RESUMO
Hepatitis C virus (HCV) infection is a major risk factor for the development of chronic liver disease. The disease typically progresses from chronic HCV to fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and death. Chronic inflammation associated with HCV infection is implicated in cirrhosis and HCC, but the molecular players and signaling pathways contributing to these processes remain largely unknown. Interferon regulatory factor 5 (IRF5) is a molecule of interest in HCV-associated HCC because it has critical roles in virus-, Toll-like receptor (TLR)-, and IFN-induced signaling pathways. IRF5 is also a tumor suppressor, and its expression is dysregulated in several human cancers. Here, we present first evidence that IRF5 expression and signaling are modulated during HCV infection. Using HCV infection of human hepatocytes and cells with autonomously replicating HCV RNA, we found that levels of IRF5 mRNA and protein expression were down-regulated. Of note, reporter assays indicated that IRF5 re-expression inhibited HCV protein translation and RNA replication. Gene expression analysis revealed significant differences in the expression of cancer pathway mediators and autophagy proteins rather than in cytokines between IRF5- and empty vector-transfected HCV replicon cells. IRF5 re-expression induced apoptosis via loss in mitochondrial membrane potential, down-regulated autophagy, and inhibited hepatocyte cell migration/invasion. Analysis of clinical HCC specimens supports a pathologic role for IRF5 in HCV-induced HCC, as IRF5 expression was down-regulated in livers from HCV-positive versus HCV-negative HCC patients or healthy donor livers. These results identify IRF5 as an important suppressor of HCV replication and HCC pathogenesis.
Assuntos
Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C/patologia , Hepatite C Crônica/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/patologia , Cirrose Hepática/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Transdução de Sinais , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
Dengue virus (DENV) infection is a significant health threat to the global population with no therapeutic option. DENV NS5 RNA-dependent RNA polymerase (RdRp) is the key replicating protein of the virus and thus an attractive target for drug development. Herein, we report on the synthesis and biological evaluation of a series of hybrid thiazolidinone-thiadiazole derivatives as a new class of DENV-2 NS5 RdRp inhibitors. This yielded compounds 12 and 21 with IC50 values of 2.3 µM and 2.1 µM, respectively, as promising leads. Limited SAR analysis indicated 3-fluorobenzylidene as the optimal substituent at C5-position of the thiazolidinone core, whereas both 2-chlorophenyl and 3-fluorophenyl substituents were equally effective at C5-position of the 1,3,4-thiadiazole core. Biophysical characterization and molecular docking studies conferred the binding site of this scaffold on DENV NS5 polymerase. Binding mode of compound 21 in Thumb pocket-II of DENV-2 NS5 polymerase will form the basis for future structure-activity relationship optimization.
Assuntos
RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/ultraestrutura , Tiadiazóis/química , Tiazolidinedionas/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/ultraestrutura , Sítios de Ligação , Combinação de Medicamentos , Descoberta de Drogas , Modelos Químicos , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Tiadiazóis/administração & dosagem , Tiazolidinedionas/administração & dosagemRESUMO
Hepatitis C virus (HCV) infection leading to chronic hepatitis is a major factor in the causation of liver cirrhosis, hepatocellular carcinoma, and liver failure. This process may involve the interplay of various host cell factors, as well as the interaction of these factors with viral RNA and proteins. We report a novel strategy using a sequence-specific biotinylated peptide nucleic acid (PNA)-neamine conjugate targeted to HCV RNA for the in situ capture of subgenomic HCV (+) RNA, along with cellular and viral factors associated with it in MH14 host cells. Using this affinity capture system in conjunction with LC/MS/MS, we have identified 83 cellular factors and three viral proteins (NS5B, NS5A, and NS3-4a protease-helicase) associated with the viral genome. The capture was highly specific. These proteins were not scored with cured MH14 cells devoid of HCV replicons because of the absence of the target sequence in cells for the PNA-neamine probe and also because, unlike oligomeric DNA, cellular proteins have no affinity for PNA. The identified cellular factors belong to different functional groups, including signaling, oncogenic, chaperonin, transcriptional regulators, and RNA helicases as well as DEAD box proteins, ribosomal proteins, translational regulators/factors, and metabolic enzymes, that represent a diverse set of cellular factors associated with the HCV RNA genome. Small interfering RNA-mediated silencing of a diverse class of selected proteins in an HCV replicon cell line either enhanced or inhibited HCV replication/translation, suggesting that these cellular factors have regulatory roles in HCV replication.
Assuntos
Cromatografia de Afinidade/métodos , Regulação Viral da Expressão Gênica , Genoma Viral , Hepacivirus/genética , Hepatócitos/virologia , RNA Viral/genética , Biotinilação , Linhagem Celular Tumoral , Cromatografia Líquida , Framicetina/química , Hepacivirus/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Ácidos Nucleicos Peptídicos/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Viral/metabolismo , Replicon , Espectrometria de Massas em Tandem , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
In continuation of our efforts to develop new derivatives as hepatitis C virus (HCV) NS5B inhibitors, we synthesized novel 5-arylidene-4-thiazolidinones. The novel compounds 29-42, together with their synthetic precursors 22-28, were tested for HCV NS5B inhibitory activity; 12 of these compounds displayed IC50 values between 25.3 and 54.1 µM. Compound 33, an arylidene derivative, was found to be the most active compound in this series with an IC50 value of 25.3 µM. Molecular docking studies were performed on the thumb pocket-II of NS5B to postulate the binding mode for these compounds.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Tiazolidinas/síntese química , Tiazolidinas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Desenho Assistido por Computador , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Hepacivirus/enzimologia , Ensaios de Triagem em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Conformação Proteica , Relação Estrutura-Atividade , Tiazolidinas/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRP) is a crucial and unique component of the HCV RNA replication machinery and a validated target for drug discovery. Multiple crystal structures of NS5B inhibitor complexes have facilitated the identification of novel compound scaffolds through in silico analysis. With the goal of discovering new NS5B inhibitor leads, HCV NS5B crystal structures bound with inhibitors in the palm and thumb allosteric pockets in combination with ligands with known inhibitory potential were explored for a comparative pharmacophore analyses. The energy-based and 3D-QSAR-based pharmacophore models were validated using enrichment analysis, and the six models thus developed were employed for high-throughput virtual screening and docking to identify nonpeptidic leads. The hits derived at each stage were analyzed for diversity based on the six pharmacophore models, followed by molecular docking and filtering based on their interaction with amino acids in the NS5B allosteric pocket and 3D-QSAR predictions. The resulting 10 hits displaying diverse scaffold were then screened employing biochemical and cell-based NS5B and anti-HCV inhibition assays. Of these, two molecules H-5 and H-6 were the most promising, exhibiting IC50 values of 28.8 and 47.3 µM against NS5B polymerase and anti-HCV inhibition of 96% and 86% at 50 µM, respectively. The identified leads comprised of benzimidazole (H-5) and pyridine (H-6) scaffolds thus constitute prototypical molecules for further optimization and development as NS5B inhibitors.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hepacivirus/enzimologia , Simulação de Acoplamento Molecular , Relação Quantitativa Estrutura-Atividade , Interface Usuário-Computador , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/efeitos adversos , Antivirais/química , Antivirais/farmacocinética , Antivirais/farmacologia , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/farmacocinética , Hepacivirus/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Conformação Proteica , Termodinâmica , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismoRESUMO
Rho-associated protein kinase (ROCK) plays a key role in regulating a variety of cellular processes, and dysregulation of ROCK signaling or expression is implicated in numerous diseases and infections. ROCK proteins have therefore emerged as validated targets for therapeutic intervention in various pathophysiological conditions such as diabetes-related complications or hepatitis C-associated pathogenesis. In this study, we report on the design and identification of novel ROCK inhibitors utilizing energy based pharmacophores and shape-based approaches. The most potent compound 8 exhibited an IC50 value of 1.5 µM against ROCK kinase activity and inhibited methymercury-induced neurotoxicity of IMR-32 cells at GI50 value of 0.27 µM. Notably, differential scanning fluorometric analysis revealed that ROCK protein complexed with compound 8 with enhanced stability relative to Fasudil, a validated nanomolar range ROCK inhibitor. Furthermore, all compounds exhibited ≥96 µM CC50 (50% cytotoxicity) in Huh7 hepatoma cells, while 6 compounds displayed anti-HCV activity in HCV replicon cells. The identified lead thus constitutes a prototypical molecule for further optimization and development as anti-ROCK inhibitor.
Assuntos
Antineoplásicos/química , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas/química , Quinases Associadas a rho/química , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/química , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Fármacos , Hepacivirus/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/patologia , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Compostos de Metilmercúrio/antagonistas & inibidores , Compostos de Metilmercúrio/toxicidade , Conformação Molecular , Simulação de Dinâmica Molecular , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Termodinâmica , Interface Usuário-Computador , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genéticaRESUMO
We have used an ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA) to modify HCV replicase in order to identify the ATP binding site in the enzyme. FSBA inactivates HCV replicase activity in a concentration-dependent manner with a binding stoichiometry of 2 moles of FSBA per mole of enzyme. The enzyme activity is protected from FSBA in the presence of rNTP substrates or double-stranded RNA template primers that do not support ATP as the incoming nucleotide but not in the presence of polyrU.rA(26). HPLC analysis of tryptic peptides of FSBA-modified enzyme revealed the presence of two distinct peptides eluted at 23 and 36 min; these were absent in the control. Further we noted that both peptides were protected from FSBA modification in the presence of Mg·ATP. The LC/MS/MS analysis of the affinity-labeled tryptic peptides purified from HPLC, identified two major modification sites at positions 382 (Tyr), and 491 (Lys) and a minor site at position 38 (Tyr). To validate the functional significance of Tyr38, Tyr382, and Lys491 in catalysis, we individually substituted these residues by alanine and examined their ability to catalyze RdRp activity. We found that both Y382A and K491A mutants were significantly affected in their ability to catalyze RdRp activity while Y38A remained unaffected. We further observed that both Y382A and K491A mutants were not affected in their ability to bind template primer but were significantly affected in their ability to photo-cross-link ATP in the absence or presence of template primer.
Assuntos
Trifosfato de Adenosina/metabolismo , Adenosina/análogos & derivados , Marcadores de Afinidade/química , Hepacivirus/enzimologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Adenosina/química , Adenosina/metabolismo , Marcadores de Afinidade/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Hepacivirus/química , Hepacivirus/genética , Hepatite C/virologia , Humanos , Simulação de Acoplamento Molecular , Mutagênese , Nucleotídeos/metabolismo , Mutação Puntual , Ligação Proteica , RNA Polimerase Dependente de RNA/genética , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Earlier, we postulated that Gln91 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) stabilizes the side chain of Tyr183 via hydrogen bonding interaction between O(H) of Tyr183 and CO of Q91 [Harris, D., et al. (1998) Biochemistry 37, 9630-9640]. To test this hypothesis, we generated mutant derivatives of Gln91 and analyzed their biochemical properties. The efficiency of reverse transcription was severely impaired by nonconservative substitution of Gln with Ala, while conservative substitution of Gln with Asn resulted in an approximately 70% loss of activity, a value similar to that observed with the Y183F mutation. The loss of polymerase activity from both Q91A and Q91N was significantly improved by a Met to Val substitution at position 184. Curiously, the Q91N mutant exhibited stringency in discriminating between correct and incorrect nucleotides, suggesting its possible interaction with residues influencing the flexibility of the dNTP binding pocket. In contrast, both double mutants, Q91A/M184V and Q91N/M184V, are found to be as error prone as the wild-type enzyme. We propose a model that suggests that subtle structural changes in the region due to mutation at position 91 may influence the stability of the side chain of Tyr183 in the catalytic YMDD motif of the enzyme, thus altering the active site geometry that may interfere in substrate recognition.
Assuntos
Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Glutamina/química , Glutamina/metabolismo , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , Motivos de Aminoácidos/genética , Substituição de Aminoácidos/genética , Sítios de Ligação/genética , Catálise , Domínio Catalítico/genética , Cristalografia por Raios X/métodos , Desoxirribonucleotídeos/genética , Glutamina/genética , Transcriptase Reversa do HIV/genética , Humanos , Estabilidade Proteica , Estrutura Secundária de Proteína , Especificidade por Substrato/genética , Tirosina/metabolismoRESUMO
In continuation of our research program on new antitubercular agents, this article is a report of the synthesis of 97 various symmetrical, unsymmetrical, and N-substituted 1,4-dihydropyridines. The synthesized molecules were tested for their activity against M. tuberculosis H (37)Rv strain with rifampin as the standard drug. The percentage inhibition was found in the range 3-93%. In an effort to understand the relationship between structure and activity, 3D-QSAR studies were also carried out on a subset that is representative of the molecules synthesized. For the generation of the QSAR models, a training set of 35 diverse molecules representing the synthesized molecules was utilized. The molecules were aligned using the atom-fit technique. The CoMFA and CoMSIA models generated on the molecules aligned by the atom-fit method show a correlation coefficient (r (2)) of 0.98 and 0.95 with cross-validated r (2)(q (2)) of 0.56 and 0.62, respectively. The 3D-QSAR models were externally validated against a test set of 19 molecules (aligned previously with the training set) for which the predictive r(2)(r(r)(pred)) is recorded as 0.74 and 0.69 for the CoMFA and CoMSIA models, respectively. The models were checked for chance correlation through y-scrambling. The QSAR models revealed the importance of the conformational flexibility of the substituents in antitubercular activity.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Di-Hidropiridinas/química , Di-Hidropiridinas/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Antituberculosos/síntese química , Simulação por Computador , Di-Hidropiridinas/síntese química , Modelos Moleculares , Relação Quantitativa Estrutura-AtividadeRESUMO
Multi-drug resistance to commonly used antitubercular drugs has propelled the development of new structural classes of antitubercular agents. This paper reports the synthesis, evaluation and 3D-QSAR analysis of a set of substituted N-phenyl-6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxamides as antitubercular agents. Substituted acetoacetanilides were reacted with various aromatic aldehydes and urea which yielded the tetrahydropyrimidine derivatives with a phenyl carbamoyl group at C5 position, and with various substitutions on the 4-phenyl and the N-phenyl aromatic rings. All compounds were screened for antitubercular activity against Mycobacterium tuberculosis H37Rv strain. The QSAR models were generated on a training set of 23 molecules. The molecules were aligned using the atom-fit and field-fit techniques. The CoMFA and CoMSIA models generated on the molecules aligned by the atom-fit method show a correlation coefficient (r2) of 0.98 and 0.95 with cross-validated r2(q2) of 0.68 and 0.58, respectively. The 3D-QSAR models were externally validated against a test set of 7 molecules for which the predictive r2 (r(pred)2) is recorded as 0.41 and 0.32 for the CoMFA and CoMSIA models, respectively. The CoMFA and CoMSIA contours helped to design some new molecules with improved activity.
Assuntos
Amidas/síntese química , Amidas/farmacologia , Antituberculosos/síntese química , Antituberculosos/farmacologia , Pirimidinas/química , Relação Quantitativa Estrutura-Atividade , Amidas/química , Antituberculosos/química , Avaliação Pré-Clínica de Medicamentos , Ligantes , Modelos Moleculares , Conformação Molecular , Mycobacterium tuberculosis/efeitos dos fármacosRESUMO
The ability of 41 1,4-diphenyl-1,4-dihydropyridine derivatives to inhibit the transport activity of P-glycoprotein were studied by flow cytometry in a multidrug-resistant human colon cancer cell line (COLO320) and in human mdr1 gene-transfected mouse lymphoma cells (L 5178 Y). The cytotoxicities of these compounds were also examined against human normal and cancer cell lines. The majority of the tested compounds proved to be effective inhibitors of rhodamine 123 outward transport, but their cytotoxicities were not negligible. Some dihydropyridine derivatives displayed cytotoxic activity against four human oral tumour cell lines and against three normal human oral cell lines. There was no clear-cut relationship between the multidrug-resistance activity or cytotoxicity and the chemical structures of the compounds. New ring substituents could prevent the oxidation of the ring of the aromatic compound.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/toxicidade , Di-Hidropiridinas/toxicidade , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Di-Hidropiridinas/farmacocinética , Humanos , Camundongos , Estrutura Molecular , TransfecçãoRESUMO
Hepatitis C virus (HCV) infection is a main cause of chronic liver disease, leading to liver cirrhosis and hepatocellular carcinoma (HCC). The objective of our research was to develop effective agents against viral replication. We have previously identiï¬ed the hydrazide-hydrazone scaffold as a promising hepatitis C virus (HCV) and hepatocelluler inhibitor. Herein we describe the design a number of 2',4'-difluoro-4-hydroxy-N'-(arylmethylidene) biphenyl-3-carbohydrazide (3a-t) as anti-HCV and anticancer agents. Results from evaluation of anti-HCV activity indicated that most of the synthesized hydrazone derivatives inhibited viral replication in the Huh7/Rep-Feo1b and Huh 7.5-FGR-JCI-Rluc2A reporter systems. Antiproliferative activities of increasing concentrations of 2',4'-difluoro-4-hydroxy-N'-(2-pyridyl methylidene)biphenyl-3-carbohydrazide 3b and diflunisal (2.5-40 µM) were assessed in liver cancer cell lines (Huh7, HepG2, Hep3B, Mahlavu, FOCUS and SNU-475) with sulforhodamine B assay for 72 h. Compound 3b with 2-pyridinyl group in the hydrazone part exhibited promising cytotoxic activity against all cell lines with IC50 values of 10, 10.34 16.21 4.74, 9.29 and 8.33 µM for Huh7, HepG2, Hep3B, Mahlavu, FOCUS and SNU-475 cells, respectively, and produced dramatic cell cycle arrest at SubG1/G0 phase as an indicator of apoptotic cell death induction.
Assuntos
Antineoplásicos/farmacologia , Antivirais/síntese química , Antivirais/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Diflunisal/análogos & derivados , Hepacivirus/efeitos dos fármacos , Hidrazinas/farmacologia , Hidrazonas/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Antineoplásicos/síntese química , Antineoplásicos/química , Antivirais/química , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Diflunisal/química , Diflunisal/farmacologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Hidrazinas/química , Hidrazonas/síntese química , Hidrazonas/química , Neoplasias Hepáticas/patologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The synthesis of a series of 5-carba-pterocarpens derivatives involving the cyclization of α-aryl-α-tetralones is described. Several compounds demonstrated potent activity and selectivity in vitro against HCV replicon reporter cells. The best profile in Huh7/Rep-Feo1b replicon reporter cells was observed with 2h (EC50 = 5.5 µM/SI = 20), while 2e was the most active in Huh7.5-FGR-JC1-Rluc2A replicon reporter cells (EC50 = 1.5 µM/SI = 70). Hydroxy groups at A- and D-rings are essential for anti-HCV activity, and substitutions in the A-ring at positions 3 and 4 resulted in enhanced activity of the compounds.
Assuntos
Antivirais/química , Antivirais/farmacologia , Guanidinas/química , Guanidinas/farmacologia , Hepacivirus/efeitos dos fármacos , Anisóis/síntese química , Anisóis/química , Anisóis/farmacologia , Antivirais/síntese química , Catálise , Linhagem Celular , Guanidinas/síntese química , Hepacivirus/genética , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Paládio/química , Replicon/efeitos dos fármacos , Tetralonas/síntese química , Tetralonas/química , Tetralonas/farmacologiaRESUMO
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
The synthesis of a novel series of 1-carba-isoflavanones through the α-arylation of α-tetralones is described. Several of these compounds demonstrated potent activity and selectivity in-vitro against HCV replicon reporter cells. Compound 10 (LQB-314) exhibited the best profile being active and selective in both replicon reporter cells (IC50 1.8 µM, SI > 111 and IC50 4.3 µM, SI > 46 in Huh7/Rep-Feo1b and Huh7.5-FGR-JC1-Rluc2A, respectively). Compound 3 (LQB-307) was the more potent and selective for Huh7.5-FGR-JC1-Rluc2A replicon reporter cells (IC50 1.5 µM, SI > 101.4).
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Desenho de Fármacos , Hepacivirus/efeitos dos fármacos , Tetralonas/síntese química , Tetralonas/farmacologia , Antivirais/efeitos adversos , Antivirais/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Genes Reporter , Genótipo , Hepacivirus/genética , Humanos , Luciferases de Renilla/genética , RNA Viral/genética , Replicon/efeitos dos fármacos , Replicon/genética , Tetralonas/efeitos adversos , Tetralonas/química , Transfecção , Replicação Viral/efeitos dos fármacosRESUMO
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
We report here the synthesis and mechanism of inhibition of pyrazolecarboxamide derivatives as a new class of HCV inhibitors. Compounds 6, 7, 8 and 16 inhibited the subgenomic HCV replicon 1b genotype at EC50 values between 5 and 8 µM and displayed an even higher potency against the infectious Jc1 HCV 2a genotype. Compound 6 exhibited an EC50 of 6.7 µM and selectivity index of 23 against HCV 1b, and reduced the RNA copies of the infectious Jc1 chimeric 2a clone by 82% at 7 µM. Evaluation of the mode of anti-HCV activity of 6 revealed that it suppressed HCV-induced COX-2 mRNA and protein expression, displaying an IC50 of 3.2 µM in COX-2 promoter-linked luciferase reporter assay. Conversely, the anti-HCV activity of 6 was abrogated upon over-expression of COX-2. These findings suggest that 6 as a representative of these pyrazolecarboxamides function as anti-HCV agents via targeting COX-2 at both the transcription and translation levels.
Assuntos
Antivirais/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/metabolismo , Hepacivirus/efeitos dos fármacos , Pirazóis/farmacologia , Pirróis/farmacologia , Antivirais/síntese química , Antivirais/química , Linhagem Celular , Inibidores de Ciclo-Oxigenase 2/síntese química , Inibidores de Ciclo-Oxigenase 2/química , Relação Dose-Resposta a Droga , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/química , Pirróis/síntese química , Pirróis/química , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
We have previously reported that the 6-aminoquinolone chemotype is a privileged scaffold to obtain antibacterial and antiviral agents. Herein we describe the design, synthesis, and enzymatic and cellular characterization of new 6-aminoquinolone derivatives as potent inhibitors of NS5B polymerase, an attractive and viable therapeutic target to develop safe anti-HCV agents. The 6-amino-7-[4-(2-pyridinyl)-1-piperazinyl]quinolone derivative 8 proved to be the best compound of this series, exhibiting an IC50 value of 0.069 µM against NS5B polymerase and selective antiviral effect (EC50 = 3.03 µM) coupled with the absence of any cytostatic effect (CC50 > 163 µM; SI > 54) in Huh 9-13 cells carrying a HCV genotype 1b, as measured by MTS assay. These results indicate that the 6-aminoquinolone scaffold is worthy of further investigation in the context of NS5B-targeted HCV drug discovery programs.
Assuntos
Aminoquinolinas/química , Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/química , Linhagem Celular , Inibidores Enzimáticos/química , Hepacivirus/enzimologia , Hepacivirus/genética , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Ressonância de Plasmônio de SuperfícieRESUMO
We have previously identified the pyrazolobenzothiazine scaffold as a promising chemotype against hepatitis C virus (HCV) NS5B polymerase, a validated and promising anti-HCV target. Herein we describe the design, synthesis, enzymatic, and cellular characterization of new pyrazolobenzothiazines as anti-HCV inhibitors. The binding site for a representative derivative was mapped to NS5B palm site I employing a mutant counterscreen assay, thus validating our previous in silico predictions. Derivative 2b proved to be the best selective anti-HCV derivative within the new series, exhibiting a IC50 of 7.9 µM against NS5B polymerase and antiviral effect (EC50 = 8.1 µM; EC90 = 23.3 µM) coupled with the absence of any antimetabolic effect (CC50 > 224 µM; SI > 28) in a cell based HCV replicon system assay. Significantly, microscopic analysis showed that, unlike the parent compounds, derivative 2b did not show any significant cell morphological alterations. Furthermore, since most of the pyrazolobenzothiazines tested altered cell morphology, this undesired aspect was further investigated by exploring possible perturbation of lipid metabolism during compound treatment.
Assuntos
Antivirais/síntese química , Pirazóis/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/farmacologia , Sítios de Ligação , Desenho de Fármacos , Hepacivirus/efeitos dos fármacos , Humanos , Pirazóis/farmacologia , Relação Estrutura-AtividadeRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Eclipta alba, traditionally known as bhringraj, has been used in Ayurvedic medicine for more than 1000 years in India. It is used for the treatment of infective hepatitis, liver cirrhosis, liver enlargement and other ailments of liver and gall bladder in India. The aim of this study was to evaluate anti-hepatitis C virus activity present in the Eclipta alba extract, perform bioassay based fractionation and identify anti-HCV phytochemicals from the active fractions. MATERIALS AND METHODS: Identification of active compounds was performed by bio-activity guided fractionation approach. Active isolates were separated by the combination of silica gel chromatography and preparative scale reverse phase HPLC. Eclipta alba extract and its isolates were examined for their ability to inhibit HCV replicase (HCV NS5B) activity in vitro and HCV replication in a cell culture system carrying replicating HCV subgenomic RNA replicon. The purified isolates were also examined for their binding affinity to HCV replicase by fluorescence quenching and their cytotoxicity by MTT assay. RESULTS: Eclipta alba extract strongly inhibited RNA dependent RNA polymerase (RdRp) activity of HCV replicase in vitro. In cell culture system, it effectively inhibited HCV replication which resulted in reduced HCV RNA titer and translation level of viral proteins. Bioassay-based fractionations of the extracts and purification of anti-HCV phytochemicals present in the active fractions have identified three compounds, wedelolactone, luteolin, and apigenin. These compounds exhibited dose dependent inhibition of HCV replicase in vitro, and anti-HCV replication activity in the cell culture system CONCLUSION: Eclipta alba extract and phytochemicals isolated from active fractions display anti-HCV activity in vitro and in cell culture system. The standardized Eclipta alba extract or its isolates can be used as an effective alternative and complementary treatment against HCV.