro inhibitors is considered a challenging work due to its active site is shallow and open-pocket. In this work, we report all advances involving peptidomimetics, peptide-derived, and peptide-hybrids found in the literature. In sense, we discuss the influence of different functional groups in the activity and selectivity. Moreover, the first inhibitors reported in the literature as covalent ligands, comprising two basic residues followed by an electrophilic moiety that binds to the catalytic serine (Ser135-O-) are also discussed in details, such as trifluoromethyl ketones, aldehydes, and boronic acids. Furthermore, it is presented the influence of introducing transition metals, providing metallopeptide inhibitors; and cyclization of linear peptides, generating cyclic and macrocyclic peptide inhibitors. Finally, we provide the most accurate state of the art found in the literature, which can be utilized to design new and effective antiviral agents.
Subject(s)
Dengue/drug therapy , Flavivirus/drug effects , Peptides/therapeutic use , Protease Inhibitors/therapeutic use , West Nile virus/drug effects , Zika Virus Infection/drug therapy , Humans , Peptides/pharmacology , Protease Inhibitors/pharmacologyABSTRACT
Background: West Nile virus is an arthropod-borne virus (arbovirus) and emerging cause of significant illness in European and Mediterranean countries. West Nile virus infection can cause severe and potentially fatal neurological illnesses, including encephalitis, meningitis, and acute flaccid paralysis. Additionally, immunosuppression, alcohol abuse, old age, and diabetes mellitus are common factors associated with West Nile neuroinvasive disease. Case Report: In August 2018, a 60-year-old male patient with a history of diffuse large B-cell lymphoma initially presented with symptoms including abdominal pain and distention, nausea, and vomiting. Three days after open abdominal surgery due to adhesive small bowel obstruction, he developed fever, prominent tremors, and rapidly progressing flaccid paralysis. The identification of West Nile virus RNA in the serum sample led to the diagnosis of West Nile neuroinvasive disease. Conclusion: Clinicians should evaluate patients with acute flaccid paralysis for the evidence of West Nile neuroinvasive disease. It is particularly important for healthcare providers to consider West Nile neuroinvasive disease in the differential diagnosis of aseptic meningitis, encephalitis, and acute paralysis cases, especially in endemic areas.
Subject(s)
Lymphoma, Large B-Cell, Diffuse/complications , West Nile Fever/etiology , Antineoplastic Combined Chemotherapy Protocols , Cyclophosphamide , Doxorubicin , Humans , Lymphoma, B-Cell/complications , Lymphoma, B-Cell/physiopathology , Lymphoma, Large B-Cell, Diffuse/physiopathology , Male , Middle Aged , Plasmapheresis/methods , Prednisone , Quadriplegia/etiology , Quadriplegia/physiopathology , Real-Time Polymerase Chain Reaction/methods , Rituximab , Turkey , Vincristine , West Nile Fever/complications , West Nile Fever/physiopathology , West Nile virus/drug effects , West Nile virus/pathogenicityABSTRACT
Inhibitors of the flaviviral serine proteases, which are crucial for the replication of dengue and West-Nile virus, have attracted much attention over the last years. A dibasic 4-guanidinobenzoate was previously reported as inhibitor of the dengue protease with potency in the low-micromolar range. In the present study, this lead structure was modified with the intent to explore structure-activity relationships and obtain compounds with increased drug-likeness. Substitutions of the guanidine moieties, the aromatic rings, and the ester with other functionalities were evaluated. All changes were accompanied by a loss of inhibition, indicating that the 4-guanidinobenzoate scaffold is an essential element of this compound class. Further experiments indicate that the target recognition of the compounds involves the reversible formation of a covalent adduct.
Subject(s)
Amides/chemistry , Antiviral Agents/chemistry , Carbamates/chemistry , Esters/chemistry , Trypsin Inhibitors/chemistry , Amides/chemical synthesis , Antiviral Agents/chemical synthesis , Carbamates/chemical synthesis , Dengue Virus/drug effects , Dengue Virus/enzymology , Drug Stability , Esters/chemical synthesis , Molecular Structure , Structure-Activity Relationship , Thrombin/antagonists & inhibitors , Trypsin Inhibitors/chemical synthesis , Viral Nonstructural Proteins/antagonists & inhibitors , West Nile virus/drug effects , West Nile virus/enzymologyABSTRACT
Flaviviruses are (re)-emerging RNA viruses strictly dependent on lipid metabolism for infection. In the search for host targeting antivirals, we explored the effect of pharmacological modulation of fatty acid metabolism during flavivirus infection. Considering the central role of acetyl-Coenzyme A carboxylase (ACC) on fatty acid metabolism, we analyzed the effect of three small-molecule ACC inhibitors (PF-05175157, PF-05206574, and PF-06256254) on the infection of medically relevant flaviviruses, namely West Nile virus (WNV), dengue virus, and Zika virus. Treatment with these compounds inhibited the multiplication of the three viruses in cultured cells. PF-05175157 induced a reduction of the viral load in serum and kidney in WNV-infected mice, unveiling its therapeutic potential for the treatment of chronic kidney disease associated with persistent WNV infection. This study constitutes a proof of concept of the reliability of ACC inhibitors to become viable antiviral candidates. These results support the repositioning of metabolic inhibitors as broad-spectrum antivirals.
Subject(s)
Acetyl-CoA Carboxylase/antagonists & inhibitors , Dengue Virus/physiology , Dengue/enzymology , Enzyme Inhibitors/administration & dosage , West Nile Fever/enzymology , West Nile virus/physiology , Zika Virus Infection/enzymology , Zika Virus/physiology , Acetyl-CoA Carboxylase/metabolism , Animals , Antiviral Agents/administration & dosage , Dengue/drug therapy , Dengue/virology , Dengue Virus/drug effects , Dengue Virus/genetics , Disease Models, Animal , Female , Humans , Male , Mice , Virus Replication/drug effects , West Nile Fever/drug therapy , West Nile Fever/virology , West Nile virus/drug effects , West Nile virus/genetics , Zika Virus/drug effects , Zika Virus/genetics , Zika Virus Infection/drug therapy , Zika Virus Infection/virologyABSTRACT
West Nile virus (WNV) is an emerging mosquito-borne flavivirus, related to dengue virus and Zika virus. To gain insight into host pathways involved in WNV infection, we performed a systematic affinity-tag purification mass spectrometry (APMS) study to identify 259 WNV-interacting human proteins. RNA interference screening revealed 26 genes that both interact with WNV proteins and influence WNV infection. We found that WNV, dengue and Zika virus capsids interact with a conserved subset of proteins that impact infection. These include the exon-junction complex (EJC) recycling factor PYM1, which is antiviral against all three viruses. The EJC has roles in nonsense-mediated decay (NMD), and we found that both the EJC and NMD are antiviral and the EJC protein RBM8A directly binds WNV RNA. To counteract this, flavivirus infection inhibits NMD and the capsid-PYM1 interaction interferes with EJC protein function and localization. Depletion of PYM1 attenuates RBM8A binding to viral RNA, suggesting that WNV sequesters PYM1 to protect viral RNA from decay. Together, these data suggest a complex interplay between the virus and host in regulating NMD and the EJC.
Subject(s)
Antiviral Agents/pharmacology , Flavivirus Infections/drug therapy , Viral Proteins/genetics , West Nile virus/drug effects , West Nile virus/genetics , Capsid Proteins , Carrier Proteins , Codon, Nonsense , Dengue Virus/genetics , Exons , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/physiology , Humans , Protein Interaction Maps , RNA Interference , RNA, Viral , RNA-Binding Proteins , Viral Proteins/physiology , West Nile virus/pathogenicity , Zika Virus/geneticsABSTRACT
Control of arbovirus transmission remains focused on vector control through application of insecticides directly to the environment. However, these insecticide applications are often reactive interventions that can be poorly-targeted, inadequate for localized control during outbreaks, and opposed due to environmental and toxicity concerns. In this study, we developed endectocide-treated feed as a systemic endectocide for birds to target blood feeding Culex tarsalis, the primary West Nile virus (WNV) bridge vector in the western United States, and conducted preliminary tests on the effects of deploying this feed in the field. In lab tests, ivermectin (IVM) was the most effective endectocide tested against Cx. tarsalis and WNV-infection did not influence mosquito mortality from IVM. Chickens and wild Eurasian collared doves exhibited no signs of toxicity when fed solely on bird feed treated with concentrations up to 200 mg IVM/kg of diet, and significantly more Cx. tarsalis that blood fed on these birds died (greater than 80% mortality) compared to controls (less than 25% mortality). Mosquito mortality following blood feeding correlated with IVM serum concentrations at the time of blood feeding, which dropped rapidly after the withdrawal of treated feed. Preliminary field testing over one WNV season in Fort Collins, Colorado demonstrated that nearly all birds captured around treated bird feeders had detectable levels of IVM in their blood. However, entomological data showed that WNV transmission was non-significantly reduced around treated bird feeders. With further development, deployment of ivermectin-treated bird feed might be an effective, localized WNV transmission control tool.