RESUMEN
The potential outcome of flavivirus and alphavirus co-infections is worrisome due to the development of severe diseases. Hundreds of millions of people worldwide live under the risk of infections caused by viruses like chikungunya virus (CHIKV, genus Alphavirus), dengue virus (DENV, genus Flavivirus), and zika virus (ZIKV, genus Flavivirus). So far, neither any drug exists against the infection by a single virus, nor against co-infection. The results described in our study demonstrate the inhibitory potential of two flavonoids derived from citrus plants: Hesperetin (HST) against NS2B/NS3pro of ZIKV and nsP2pro of CHIKV and, Hesperidin (HSD) against nsP2pro of CHIKV. The flavonoids are noncompetitive inhibitors and the determined IC50 values are in low µM range for HST against ZIKV NS2B/NS3pro (12.6 ± 1.3 µM) and against CHIKV nsP2pro (2.5 ± 0.4 µM). The IC50 for HSD against CHIKV nsP2pro was 7.1 ± 1.1 µM. The calculated ligand efficiencies for HST were > 0.3, which reflect its potential to be used as a lead compound. Docking and molecular dynamics simulations display the effect of HST and HSD on the protease 3D models of CHIKV and ZIKV. Conformational changes after ligand binding and their effect on the substrate-binding pocket of the proteases were investigated. Additionally, MTT assays demonstrated a very low cytotoxicity of both the molecules. Based on our results, we assume that HST comprise a chemical structure that serves as a starting point molecule to develop a potent inhibitor to combat CHIKV and ZIKV co-infections by inhibiting the virus proteases.
Asunto(s)
Virus Chikungunya/enzimología , Citrus/química , Hesperidina/farmacología , Péptido Hidrolasas/metabolismo , Virus Zika/enzimología , Animales , Virus Chikungunya/efectos de los fármacos , Chlorocebus aethiops , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Simulación del Acoplamiento Molecular , Péptido Hidrolasas/química , Extractos Vegetales/química , Conformación Proteica , Células Vero , Proteínas Virales/química , Proteínas Virales/metabolismo , Virus Zika/efectos de los fármacosRESUMEN
Chikungunya virus; the pathogen for chikungunya febrile and arthritic disease, having 11.8 kb positive-sense RNA genome encodes polyproteins for structural and non-structural regions. The polyprotein (P1234) corresponding to the non-structural part from 5' end gets auto-cleaved by the action of nsP2 protease, which leads to the generation of individual functional enzymatic proteins like nsP4, nsP1, nsP2 and nsP3. Thus, nsP2 protein initiates viral replication. Targeting nsP2 to block virus replication has always been the foremost strategy to develop antivirals. Plant-based molecules are one of the top choices to develop as inhibitor due to their less toxicity and wide availability. Using a combination of receptor-based docking and MD simulations, we identified a flavanone glycoside- naringin, which binds to nsP2 protease at nM affinity. The biomolecular interaction between naringin and nsP2 was established through SPR. As discerned through FTIR and intrinsic fluorescence studies, upon binding with naringin, a global structural change in nsP2 occurs. This structural modulation in nsP2 due to binding of naringin is likely to interfere with the normal functioning of this enzyme during the viral life cycle. In conclusion, this report highlights the potential of naringin as an anti-viral agent against Chikungunya.
Asunto(s)
Virus Chikungunya/enzimología , Cisteína Endopeptidasas/química , Flavanonas/química , Simulación del Acoplamiento Molecular , Sitios de Unión , Cisteína Endopeptidasas/metabolismo , Flavanonas/metabolismo , Unión ProteicaRESUMEN
Chikungunya virus (CHIKV) causes Chikungunya fever (CHIKF) and till date no effective medicine for its cure is available in market. Different research groups find various possible interactions between small molecules and non-structural proteins, viz. nsP3, one of the most important viral elements in CHIKV. In this work, authors have studied the interactions of nsP3 protease of CHIKV with pyranooxazoles. Initially, a one-pot three-component reaction was designed using oxazolidine-2,4-dione, benzaldehyde and cyanoethylacetate to get a proposed biological active molecule, i.e. based on pyranooxazoles. The mechanism for the synthesis of the product based on pyranooxazole was studied through density functional theory (DFT) using Gaussian. Then, a library of the obtained pyranooxazole was created through computational tools by varying the substituents. Further, virtual screening of the designed library of pyranooxazoles (200 compounds) against nsP3 protease of CHIKV was performed. Herein, CMPD 104 showed strongest binding affinity toward the targeted nsP3 protease of CHIKV, based on the least binding energy obtained from docking. Based on docking results, the pharmacological, toxicity, biological score and Lipinski's filters were studied. Further, DFT studies of top five compounds were done using Gaussian. Molecular dynamics (MD) simulation of nsP3 protease of CHIKV with and without 104 was performed using AMBER18 utilizing ff14SB force field in three steps (minimization, equilibration and production). This work is emphasized to designing of one-pot three-component synthesis and to develop a theoretical model to inhibit the nsP3 protease of CHIKV. AbbreviationsCHIKFChikungunya feverCHIKVChikungunya virusDFTdensity functional theoryDSDiscovery StudioMDmolecular dynamicsMM-GBSAmolecular mechanics-generalized born surface areaMMVMolegro molecular viewerCommunicated by Ramaswamy H. Sarma.
Asunto(s)
Antivirales/química , Virus Chikungunya , Inhibidores de Proteasas/química , Virus Chikungunya/efectos de los fármacos , Virus Chikungunya/enzimología , Simulación de Dinámica Molecular , Péptido Hidrolasas , Proteínas no Estructurales Virales , Replicación ViralRESUMEN
Alphaviruses, including Chikungunya (CHIKV) and Venezuelan equine encephalitis virus (VEEV), are among the leading causes of recurrent epidemics all over the world. Alphaviral nonstructural protein 1 (nsP1) orchestrates the capping of nascent viral RNA via its S-adenosyl methionine-dependent N-7-methyltransferase (MTase) and guanylyltransferase activities. Here, we developed and validated a novel capillary electrophoresis (CE)-based assay for measuring the MTase activity of purified VEEV and CHIKV nsP1. We employed the assay to assess the MTase inhibition efficiency of a few adenosine analogs and identified 5-iodotubercidin (5-IT) as an inhibitor of nsP1. The antiviral potency of 5-IT was evaluated in vitro using a combination of cell-based assays, which suggest that 5-IT is efficacious against CHIKV in cell culture (EC50 : 0.409 µm).
Asunto(s)
Adenosina/análogos & derivados , Adenosina/farmacología , Antivirales/química , Antivirales/farmacología , Virus Chikungunya/efectos de los fármacos , Metiltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Virus Chikungunya/enzimología , Virus Chikungunya/fisiología , Chlorocebus aethiops , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
Alphavirus non-structural protein, nsP1â¯has a distinct molecular mechanism of capping the viral RNAs than the conventional capping mechanism of host. Thus, alphavirus capping enzyme nsP1 is a potential drug target. nsP1 catalyzes the methylation of guanosine triphosphate (GTP) by transferring the methyl group from S-adenosylmethionine (SAM) to a GTP molecule at its N7 position with the help of nsP1 methyltransferase (MTase) followed by guanylylation (GT) reaction which involves the formation of m7GMP-nsP1 covalent complex by nsP1 guanylyltransferase (GTase). In subsequent reactions, m7GMP moiety is added to the 5' end of the viral ppRNA by nsP1 GTase resulting in the formation of cap0 structure. In the present study, chikungunya virus (CHIKV) nsP1 MTase and GT reactions were confirmed by an indirect non-radioactive colorimetric assay and western blot assay using an antibody specific for the m7G cap, respectively. The purified recombinant CHIKV nsP1â¯has been used for the development of a rapid and sensitive non-radioactive enzyme linked immunosorbent assay (ELISA) to identify the inhibitors of CHIKV nsP1. The MTase reaction is followed by GT reaction and resulted in m7GMP-nsP1 covalent complex formation. The developed ELISA nsP1 assay measures this m7GMP-nsP1 complex by utilizing anti-m7G cap monoclonal antibody. The mutation of a conserved residue Asp63 to Ala revealed its role in nsP1 enzyme reaction. Inductively coupled plasma mass spectroscopy (ICP-MS) was used to determine the presence of magnesium ions (Mg2+) in the purified nsP1 protein. The divalent metal ion selectivity and investigation show preference for Mg2+ ion by CHIKV nsP1. Additionally, using the developed ELISA nsP1 assay, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA) and ribavirin were determined and the IC50 values were estimated to be 2.69 µM, 5.72 µM and 1.18â¯mM, respectively.
Asunto(s)
Antivirales/farmacología , Virus Chikungunya/enzimología , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática/métodos , Metiltransferasas/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Adenosina/análogos & derivados , Adenosina/farmacología , Ácido Aurintricarboxílico/farmacología , Cationes Bivalentes/metabolismo , Coenzimas/metabolismo , Concentración 50 Inhibidora , Magnesio/metabolismo , Ribavirina/farmacologíaRESUMEN
Chikungunya virus has emerged as one of the most important global arboviral threats over the last decade. Inspite of large scale morbidity, with long lasting polyarthralgia, so far no licensed vaccine or antiviral is available. CHIKV nsP2 protease is crucial for processing of viral nonstructural polypeptide precursor to release enzymes required for viral replication, thus making it a promising drug target. In this study, high cell density cultivation (HCDC) of Escherichia coli in batch process was carried out to produce rCHIKV nsP2pro in a cost-effective manner. The purified nsP2pro and fluorogenic peptide substrate have been adapted for fluorescence resonance energy transfer (FRET) based high throughput screening (HTS) assay with Z' value and CV of 0.67 ± 0.054 and <10% respectively. We used this cell free HTS system to screen panel of metal ions and its conjugate which revealed zinc acetate as a potential candidate, which was further found to inhibit CHIKV in Vero cells. Scale-up process has not been previously reported for any of the arboviral nonstructural enzymes. The successful scale-up method for viral protease together with a HTS assay could lead to the development of industrial level large-scale screening platform for identification of protease inhibitors against emerging and re-emerging viruses.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores de Proteasas/farmacología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/farmacología , Virus Chikungunya/enzimología , Virus Chikungunya/fisiología , Chlorocebus aethiops , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Especificidad por Sustrato , Células Vero , Acetato de Zinc/farmacologíaRESUMEN
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has evolved effective mechanisms to counteract the type I interferon (IFN) response. Upon recognition of the virus, cells secrete IFNs, which signal through transmembrane receptors (IFNAR) to phosphorylate STAT proteins (pSTAT). pSTAT dimers are transported into the nucleus by importin-α5 and activate the transcription of IFN-stimulated genes (ISGs), increasing cellular resistance to infection. Subsequently, STAT proteins are shuttled back into the cytoplasm by the exportin CRM1. CHIKV nonstructural protein 2 (nsP2) reduces ISG expression by inhibiting general host cell transcription and by specifically reducing the levels of nuclear pSTAT1 via an unknown mechanism. To systematically examine where nsP2 acts within the JAK/STAT signaling cascade, we used two well-characterized mutants of nsP2, P718S and KR649AA. Both mutations abrogate nsP2's ability to shut off host transcription, but only the KR649AA mutant localizes exclusively to the cytoplasm and no longer specifically inhibits JAK/STAT signaling. These mutant nsP2 proteins did not differentially affect IFNAR expression levels or STAT1 phosphorylation in response to IFNs. Coimmunoprecipitation experiments showed that in the presence of nsP2, STAT1 still effectively bound importin-α5. Chemically blocking CRM1-mediated nuclear export in the presence of nsP2 additionally showed that nuclear translocation of STAT1 is not affected by nsP2. nsP2 putatively has five domains. Redirecting the nsP2 KR649AA mutant or just nsP2's C-terminal methyltransferase-like domain into the nucleus strongly reduced nuclear pSTAT in response to IFN stimulation. This demonstrates that the C-terminal domain of nuclear nsP2 specifically inhibits the IFN response by promoting the nuclear export of STAT1.IMPORTANCE Chikungunya virus is an emerging pathogen associated with large outbreaks on the African, Asian, European, and both American continents. In most patients, infection results in high fever, rash, and incapacitating (chronic) arthralgia. CHIKV effectively inhibits the first line of defense, the innate immune response. As a result, stimulation of the innate immune response with interferons (IFNs) is ineffective as a treatment for CHIKV disease. The IFN response requires an intact downstream signaling cascade called the JAK/STAT signaling pathway, which is effectively inhibited by CHIKV nonstructural protein 2 (nsP2) via an unknown mechanism. The research described here specifies where in the JAK/STAT signaling cascade the IFN response is inhibited and which protein domain of nsP2 is responsible for IFN inhibition. The results illuminate new aspects of antiviral defense and CHIKV counterdefense strategies and will direct the search for novel antiviral compounds.
Asunto(s)
Virus Chikungunya/enzimología , Cisteína Endopeptidasas/genética , Inmunidad Innata , Interferón Tipo I/antagonistas & inhibidores , Factor de Transcripción STAT1/genética , Línea Celular , Virus Chikungunya/genética , Virus Chikungunya/fisiología , Simulación por Computador , Cisteína Endopeptidasas/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunoprecipitación , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Metiltransferasas/metabolismo , Mutación , Transducción de Señal/genética , Transducción de Señal/inmunología , Replicación ViralRESUMEN
Chikungunya virus (CHIKV), a mosquito-borne pathogenic virus that reemerged and caused epidemic in the Indian Ocean island of La Réunion, is a potential public health threat. Currently there is no antiviral drug or vaccine commercially available for the treatment of chikungunya fever, which necessitates the urge for an effective antiviral therapy for chikungunya treatment. In the present study, a FRET based protease assay was used to analyze the proteolytic activity of chikungunya nsP2 protease (CHIKV nsP2pro) - an essential viral enzyme, with fluorogenic substrate peptide. This protease assay was used to assess the inhibitory activity of Pep-I (MMsINC® database ID MMs03131094) and Pep-II (MMsINC® database ID MMs03927237), peptidomimetic compounds identified in a previous study by our group. Both compounds inhibited CHIKV nsP2pro with half maximal inhibition concentration (IC50) values of â¼34⯵M and â¼42⯵M, respectively. Kinetic studies showed that the inhibition constant (Ki) value is 33.34⯱â¯2.53⯵M for Pep-I and 45.89⯱â¯4.38⯵M for Pep-II. Additionally, these two compounds significantly inhibited CHIKV replication in BHK-21â¯cells at concentrations much lower than their cytotoxic concentrations. Intriguingly, these compounds did not show inhibitory effect on Sindbis virus. This suggests that Pep-I and Pep-II compounds identified as CHIKV nsP2 substrate peptidomimetics, specifically inhibit CHIKV replication.
Asunto(s)
Fiebre Chikungunya/tratamiento farmacológico , Virus Chikungunya/enzimología , Proteasas de Cisteína/química , Peptidomiméticos/farmacología , Fiebre Chikungunya/enzimología , Fiebre Chikungunya/virología , Virus Chikungunya/efectos de los fármacos , Virus Chikungunya/patogenicidad , Proteasas de Cisteína/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Humanos , Cinética , Peptidomiméticos/química , Replicación Viral/efectos de los fármacosRESUMEN
Chikungunya virus is a re-emerging arbovirus transmitted to humans by mosquitoes, responsible for an acute flu-like illness associated with debilitating arthralgia, which can persist for several months or become chronic. In recent years, this viral infection has spread worldwide with a previously unknown virulence. To date, no specific antivirals treatments nor vaccines are available against this important pathogen. Starting from the structures of two antiviral hits previously identified in our research group with in silico techniques, this work describes the design and preparation of 31 novel structural analogues, with which different pharmacophoric features of the two hits have been explored and correlated with the inhibition of Chikungunya virus replication in cells. Structure-activity relationships were elucidated for the original scaffolds, and different novel antiviral compounds with EC50 values in the low micromolar range were identified. This work provides the foundation for further investigation of these promising novel structures as antiviral agents against Chikungunya virus.
Asunto(s)
Antivirales/síntesis química , Virus Chikungunya/fisiología , Diseño de Fármacos , Bibliotecas de Moléculas Pequeñas/química , Animales , Antivirales/química , Antivirales/farmacología , Sitios de Unión , Dominio Catalítico , Virus Chikungunya/enzimología , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Termodinámica , Proteínas Virales/química , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
PURPOSE: Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus that causes chikungunya fever in humans. The CHIKV non-structural protein 2 (nsP2) is a multifunctional protein that additionally modulates the host cell to dampen the innate immune response and inhibit other cellular processes. EXPERIMENTAL DESIGN: To further investigate the interactions of nsP2 with host cells, the protease domain of CHIKV nsP2 (nsP2-pro) is transfected into Hela cells, and differential protein expression is detected by 2D polyacrylamide gel electrophoresis. RESULTS: A total of 21 differentially regulated (six upregulated, 15 downregulated) spots are observed, of which five are identified by mass spectrometry. The downregulation of one of the identified proteins, ubiquitin-conjugating enzyme E2 L3 (UBE2L3) is confirmed by western blotting of both nsP2-pro transfection and CHIKV natural infection, and the downregulation of UBE2L3 is additionally shown to require an enzymatically active nsP2 protease domain. Transfection of full length UBE2L3 into HEK293T/17 cells prior to CHIKV infection reduce levels of infection and E protein expression but do not alter RNA genome levels. CONCLUSION: These results suggest that UBE2L3 is a cellular target of the CHIKV nsP2 protease, and this possibly mediates the pathogenesis of chikungunya fever.
Asunto(s)
Fiebre Chikungunya/metabolismo , Virus Chikungunya/enzimología , Cisteína Endopeptidasas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Replicación Viral , Fiebre Chikungunya/virología , Regulación hacia Abajo , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Transducción de Señal , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidoresRESUMEN
Chikungunya virus (CHIKV) is an important arboviral infectious agent in tropical and subtropical regions, often causing persistent and debilitating disease. The viral enzyme non-structural protein 4 (nsP4), as RNA-dependent RNA polymerase (RdRP), catalyzes the formation of negative-sense, genomic and subgenomic viral RNAs. Here we report a truncated nsP4 construct that is soluble, stable and purified recombinantly from Escherichia coli. Sequence analyses and homology modelling indicate that all necessary RdRP elements are included. Hydrogen/deuterium exchange with mass spectrometry was used to analyze solvent accessibility and flexibility of subdomains. Fluorophore-conjugated RNA ligands were designed and screened by using fluorescence anisotropy to select a suitable substrate for RdRP assays. Assay trials revealed that nsP4 core domain is conditionally active upon choice of detergent species, and carries out both primed extension and terminal adenylyltransferase activities. The polymerization assay can be further developed to screen for antiviral compounds in vitro.
Asunto(s)
Virus Chikungunya/enzimología , Detergentes/farmacología , ARN Polimerasa Dependiente del ARN/efectos de los fármacos , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/química , Antivirales , Dominio Catalítico , Fiebre Chikungunya/virología , Virus Chikungunya/genética , Clonación Molecular , Detergentes/química , Pruebas de Enzimas , Escherichia coli/genética , Polarización de Fluorescencia , Cinética , Nucleotidiltransferasas/genética , Proteínas con Motivos de Reconocimiento de ARN/química , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alineación de Secuencia , Análisis de Secuencia , Homología Estructural de Proteína , Proteínas no Estructurales Virales/genéticaRESUMEN
Dengue virus (DENV) and chikungunya virus (CHIKV) are reemergent arboviruses that are transmitted by mosquitoes of the Aedes genus. During the last several decades, these viruses have been responsible for millions of cases of infection and thousands of deaths worldwide. Therefore, several investigations were conducted over the past few years to find antiviral compounds for the treatment of DENV and CHIKV infections. One attractive strategy is the screening of compounds that target enzymes involved in the replication of both DENV and CHIKV. In this review, we describe advances in the evaluation of natural products targeting the enzymes involved in the replication of these viruses.
Asunto(s)
Antivirales/farmacología , Productos Biológicos/farmacología , Virus Chikungunya/efectos de los fármacos , Virus Chikungunya/enzimología , Virus del Dengue/efectos de los fármacos , Virus del Dengue/enzimología , Inhibidores Enzimáticos/farmacología , Antivirales/química , Productos Biológicos/química , Virus Chikungunya/fisiología , Virus del Dengue/fisiología , Inhibidores Enzimáticos/química , Estructura Molecular , Extractos Vegetales/química , Extractos Vegetales/farmacología , Proteínas Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacosRESUMEN
Chikungunya virus is a growing human pathogen transmitted by mosquito bite. It causes fever, chills, nausea, vomiting, joint pain, headache, and swelling in the joints. Its replication and propagation depend on the protease activity of the Chikungunya virus-nsP2 protein, which cleaves the nsP1234 polyprotein replication complex into individual functional units. The N-terminal segment of papain is structurally identical with the Chikungunya virus-nsP2 protease. Hence, molecular dynamics simulations were performed to compare molecular mechanism of these proteases. The Chikungunya virus-snP2 protease shows more conformational changes and adopts an alternate conformation. However, N-terminal segment of these two proteases has identical active site scaffold with the conserved catalytic diad. Hence, some of the non-peptide inhibitors of papain were used for induced fit docking at the active site of the nsP2 to assess the binding mode. In addition, the peptides that connect different domains/protein in Chikungunya virus poly-protein were also subjected for docking. The overall results suggest that the active site scaffold is the same in both the proteases and a possibility exists to experimentally assess the efficacy of some of the papain inhibitors to inhibit the Chikungunya virus-nsP2.
Asunto(s)
Virus Chikungunya/enzimología , Cisteína Endopeptidasas/química , Papaína/química , Proteínas Virales/química , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Fiebre Chikungunya/patología , Fiebre Chikungunya/virología , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Papaína/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Relación Estructura-Actividad , Termodinámica , Proteínas Virales/metabolismoRESUMEN
Chikungunya virus nsP2 replication protein is a cysteine protease, which cleaves the nonstructural nsP1234 polyprotein into functional replication components. The cleavage and processing of nsP1234 by nsP2 protease is essential for the replication and proliferation of the virus. Thus, ChikV nsP2 protease is a promising target for antiviral drug discovery. In this study, the crystal structure of the C-terminal domain of ChikV nsP2 protease (PDB ID: 4ZTB) was used for structure based identification and rational designing of peptidomimetic inhibitors against nsP2 protease. The interactions of the junction residues of nsP3/4 polyprotein in the active site of nsP2 protease have been mimicked to identify and design potential inhibitory molecules. Molecular docking of the nsP3/4 junction peptide in the active site of ChikV nsP2 protease provided the structural insight of the probable binding mode of nsP3/4 peptide and pigeonholed the molecular interactions critical for the substrate binding. Further, the shape and pharmacophoric properties of the viral nsP3/4 substrate peptide were taken into consideration and the mimetic molecules were identified and designed. The designed mimetic compounds were then analyzed by docking and their binding affinity was assessed by molecular dynamics simulations.
Asunto(s)
Antivirales/química , Virus Chikungunya/química , Cisteína Endopeptidasas/química , Peptidomiméticos/química , Inhibidores de Proteasas/química , Proteínas no Estructurales Virales/química , Secuencias de Aminoácidos , Dominio Catalítico , Virus Chikungunya/enzimología , Cristalografía por Rayos X , Diseño de Fármacos , Cinética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad , Especificidad por Sustrato , Termodinámica , Proteínas no Estructurales Virales/antagonistas & inhibidoresRESUMEN
Chikungunya virus (CHIKV; genus Alphavirus) is the causative agent of chikungunya fever. CHIKV replication can be inhibited by some broad-spectrum antiviral compounds; in contrast, there is very little information about compounds specifically inhibiting the enzymatic activities of CHIKV replication proteins. These proteins are translated in the form of a nonstructural (ns) P1234 polyprotein precursor from the CHIKV positive-strand RNA genome. Active forms of replicase enzymes are generated using the autoproteolytic activity of nsP2. The available three-dimensional (3D) structure of nsP2 protease has made it a target for in silico drug design; however, there is thus far little evidence that the designed compounds indeed inhibit the protease activity of nsP2 and/or suppress CHIKV replication. In this study, a set of 12 compounds, predicted to interact with the active center of nsP2 protease, was designed using target-based modeling. The majority of these compounds were shown to inhibit the ability of nsP2 to process recombinant protein and synthetic peptide substrates. Furthermore, all compounds found to be active in these cell-free assays also suppressed CHIKV replication in cell culture, the 50% effective concentration (EC50) of the most potent inhibitor being â¼1.5 µM. Analysis of stereoisomers of one compound revealed that inhibition of both the nsP2 protease activity and CHIKV replication depended on the conformation of the inhibitor. Combining the data obtained from different assays also indicates that some of the analyzed compounds may suppress CHIKV replication using more than one mechanism.
Asunto(s)
Antivirales/síntesis química , Virus Chikungunya/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , Genoma Viral , Poliproteínas/antagonistas & inhibidores , Inhibidores de Proteasas/síntesis química , ARN Helicasas/antagonistas & inhibidores , Animales , Ácidos Carboxílicos/síntesis química , Ácidos Carboxílicos/farmacología , Dominio Catalítico , Línea Celular , Virus Chikungunya/enzimología , Virus Chikungunya/genética , Virus Chikungunya/crecimiento & desarrollo , Cricetinae , Cristalografía por Rayos X , Ciclopropanos/síntesis química , Ciclopropanos/farmacología , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Diseño de Fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Poliproteínas/química , Poliproteínas/genética , Poliproteínas/metabolismo , Inhibidores de Proteasas/farmacología , ARN Helicasas/química , ARN Helicasas/genética , ARN Helicasas/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus that causes severe and debilitating disease symptoms. Alarmingly, transmission rates of CHIKV have increased dramatically over the last decade resulting in 1.7 million suspected cases in the Western hemisphere alone. There are currently no antivirals for treatment of CHIKV infection and novel anti-alphaviral compounds are badly needed. nsP1 is the alphavirus protein responsible for the methyltransferase and guanylyltransferase activities necessary for formation of the 5' type 0 cap structure added to newly formed viral RNA. Formation of this cap depends on nsP1 binding GTP and transferring a methylated GMP to nascent viral RNA. We have developed a fluorescence polarization-based assay that monitors displacement of a fluorescently-labeled GTP analog in real time. Determining the relative affinities of 15 GTP analogs for nsP1 GTP revealed important structural aspects of GTP that will inform identification of inhibitors able to outcompete GTP for the nsP1 binding site. Validation of the assay for HTS was completed and a secondary orthogonal assay that measures guanylation activity was developed in order to evaluate hits from future drug screens. This platform provides an avenue for identification of potent nsP1 inhibitors, which would potentially provide compounds capable of treating disease caused by CHIKV infection.
Asunto(s)
Fiebre Chikungunya/virología , Virus Chikungunya/enzimología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Metiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/antagonistas & inhibidores , Antivirales/química , Antivirales/farmacología , Fiebre Chikungunya/tratamiento farmacológico , Virus Chikungunya/efectos de los fármacos , Virus Chikungunya/metabolismo , Guanosina Trifosfato/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Metiltransferasas/metabolismo , Nucleotidiltransferasas/metabolismo , Caperuzas de ARN/metabolismoRESUMEN
Chikungunya virus (CHIKV) is a re-emerging alphavirus which causes severe and prolonged arthralgic febrile illness. The recent global spread of the virus and lack of approved therapeutic options makes it imperative to gain greater insight into the molecular mechanisms underlying CHIKV pathogenesis, in particular host factors recruited by the virus. In the current study, we identify sphingosine kinase 2 (SK2) as a CHIKV host factor co-localized with the viral replication complex (VRC) during infection. SK2 was demonstrated to co-localize with viral RNA and nonstructural proteins. Targeted impairment of SK2 expression or function significantly inhibited CHIKV infection. Furthermore, affinity purification-mass spectrometry studies revealed that SK2 associates with a number of proteins involved in cellular gene expression specifically during viral infection, suggesting a role in replication. Collectively these results identify SK2 as a novel CHIKV host factor.
Asunto(s)
Infecciones por Alphavirus/virología , Virus Chikungunya/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Infecciones por Alphavirus/prevención & control , Animales , Línea Celular , Células Cultivadas , Virus Chikungunya/genética , Regulación Enzimológica de la Expresión Génica , Regulación Viral de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Músculo Esquelético/citología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Interferencia de ARN , ARN Viral/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiologíaRESUMEN
Chikungunya virus is the pathogenic alphavirus that causes chikungunya fever in humans. In the last decade millions of cases have been reported around the world from Africa to Asia to the Americas. The alphavirus nsP2 protein is multifunctional and is considered to be pivotal to viral replication, as the nsP2 protease activity is critical for proteolytic processing of the viral polyprotein during replication. Classically the alphavirus nsP2 protease is thought to be papain-like with the enzyme reaction proceeding through a cysteine/histidine catalytic dyad. We performed structure-function studies on the chikungunya nsP2 protease and show that the enzyme is not papain-like. Characterization of the catalytic dyad cysteine residue enabled us to identify a nearby serine that is catalytically interchangeable with the dyad cysteine residue. The enzyme retains activity upon alanine replacement of either residue but a replacement of both cysteine and serine residues results in no detectable activity. Protein dynamics appears to allow the use of either the cysteine or the serine residue in catalysis. This switchable dyad residue has not been previously reported for alphavirus nsP2 proteases and would have a major impact on the nsP2 protease as an anti-viral target.
Asunto(s)
Virus Chikungunya/enzimología , Cisteína Endopeptidasas/química , Proteínas Virales/química , Secuencia de Aminoácidos , Dominio Catalítico , Estabilidad de Enzimas , Cinética , Modelos Moleculares , Papaína/química , Proteolisis , Serina/químicaRESUMEN
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. Recent outbreaks of CHIKV infections have been reported in Asia, Africa, and Europe. The symptoms of CHIKV infection include fever, headache, nausea, vomiting, myalgia, rash, and chronic persistent arthralgia. To date, no vaccines or selective antiviral drugs against this important emerging virus have been reported. In this study, the design, synthesis, and antiviral activity screening of new topographical peptidomimetics revealed three potential prototype agents 3a, 4b, and 5d showing 93-100% maximum inhibition of CHIKV replication in cell-based assay having EC90 of 8.76-9.57 µg/mL. Intensive molecular modeling studies including covalent docking, lowest unoccupied molecular orbital energies, and the atomic condensed Fukui functions calculations strongly suggested the covalent binding of peptidomimetics 3a, 4b, and 5d to CHIKV nsP2 protease leading to permanent enzyme inactivation via Michael adduct formation between α/ß-unsaturated ketone functionality in our designed peptidomimetics and active site catalytic cysteine1013. Furthermore, small molecular weight peptidomimetics 3a and 4b satisfied the Lipinski rule of five for drug-likeness and showed promising intestinal absorption and aqueous solubility via computational admet studies making them promising hits for further optimization.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Virus Chikungunya/efectos de los fármacos , Virus Chikungunya/enzimología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Animales , Antivirales/síntesis química , Dominio Catalítico , Fiebre Chikungunya/tratamiento farmacológico , Chlorocebus aethiops , Simulación por Computador , Diseño de Fármacos , Humanos , Absorción Intestinal , Ligandos , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Peptidomiméticos/síntesis química , Peptidomiméticos/química , Peptidomiméticos/farmacología , Inhibidores de Proteasas/síntesis química , Teoría Cuántica , Solubilidad , Relación Estructura-Actividad , Células VeroRESUMEN
Alphavirus nsP2 proteins are multifunctional and essential for viral replication. The protease role of nsP2 is critical for virus replication as only the virus protease activity is used for processing of the viral non-structural polypeptide. Chikungunya virus is an emerging disease problem that is becoming a world-wide health issue. We have generated purified recombinant chikungunya virus nsP2 proteins, both full length and a truncated protease domain from the C-terminus of the nsP2 protein. Enzyme characterization shows that the protease domain alone has different properties compared with the full length nsP2 protease. We also show chikungunya nsP2 protease possesses different substrate specificity to the canonical alphavirus nsP2 polyprotein cleavage specificity. Moreover, the chikungunya nsP2 also appears to differ from other alphavirus nsP2 in its distinctive ability to recognize small peptide substrates.