RESUMO
Histone acetylation is a prominent epigenetic modification linked to the memory loss symptoms associated with neurodegenerative disease. The use of existing histone deacetylase inhibitor (HDACi) drugs for treatment is precluded by their weak blood-brain barrier (BBB) permeability and undesirable toxicity. Here, we address these shortcomings by developing a new class of disulfide-based compounds, inspired by the scaffold of the FDA-approved HDACi romidepsin (FK288). Our findings indicate that our novel compound MJM-1 increases the overall level of histone 3 (H3) acetylation in a prostate cancer cell line. In mice, MJM-1 injected intraperitoneally (i.p.) crossed the BBB and could be detected in the hippocampus, a brain region that mediates memory. Consistent with this finding, we found that the post-training i.p. administration of MJM-1 enhanced hippocampus-dependent spatial memory consolidation in male mice. Therefore, MJM-1 represents a potential lead for further optimization as a therapeutic strategy for ameliorating cognitive deficits in aging and neurodegenerative diseases.
Assuntos
Encéfalo/metabolismo , Inibidores de Histona Desacetilases/síntese química , Memória Espacial/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/farmacocinética , Inibidores de Histona Desacetilases/farmacologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Small molecules that bind the SARS-CoV-2 nonstructural protein 3 Mac1 domain in place of ADP-ribose could be useful as molecular probes or scaffolds for COVID-19 antiviral drug discovery because Mac1 has been linked to the ability of coronaviruses to evade cellular detection. A high-throughput assay based on differential scanning fluorimetry (DSF) was therefore optimized and used to identify possible Mac1 ligands in small libraries of drugs and drug-like compounds. Numerous promising compounds included nucleotides, steroids, ß-lactams, and benzimidazoles. The main drawback to this approach was that a high percentage of compounds in some libraries were found to influence the observed Mac1 melting temperature. To prioritize DSF screening hits, the shapes of the observed melting curves and initial assay fluorescence were examined, and the results were compared with virtual screens performed using AutoDock Vina. The molecular basis for alternate ligand binding was also examined by determining a structure of one of the hits, cyclic adenosine monophosphate, with atomic resolution.
Assuntos
Proteases Semelhantes à Papaína de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Ensaios de Triagem em Larga Escala/métodos , SARS-CoV-2/química , Antivirais/química , Antivirais/farmacologia , Sítios de Ligação , Proteases Semelhantes à Papaína de Coronavírus/genética , AMP Cíclico/química , AMP Cíclico/metabolismo , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , Domínios Proteicos , SARS-CoV-2/efeitos dos fármacosRESUMO
Small molecules that bind the SARS-CoV-2 non-structural protein 3 Mac1 domain in place of ADP-ribose could be useful as molecular probes or scaffolds for COVID-19 antiviral drug discovery because Mac1 has been linked to coronavirus' ability to evade cellular detection. A high-throughput assay based on differential scanning fluorimetry (DSF) was therefore optimized and used to identify possible Mac1 ligands in small libraries of drugs and drug-like compounds. Numerous promising compounds included nucleotides, steroids, beta-lactams, and benzimidazoles. The main drawback to this approach was that a high percentage of compounds in some libraries were found to influence the observed Mac1 melting temperature. To prioritize DSF screening hits, the shapes of the observed melting curves and initial assay fluorescence were examined, and the results were compared with virtual screens performed using Autodock VINA. The molecular basis for alternate ligand binding was also examined by determining a structure of one of the hits, cyclic adenosine monophosphate, with atomic resolution.
RESUMO
The virus that causes COVID-19, SARS-CoV-2, has a large RNA genome that encodes numerous proteins that might be targets for antiviral drugs. Some of these proteins, such as the RNA-dependent RNA polymerase, helicase, and main protease, are well conserved between SARS-CoV-2 and the original SARS virus, but several others are not. This study examines one of the proteins encoded by SARS-CoV-2 that is most different, a macrodomain of nonstructural protein 3 (nsp3). Although 26% of the amino acids in this SARS-CoV-2 macrodomain differ from those observed in other coronaviruses, biochemical and structural data reveal that the protein retains the ability to bind ADP-ribose, which is an important characteristic of beta coronaviruses and a potential therapeutic target.
Assuntos
Betacoronavirus/química , Proteínas não Estruturais Virais/química , Adenosina Difosfato Ribose/metabolismo , COVID-19 , Coronavirus/química , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Proteases Semelhantes à Papaína de Coronavírus , Cristalografia por Raios X , Sistemas de Liberação de Medicamentos , Humanos , Modelos Moleculares , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Domínios Proteicos , SARS-CoV-2 , Termodinâmica , Proteínas não Estruturais Virais/metabolismoRESUMO
Nudix proteins are members of a large family of homologous enzymes that hydrolyze nucleoside diphosphates linked to other compounds. The substrates for a subset of Nudix enzymes are all nucleotides linked to RNA, like the m7G mRNA caps and the more recently discovered NAD(H) RNA caps. However, the RNA affinity and nucleic acid specificity of Nudix proteins has not yet been explored in depth. In this study we designed new fluorescence-based assays to examine the interaction of purified recombinant E. coli NudC and human Nudt1 (aka MTH1) Nudt3, Nudt12, Nudt16, and Nudt20 (aka Dcp2). All Nudix proteins except Nudt1 and Nudt12 bound both RNA and DNA stoichiometrically with high affinity (dissociation constants in the nanomolar range) and no clear sequence specificity. In stark contrast, Nudt12 binds RNA but not similar DNA oligonucleotides. Nudt12 also bound RNAs with 5' NAD+ caps more tightly than those with NADH or m7G cap. NudC was similarly selective against m7G caps but did not differentiate between NAD+ and NADH capped RNA. Nudt3, Nudt16, and Nudt20 bound m7G capped RNA more tightly than RNA with NADH caps.
Assuntos
Enzimas Reparadoras do DNA/análise , DNA/química , Corantes Fluorescentes/química , Monoéster Fosfórico Hidrolases/análise , Pirofosfatases/análise , RNA/química , Sítios de Ligação , Escherichia coli/enzimologia , Humanos , Proteínas Recombinantes/análise , Nudix HidrolasesRESUMO
This review discusses new developments in Förster resonance energy transfer (FRET) microscopy and its application to cellular receptors. The method is based on the kinetic theory of FRET, which can be used to predict FRET not only in dimers, but also higher order oligomers of donor and acceptor fluorophores. Models based on such FRET predictions can be fit to observed FRET efficiency histograms (also called FRET spectrograms) and used to estimate intracellular binding constants, free energy values, and stoichiometries. These "FRET spectrometry" methods have been used to analyze oligomers formed by various receptors in cell signaling pathways, but until recently such studies were limited to receptors residing on the cell surface. To study complexes residing inside the cell, a technique called Quantitative Micro-Spectroscopic Imaging (Q-MSI) was developed. Q-MSI combines determination of quaternary structure from pixel-level apparent FRET spectrograms with the determination of both donor and acceptor concentrations at the organelle level. This is done by resolving and analyzing the spectrum of a third fluorescent marker, which does not participate in FRET. Q-MSI was first used to study the interaction of a class of cytoplasmic receptors that bind viral RNA and signal an antiviral response via complexes formed mainly on mitochondrial membranes. Q-MSI revealed previously unknown RNA mitochondrial receptor orientations, and the interaction between the viral RNA receptor called LGP2 with the RNA helicase encoded by the hepatitis virus. The biological importance of these new observations is discussed.
Assuntos
Sobrevivência Celular , Proteína DEAD-box 58/metabolismo , Transferência Ressonante de Energia de Fluorescência , Transdução de SinaisRESUMO
DECH-box proteins are a subset of DExH/D-box superfamily 2 helicases possessing a conserved Asp-Glu-Cys-His motif in their ATP binding site. The conserved His helps position the Asp and Glu residues, which coordinate the divalent metal cation that connects the protein to ATP and activate the water molecule needed for ATP hydrolysis, but the role of the Cys is still unclear. This study uses site-directed mutants of the model DECH-box helicase encoded by the hepatitis C virus (HCV) to examine the role of the Cys in helicase action. Proteins lacking a Cys unwound DNA less efficiently than wild-type proteins did. For example, at low protein concentrations, a helicase harboring a Gly instead of the DECH-box Cys unwound DNA more slowly than the wild-type helicase did, but at higher protein concentrations, the two proteins unwound DNA at similar rates. All HCV proteins analyzed had similar affinities for ATP and nucleic acids and hydrolyzed ATP in the presence of RNA at similar rates. However, in the absence of RNA, all proteins lacking a DECH-box cysteine hydrolyzed ATP 10-15 times faster with higher Km values, and lower apparent affinities for metal ions, compared to those observed with wild-type proteins. These differences were observed with proteins isolated from HCV genotypes 2a and 1b, suggesting that this role is conserved. These data suggest the helicase needs Cys292 to bind ATP in a state where ATP is not hydrolyzed until RNA binds.
Assuntos
Trifosfato de Adenosina/metabolismo , Cisteína/química , DNA/metabolismo , Hepacivirus/enzimologia , RNA/química , RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sítios de Ligação , Catálise , Cisteína/genética , Cisteína/metabolismo , DNA/química , Humanos , Hidrólise , Mutagênese Sítio-Dirigida , Mutação , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
Human cells detect RNA viruses through a set of helicases called RIG-I-like receptors (RLRs) that initiate the interferon response via a mitochondrial signaling complex. Many RNA viruses also encode helicases, which are sometimes covalently linked to proteases that cleave signaling proteins. One unresolved question is how RLRs interact with each other and with viral proteins in cells. This study examined the interactions among the hepatitis C virus (HCV) helicase and RLR helicases in live cells with quantitative microspectroscopic imaging (Q-MSI), a technique that determines FRET efficiency and subcellular donor and acceptor concentrations. HEK293T cells were transfected with various vector combinations to express cyan fluorescent protein (CFP) or YFP fused to either biologically active HCV helicase or one RLR (i.e. RIG-I, MDA5, or LGP2), expressed in the presence or absence of polyinosinic-polycytidylic acid (poly(I:C)), which elicits RLR accumulation at mitochondria. Q-MSI confirmed previously reported RLR interactions and revealed an interaction between HCV helicase and LGP2. Mitochondria in CFP-RIG-I:YFP-RIG-I cells, CFP-MDA5:YFP-MDA5 cells, and CFP-MDA5:YFP-LGP2 cells had higher FRET efficiencies in the presence of poly(I:C), indicating that RNA causes these proteins to accumulate at mitochondria in higher-order complexes than those formed in the absence of poly(I:C). However, mitochondria in CFP-LGP2:YFP-LGP2 cells had lower FRET signal in the presence of poly(I:C), suggesting that LGP2 oligomers disperse so that LGP2 can bind MDA5. Data support a new model where an LGP2-MDA5 oligomer shuttles NS3 to the mitochondria to block antiviral signaling.
Assuntos
Hepacivirus/enzimologia , Helicase IFIH1 Induzida por Interferon/metabolismo , Mitocôndrias/enzimologia , Modelos Biológicos , RNA Helicases/metabolismo , Transdução de Sinais , Proteínas não Estruturais Virais/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Hepacivirus/genética , Humanos , Helicase IFIH1 Induzida por Interferon/genética , Microscopia de Fluorescência/métodos , Mitocôndrias/genética , Poli I-C/farmacologia , RNA Helicases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas não Estruturais Virais/genéticaRESUMO
A ligand-based approach was applied to screen in silico a library of commercially available compounds, with the aim to find novel inhibitors of the HCV replication starting from the study of the viral NS3 helicase. Six structures were selected for evaluation in the HCV subgenomic replicon assay and one hit was found to inhibit the HCV replicon replication in the low micromolar range. A small series of new pyrrolone compounds was designed and synthesised, and novel structures were identified with improved antiviral activity.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Pirróis/farmacologia , Antivirais/química , Avaliação Pré-Clínica de Medicamentos , Hepacivirus/fisiologia , Pirróis/química , Replicação Viral/efeitos dos fármacosRESUMO
A structure-based virtual screening of commercial compounds was carried out on the HCV NS3 helicase structure, with the aim to identify novel inhibitors of HCV replication. Among a selection of 13 commercial structures, one compound was found to inhibit the subgenomic HCV replicon in the low micromolar range. Different series of new piperazine-based analogues were designed and synthesised, and among them, several novel structures exhibited antiviral activity in the HCV replicon assay. Some of the new compounds were also found to inhibit HCV NS3 helicase function in vitro, and one directly bound NS3 with a dissociation constant of 570 ± 270 nM.
Assuntos
Antivirais/química , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Piperazinas/química , Piperazinas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Linhagem Celular , Desenho de Fármacos , Hepacivirus/genética , Hepacivirus/fisiologia , Hepatite C/virologia , Humanos , Simulação de Acoplamento Molecular , Replicon/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
We report the discovery of the bicyclic octahydrocyclohepta[b]pyrrol-4(1H)-one scaffold as a new chemotype with anti-HCV activity on genotype 1b and 2a subgenomic replicons. The most potent compound 34 displayed EC50 values of 1.8 µM and 4.5 µM in genotype 1b and 2a, respectively, coupled with the absence of any antimetabolic effect (gt 1b SI = 112.4; gt 2a SI = 44.2) in a cell-based assay. Compound 34 did not target HCV NS5B, IRES, NS3 helicase, or selected host factors, and thus future work will involve the unique mechanism of action of these new antiviral compounds.
Assuntos
Antivirais/química , Antivirais/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Hepacivirus/efeitos dos fármacos , Linhagem Celular , Genótipo , Hepacivirus/genética , Hepacivirus/fisiologia , Humanos , Replicação Viral/efeitos dos fármacosRESUMO
A structure-based virtual screening technique was applied to the study of the HCV NS3 helicase, with the aim to find novel inhibitors of the HCV replication. A library of â¼450000 commercially available compounds was analysed in silico and 21 structures were selected for biological evaluation in the HCV replicon assay. One hit characterized by a substituted thieno-pyrimidine scaffold was found to inhibit the viral replication with an EC50 value in the sub-micromolar range and a good selectivity index. Different series of novel thieno-pyrimidine derivatives were designed and synthesised; several new structures showed antiviral activity in the low or sub-micromolar range.
Assuntos
Antivirais/química , Desenho Assistido por Computador , Pirimidinas/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Desenho de Fármacos , Hepacivirus/efeitos dos fármacos , Hepacivirus/crescimento & desenvolvimento , Hepatite C/tratamento farmacológico , Bibliotecas Digitais , Pirimidinas/química , Pirimidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
The hepatitis C virus (HCV) is a species of diverse genotypes that infect over 170 million people worldwide, causing chronic inflammation, cirrhosis and hepatocellular carcinoma. HCV genotype 3a is common in Brazil, and it is associated with a relatively poor response to current direct-acting antiviral therapies. The HCV NS3 protein cleaves part of the HCV polyprotein, and cellular antiviral proteins. It is therefore the target of several HCV drugs. In addition to its protease activity, NS3 is also an RNA helicase. Previously, HCV present in a relapse patient was found to harbor a mutation known to be lethal to HCV genotype 1b. The point mutation encodes the amino acid substitution W501R in the helicase RNA binding site. To examine how the W501R substitution affects NS3 helicase activity in a genotype 3a background, wild type and W501R genotype 3a NS3 alleles were sub-cloned, expressed in E. coli, and the recombinant proteins were purified and characterized. The impact of the W501R allele on genotype 2a and 3a subgenomic replicons was also analyzed. Assays monitoring helicase-catalyzed DNA and RNA unwinding revealed that the catalytic efficiency of wild type genotype 3a NS3 helicase was more than 600 times greater than the W501R protein. Other assays revealed that the W501R protein bound DNA less than 2 times weaker than wild type, and both proteins hydrolyzed ATP at similar rates. In Huh7.5 cells, both genotype 2a and 3a subgenomic HCV replicons harboring the W501R allele showed a severe defect in replication. Since the W501R allele is carried as a minor variant, its replication would therefore need to be attributed to the trans-complementation by other wild type quasispecies.
Assuntos
DNA Helicases/genética , Hepacivirus/genética , Hepatite C/patologia , Mutação de Sentido Incorreto , Proteínas não Estruturais Virais/genética , Antivirais/uso terapêutico , Sítios de Ligação/genética , Biocatálise , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , DNA Helicases/metabolismo , Escherichia coli/genética , Genótipo , Hepacivirus/enzimologia , Hepacivirus/fisiologia , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Interferon-alfa/uso terapêutico , Pessoa de Meia-Idade , RNA/genética , RNA/metabolismo , Proteínas Recombinantes/metabolismo , Recidiva , Ribavirina/uso terapêutico , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/genéticaRESUMO
The flavivirus nonstructural protein 3 (NS3) is a protease and helicase, and on the basis of its similarity to its homologue encoded by the hepatitis C virus (HCV), the flavivirus NS3 might be a promising drug target. Few flavivirus helicase inhibitors have been reported, in part, because few specific inhibitors have been identified when nucleic acid unwinding assays have been used to screen for helicase inhibitors. To explore the possibility that compounds inhibiting NS3-catalyzed ATP hydrolysis might function as antivirals even if they do not inhibit RNA unwinding in vitro, we designed a robust dengue virus (DENV) NS3 ATPase assay suitable for high-throughput screening. Members of two classes of inhibitory compounds were further tested in DENV helicase-catalyzed RNA unwinding assays, assays monitoring HCV helicase action, subgenomic DENV replicon assays, and cell viability assays and for their ability to inhibit West Nile virus (Kunjin subtype) replication in cells. The first class contained analogues of NIH molecular probe ML283, a benzothiazole oligomer derived from the dye primuline, and they also inhibited HCV helicase and DENV NS3-catalyzed RNA unwinding. The most intriguing ML283 analogue inhibited DENV NS3 with an IC50 value of 500 nM and was active against the DENV replicon. The second class contained specific DENV ATPase inhibitors that did not inhibit DENV RNA unwinding or reactions catalyzed by HCV helicase. Members of this class contained a 4-hydroxy-3-(5-methylfuran-2-carbonyl)-2H-pyrrol-5-one scaffold, and about 20 µM of the most potent pyrrolone inhibited both DENV replicons and West Nile virus replication in cells by 50%.
RESUMO
This study examines the specificity and mechanism of action of a recently reported hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase-protease inhibitor (HPI), and the interaction of HPI with the NS3 protease inhibitors telaprevir, boceprevir, danoprevir, and grazoprevir. HPI most effectively reduced cellular levels of subgenomic genotype 4a replicons, followed by genotypes 3a and 1b replicons. HPI had no effect on HCV genotype 2a or dengue virus replicon levels. Resistance evolved more slowly to HPI than telaprevir, and HPI inhibited telaprevir-resistant replicons. Molecular modeling and analysis of the ability of HPI to inhibit peptide hydrolysis catalyzed by a variety of wildtype and mutant NS3 proteins suggested that HPI forms a bridge between the NS3 RNA-binding cleft and an allosteric site previously shown to bind other protease inhibitors. In most combinations, the antiviral effect of HPI was additive with telaprevir and boceprevir, minor synergy was observed with danoprevir, and modest synergy was observed with grazoprevir.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Hepacivirus/química , Hepacivirus/metabolismo , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Modelos Moleculares , Terapia de Alvo Molecular , Oligopeptídeos/farmacologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismoRESUMO
Although all-oral direct-acting antiviral (DAA) therapy for hepatitis C virus (HCV) treatment is now a reality, today's HCV drugs are expensive, and more affordable drugs are still urgently needed. In this work, we report the identification of the 2-phenyl-4,5,6,7-Tetrahydro-1H-indole chemical scaffold that inhibits cellular replication of HCV genotype 1b and 2a subgenomic replicons. The anti-HCV genotype 1b and 2a profiling and effects on cell viability of a selected representative set of derivatives as well as their chemical synthesis are described herein. The most potent compound 39 displayed EC50 values of 7.9 and 2.6 µM in genotype 1b and 2a, respectively. Biochemical assays showed that derivative 39 had no effect on HCV NS5B polymerase, NS3 helicase, IRES mediated translation and selected host factors. Thus, future work will involve both the chemical optimization and target identification of 2-phenyl-4,5,6,7-Tetrahydro-1H-indoles as new anti-HCV agents.
Assuntos
Antivirais/farmacologia , Descoberta de Drogas , Hepacivirus/efeitos dos fármacos , Indóis/farmacologia , Antivirais/síntese química , Antivirais/química , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Indóis/síntese química , Indóis/química , Testes de Sensibilidade Microbiana , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Despite the great progress made in the last 10 years, alternative strategies might help improving definitive treatment options against hepatitis C virus infection. METHODS: With the aim of identifying novel inhibitors of the hepatitis C virus-1b replication targeting the viral NS3 helicase, the structures of previously reported symmetrical inhibitors of this enzyme were rationally modified, and according to docking-based studies, four novel scaffolds were selected for synthesis and evaluation in the hepatitis C virus-1b subgenomic replicon assay. RESULTS: Among the newly designed compounds, one new structural family was found to inhibit the hepatitis C virus-1b replication in the micromolar range. This scaffold was chosen for further exploration and different novel analogues were synthesised and evaluated. CONCLUSIONS: Different new inhibitors of the hepatitis C virus genotype 1b replication were identified. Some of the new compounds show mild inhibition of the NS3 helicase enzyme.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Fenilenodiaminas/síntese química , Fenilenodiaminas/química , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 µM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 µM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure-activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines.
Assuntos
Antivirais/farmacologia , Azóis/farmacologia , Hepatite C/virologia , Ácidos Nucleicos/metabolismo , Compostos Organosselênicos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Antioxidantes/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virologia , Proliferação de Células , Ensaio de Desvio de Mobilidade Eletroforética , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Hepatite C/metabolismo , Humanos , Hidrólise , Isoindóis , Cinética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virologia , Modelos Moleculares , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Hepatitis C (HCV) protein 3/4A (NS3/4A) is a bifunctional enzyme comprising two separate domains with protease and helicase activities, which are essential for viral propagation. Both domains are stable and have enzymatic activity separately, and the relevance and implications of having protease and helicase together as a single protein remains to be explored. Altered in vitro activities of isolated domains compared with the full-length NS3/4A protein suggest the existence of interdomain communication. The molecular mechanism and extent of this communication was investigated by probing the domain-domain interface observed in HCV NS3/4A crystal structures. We found in molecular dynamics simulations that the two domains of NS3/4A are dynamically coupled through the interface. Interestingly, mutations designed to disrupt this interface did not hinder the catalytic activities of either domain. In contrast, substrate cleavage and DNA unwinding by these mutants were mostly enhanced compared with the wild-type protein. Disrupting the interface did not significantly alter RNA unwinding activity; however, the full-length protein was more efficient in RNA unwinding than the isolated protease domain, suggesting a more direct role in RNA processing independent of the interface. Our findings suggest that HCV NS3/4A adopts an "extended" catalytically active conformation, and interface formation acts as a switch to regulate activity. We propose a unifying model connecting HCV NS3/4A conformational states and protease and helicase function, where interface formation and the dynamic interplay between the two enzymatic domains of HCV NS3/4A potentially modulate the protease and helicase activities in vivo.