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1.
Antimicrob Agents Chemother ; 67(5): e0169422, 2023 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-37039636

RESUMO

GSK878 is a newly described HIV-1 inhibitor that binds to the mature capsid (CA) hexamer in a pocket originally identified as the binding site of the well-studied CA inhibitor PF-74. Here, we show that GSK878 is highly potent, inhibiting an HIV-1 reporter virus in MT-2 cells with a mean 50% effective concentration (EC50) of 39 pM and inhibiting a panel of 48 chimeric viruses containing diverse CA sequences with a mean EC50 of 94 pM. CA mutations associated with reduced susceptibility to other inhibitors that bind to PF-74 binding site (L56I, M66I, Q67H, N74D, T107N, and Q67H/N74D) also reduced susceptibility to GSK878, with M66I, Q67H/N74D, and L56I having the greatest impact on antiviral activity. Amino acid substitutions in the CA cyclophilin A (CypA) binding loop (H87P and P90A), distal from the inhibitor binding site and associated with reduced CA-CypA binding, subtly, but reproducibly, also decreased GSK878 potency. Mechanism-of-action studies showed that GSK878 blocked both early (preintegration) and late (postintegration) steps in HIV-1 replication, with the early inhibition primarily determining the compound's antiviral activity. The early inhibition results from blocks to HIV-1 nuclear import and proviral integration and is associated with altered stability of the HIV-1 CA core.


Assuntos
Capsídeo , HIV-1 , Capsídeo/metabolismo , Antivirais/farmacologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Sítios de Ligação , Ciclofilina A/metabolismo
2.
Antimicrob Agents Chemother ; 58(9): 5155-63, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24936600

RESUMO

A comparison of the daclatasvir (DCV [BMS-790052]) resistance barrier on authentic or hybrid replicons containing NS5A from hepatitis C virus (HCV) genotypes 1 to 6 (GT-1 to -6) was completed using a replicon elimination assay. The data indicated that genotype 1b (GT-1b) has the highest relative resistance barrier and genotype 2a (GT-2a M31) has the lowest. The rank order of resistance barriers to DCV was 1b>4a≥5a>6a≅1a>2a JFH>3a>2a M31. Importantly, DCV in combination with a protease inhibitor (PI) eliminated GT-2a M31 replicon RNA at a clinically relevant concentration. Previously, we reported the antiviral activity and resistance profiles of DCV on HCV genotypes 1 to 4 evaluated in the replicon system. Here, we report the antiviral activity and resistance profiles of DCV against hybrid replicons with NS5A sequences derived from HCV GT-5a and GT-6a clinical isolates. DCV was effective against both GT-5a and -6a hybrid replicon cell lines (50% effective concentrations [EC50s] ranging from 3 to 7 pM for GT-5a, and 74 pM for GT-6a). Resistance selection identified amino acid substitutions in the N-terminal domain of NS5A. For GT-5a, L31F and L31V, alone or in combination with K56R, were the major resistance variants (EC50s ranging from 2 to 40 nM). In GT-6a, Q24H, L31M, P32L/S, and T58A/S were identified as resistance variants (EC50s ranging from 2 to 250 nM). The in vitro data suggest that DCV has the potential to be an effective agent for HCV genotypes 1 to 6 when used in combination therapy.


Assuntos
Farmacorresistência Viral/genética , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Imidazóis/farmacologia , Proteínas não Estruturais Virais/genética , Substituição de Aminoácidos/efeitos dos fármacos , Substituição de Aminoácidos/genética , Antivirais/farmacologia , Carbamatos , Farmacorresistência Viral/efeitos dos fármacos , Genótipo , Inibidores de Proteases/farmacologia , Pirrolidinas , Replicon/efeitos dos fármacos , Replicon/genética , Valina/análogos & derivados
3.
J Virol ; 87(4): 2320-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23236071

RESUMO

Hepatitis C virus NS5A has three structural domains, is required for RNA replication and virion assembly, and exists in hypo- and hyperphosphorylated forms. Accumulated data suggest that phosphorylation is involved in modulating NS5A functions. We performed a mutational analysis of highly conserved serine residues in the linker region between domains I and II of genotype 2a JFH1 NS5A. As with genotype 1b Con1 NS5A, we found that specific serine residues were important for efficient hyperphosphorylation of JFH1 NS5A. However, in contrast with Con1 replicons, we observed a positive correlation between hyperphosphorylation and JFH1 replicon replication. We previously demonstrated trans-complementation of a hyperphosphorylation-deficient, replication-defective JFH1 replicon. Our results suggested that the defective NS5A encoded by this replicon, while lacking one NS5A function, was capable of performing a separate replication function. In this report, we examined an additional set of replication-defective NS5A mutations in trans-complementation assays. While some behaved similarly to the S232I replicon, others displayed a unique trans-complementation phenotype, suggesting that NS5A trans-complementation can occur by two distinct modes. Moreover, we were able, for the first time, to demonstrate intragenic complementation of replication-defective NS5A alleles. Our results identified three complementation groups: group A, comprising mutations within NS5A domain I; group B, comprising mutations affecting serine residues important for hyperphosphorylation and a subset of the domain I mutations; and group C, comprising a single mutation within the C-terminal region of domain II. We postulate that these complementation groups define three distinct and genetically separable functions of NS5A in RNA replication.


Assuntos
Teste de Complementação Genética , Hepacivirus/fisiologia , Proteínas não Estruturais Virais/deficiência , Proteínas não Estruturais Virais/genética , Replicação Viral , Alelos , Linhagem Celular , Análise Mutacional de DNA , Hepacivirus/genética , Hepatócitos/virologia , Humanos , Fosforilação , Processamento de Proteína Pós-Traducional , Serina/genética , Serina/metabolismo
4.
Antimicrob Agents Chemother ; 57(1): 611-3, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23089758

RESUMO

The NS5A replication complex inhibitor daclatasvir (DCV; BMS-790052) inhibits hybrid replicons containing hepatitis C virus (HCV) genotype 3a (HCV3a) NS5A genes with 50% effective concentrations (EC(50)s) ranging from 120 to 870 pM. Selection studies with a hybrid HCV3a replicon identified NS5A residues 31 and 93 as sites for DCV-selected resistance. Our results support the potential use of DCV as a component in combination therapies for HCV3a chronic infection.


Assuntos
Substituição de Aminoácidos/genética , Antivirais/farmacologia , Hepacivirus/genética , Imidazóis/farmacologia , Vírus Reordenados/genética , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/efeitos dos fármacos , Aminoácidos/genética , Carbamatos , Genótipo , Hepacivirus/efeitos dos fármacos , Dados de Sequência Molecular , Pirrolidinas , Vírus Reordenados/efeitos dos fármacos , Replicon/efeitos dos fármacos , Valina/análogos & derivados
5.
Antimicrob Agents Chemother ; 57(5): 2054-65, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23403428

RESUMO

Daclatasvir (DCV; BMS-790052) is a hepatitis C virus (HCV) NS5A replication complex inhibitor (RCI) with picomolar to low nanomolar potency and broad genotypic coverage in vitro. Viral RNA declines have been observed in the clinic for both alpha interferon-ribavirin (IFN-α-RBV) and IFN-RBV-free regimens that include DCV. Follow-up specimens (up to 6 months) from selected subjects treated with DCV in 14-day monotherapy studies were analyzed for genotype and phenotype. Variants were detected by clonal sequencing in specimens from baseline and were readily detected by population sequencing following viral RNA breakthrough and posttreatment. The major amino acid substitutions generating resistance in vivo were at residues M28, Q30, L31, and Y93 for genotype 1a (GT-1a) and L31 and Y93 for GT-1b, similar to the resistance substitutions observed with the in vitro replicon system. The primary difference in the resistance patterns observed in vitro and in vivo was the increased complexity of linked variant combinations observed in clinical specimens. Changes in the percentage of individual variants were observed during follow-up; however, the overall percentage of variants in the total population persisted up to 6 months. Our results suggest that during the 14-day monotherapy, most wild-type virus was eradicated by DCV. After the end of DCV treatment, viral fitness, rather than DCV resistance, probably determines which viral variants emerge as dominant in populations.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Imidazóis/farmacologia , RNA Viral/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Substituição de Aminoácidos , Carbamatos , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , Farmacorresistência Viral/genética , Feminino , Genótipo , Hepacivirus/genética , Hepacivirus/crescimento & desenvolvimento , Hepatite C Crônica/virologia , Humanos , Masculino , Tipagem Molecular , Fenótipo , Pirrolidinas , RNA Viral/sangue , Valina/análogos & derivados , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
6.
Hepatology ; 55(6): 1692-9, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22234905

RESUMO

UNLABELLED: The influence of naturally occurring polymorphisms on the potency of the HCV nonstructural protein 5A (NS5A) replication complex inhibitor, BMS-790052, was investigated by evaluating hybrid replicons in which the entire NS5A coding region of genotype (GT) la and 1b laboratory (lab) strains (H77c and Con1) were replaced with the corresponding regions of specimens collected from 10 GT-1a- and 6 GT-1b-infected subjects. For baseline (BL) specimens, with no previously observed resistance variants identified by population sequencing, the median 50% effective concentration (EC(50) ) values for BMS-790052 were similar for the clinically derived and lab strains. A Q30R variant was observed at viral breakthrough (VBT) in one of the GT-1a-infected subjects. Because the lowest plasma exposure of BMS-790052 observed in this subject was 117 nM and the median 50% effective concentration value for a GT-1a H77c replicon containing a Q30R substitution is ~7 nM, a rigorous investigation was initiated to determine the basis for resistance. Three approaches were used: (1) replacement of the entire H77c NS5A or (2) replacement of the N-terminal region of NS5A, with sequence from BL and day 14, and (3) substitution of specific amino acids. A BL polymorphism (E62D) did not contribute resistance to BMS-790052; however, the linked variant, Q30R-E62D, conferred high-level resistance in vitro and is likely responsible for VBT in vivo. CONCLUSION: Our data show that a BL polymorphism with minimal effect on the anti-HCV effect of BMS-790052 can affect the emergence of resistance and significantly affect clinical outcome. This work establishes a clear, systematic approach to monitor resistance to NS5A inhibitors in the clinic.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Imidazóis/farmacologia , Polimorfismo Genético , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Carbamatos , Linhagem Celular , Genótipo , Humanos , Pirrolidinas , RNA Viral/biossíntese , Valina/análogos & derivados
7.
Bioorg Med Chem Lett ; 23(15): 4428-35, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23803586

RESUMO

The isoquinolinamide series of HCV NS5A inhibitors exemplified by compounds 2b and 2c provided the first dual genotype-1a/1b (GT-1a/1b) inhibitor class that demonstrated a significant improvement in potency toward GT-1a replicons compared to that of the initial program lead, stilbene 2a. Structure-activity relationship (SAR) studies that uncovered an alternate phenylglycine-based cap series that exhibit further improvements in virology profile, along with some insights into the pharmacophoric elements associated with the GT-1a potency, are described.


Assuntos
Antivirais/química , Glicina/análogos & derivados , Hepacivirus/enzimologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/síntese química , Antivirais/farmacocinética , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Genótipo , Glicina/síntese química , Glicina/química , Glicina/farmacocinética , Meia-Vida , Hepacivirus/genética , Hepacivirus/fisiologia , Microssomos Hepáticos/metabolismo , Conformação Molecular , Ratos , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
8.
Bioorg Med Chem Lett ; 23(3): 779-84, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23273521

RESUMO

In a recent disclosure, we described the discovery of dimeric, prolinamide-based NS5A replication complex inhibitors exhibiting excellent potency towards an HCV genotype 1b replicon. That disclosure dealt with the SAR exploration of the peripheral region of our lead chemotype, and herein is described the SAR uncovered from a complementary effort that focused on the central core region. From this effort, the contribution of the core region to the overall topology of the pharmacophore, primarily vector orientation and planarity, was determined, with a set of analogs exhibiting <10 nM EC(50) in a genotype 1b replicon assay.


Assuntos
Antivirais/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/farmacologia , Carbamatos , Hepacivirus/efeitos dos fármacos , Imidazóis/química , Imidazóis/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Prolina/análogos & derivados , Prolina/química , Prolina/farmacologia , Pirrolidinas , Relação Estrutura-Atividade , Valina/análogos & derivados , Proteínas não Estruturais Virais/química , Replicação Viral/efeitos dos fármacos
9.
J Med Chem ; 66(3): 1941-1954, 2023 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-36719971

RESUMO

Long-acting (LA) human immunodeficiency virus-1 (HIV-1) antiretroviral therapy characterized by a ≥1 month dosing interval offers significant advantages over daily oral therapy. However, the criteria for compounds that enter clinical development are high. Exceptional potency and low plasma clearance are required to meet dose size requirements; excellent chemical stability and/or crystalline form stability is required to meet formulation requirements, and new antivirals in HIV-1 therapy need to be largely free of side effects and drug-drug interactions. In view of these challenges, the discovery that capsid inhibitors comprising a quinazolinone core tolerate a wide range of structural modifications while maintaining picomolar potency against HIV-1 infection in vitro, are assembled efficiently in a multi-component reaction, and can be isolated in a stereochemically pure form is reported herein. The detailed characterization of a prototypical compound, GSK878, is presented, including an X-ray co-crystal structure and subcutaneous and intramuscular pharmacokinetic data in rats and dogs.


Assuntos
Fármacos Anti-HIV , Infecções por HIV , HIV-1 , Humanos , Ratos , Animais , Cães , Capsídeo , Proteínas do Capsídeo , Quinazolinonas/farmacologia , Quinazolinonas/uso terapêutico , Fármacos Anti-HIV/farmacocinética , Infecções por HIV/tratamento farmacológico
10.
Antimicrob Agents Chemother ; 56(3): 1350-8, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22214777

RESUMO

BMS-790052, a first-in-class hepatitis C virus (HCV) replication complex inhibitor, targeting nonstructural protein 5A (NS5A), displays picomolar to nanomolar potency against genotypes 1 to 5. This exceptional potency translated into robust anti-HCV activity in clinical studies with HCV genotype 1-infected subjects. To date, all BMS-790052-associated resistance mutations have mapped to the N-terminal region of NS5A. To further characterize the antiviral activity of BMS-790052, HCV replicon elimination and colony formation assays were performed. Replicon was cleared from genotype 1a and 1b replicon cells in a time- and dose-dependent manner. Elimination of the genotype 1a replicon required longer treatment durations and higher concentrations of BMS-790052 than those for the genotype1b replicon. Single amino acid substitutions that conferred relatively low levels of resistance were observed at early time points and at low doses. Higher doses and longer treatment durations yielded mutations that conferred greater levels of resistance, including linked amino acid substitutions. Replicon cells that survived inhibitor treatment remained fully sensitivity to pegylated alpha interferon (pegIFN-α) and other HCV inhibitors. Moreover, genotype 1a replicon elimination was markedly enhanced when pegIFN-α and BMS-790052 were combined. Resistant variants observed in this study were very similar to those observed in a multiple ascending dose (MAD) monotherapy trial of BMS-790052, validating replicon elimination studies as a model to predict clinical resistance. Insights gained from the in vitro anti-HCV activity and resistance profiles of BMS-790052 will be used to help guide the clinical development of this novel HCV inhibitor.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral/genética , Hepacivirus/efeitos dos fármacos , Pironas/administração & dosagem , Replicon/genética , Triazóis/administração & dosagem , Proteínas não Estruturais Virais/genética , Substituição de Aminoácidos , Linhagem Celular Tumoral , Farmacorresistência Viral/efeitos dos fármacos , Genótipo , Hepacivirus/fisiologia , Humanos , Concentração Inibidora 50 , Interferon-alfa/farmacologia , Fenótipo , Polietilenoglicóis/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes/farmacologia , Deleção de Sequência , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
11.
Antimicrob Agents Chemother ; 56(3): 1588-90, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22203595

RESUMO

The antiviral profile of BMS-790052, a potent hepatitis C virus (HCV) replication complex inhibitor targeting nonstructural protein NS5A, is well characterized for HCV genotype-1. Here, we report that BMS-790052 inhibits hybrid replicons containing HCV genotype-4 NS5A genes with 50% effective concentrations (EC(50)s) ranging from 7 to 13 pM. NS5A residue 30 was an important site for BMS-790052-selected resistance in the hybrid replicons. Our results support the potential of BMS-790052 as a valuable component of combination therapy for HCV genotype-4 chronic infection.


Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Imidazóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Carbamatos , Linhagem Celular , Farmacorresistência Viral , Genes Reporter , Genótipo , Hepacivirus/fisiologia , Humanos , Concentração Inibidora 50 , Luciferases/genética , Dados de Sequência Molecular , Pirrolidinas , Replicon/genética , Valina/análogos & derivados , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
12.
J Virol ; 85(14): 7312-20, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21593143

RESUMO

BMS-790052, targeting nonstructural protein 5A (NS5A), is the most potent hepatitis C virus (HCV) inhibitor described to date. It is highly effective against genotype 1 replicons and also displays robust genotype 1 anti-HCV activity in the clinic (M. Gao et al., Nature 465:96-100, 2010). BMS-790052 inhibits genotype 2a JFH1 replicon cells and cell culture infectious virus with 50% effective concentrations (EC(50)s) of 46.8 and 16.1 pM, respectively. Resistance selection studies with the JFH1 replicon and virus systems identified drug-induced mutations within the N-terminal region of NS5A. F28S, L31M, C92R, and Y93H were the major resistance mutations identified; the impact of these mutations on inhibitor sensitivity between the replicon and virus was very similar. The C92R and Y93H mutations negatively impacted fitness of the JFH1 virus. Second-site replacements at NS5A residue 30 (K30E/Q) restored efficient replication of the C92R viral variant, thus demonstrating a genetic interaction between NS5A residues 30 and 92. By using a trans-complementation assay with JFH1 replicons encoding inhibitor-sensitive and inhibitor-resistant NS5A proteins, we provide genetic evidence that NS5A performs the following two distinct functions in HCV RNA replication: a cis-acting function that likely occurs as part of the HCV replication complex and a trans-acting function that may occur outside the replication complex. The cis-acting function is likely performed by basally phosphorylated NS5A, while the trans-acting function likely requires hyperphosphorylation. Our data indicate that BMS-790052 blocks the cis-acting function of NS5A. Since BMS-790052 also impairs JFH1 NS5A hyperphosphorylation, it likely also blocks the trans-acting function.


Assuntos
Hepacivirus/genética , Imidazóis/farmacologia , RNA Viral/biossíntese , Proteínas não Estruturais Virais/fisiologia , Sequência de Bases , Carbamatos , Linhagem Celular Tumoral , Primers do DNA , Humanos , Mutação , Fosforilação , Pirrolidinas , Valina/análogos & derivados , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
13.
Hepatology ; 54(6): 1924-35, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21809362

RESUMO

UNLABELLED: The NS5A replication complex inhibitor, BMS-790052, inhibits hepatitis C virus (HCV) replication with picomolar potency in preclinical assays. This potency translated in vivo to a substantial antiviral effect in a single-ascending dose study and a 14-day multiple-ascending dose (MAD) monotherapy study. However, HCV RNA remained detectable in genotype 1a-infected patients at the end of the MAD study. In contrast, viral breakthrough was observed less often in patients infected with genotype 1b, and, in several patients, HCV RNA declined and remained below the level of quantitation (<25 IU/mL) through the duration of treatment. Here, we report on the results of the genotypic and phenotypic analyses of resistant variants in 24 genotype 1-infected patients who received BMS-790052 (1, 10, 30, 60, and 100 mg, once-daily or 30 mg twice-daily) in the 14-day MAD study. Sequence analysis was performed on viral complementary DNA isolated from serum specimens collected at baseline and days 1 (4, 8, and 12 hours), 2, 4, 7, and 14 postdosing. Analyses of the sequence variants (1) established a correlation between resistant variants emerging in vivo with BMS-790052 treatment and those observed in the in vitro replicon system (major substitutions at residues 28, 30, 31, and 93 for genotype 1a and residues 31 and 93 for genotype 1b); (2) determined the prevalence of variants at baseline and the emergence of resistance at different times during dosing; and (3) revealed the resistance profile and replicative ability (i.e., fitness) of the variants. CONCLUSION: Although resistance emerged during monotherapy with BMS-790052, the substantial anti-HCV effect of this compound makes it an excellent candidate for effective combination therapy.


Assuntos
Hepacivirus/genética , Hepatite C/tratamento farmacológico , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Proteínas não Estruturais Virais/fisiologia , Carbamatos , Método Duplo-Cego , Genótipo , Hepacivirus/efeitos dos fármacos , Humanos , Imidazóis/administração & dosagem , Fenótipo , Pirrolidinas , RNA Viral/efeitos dos fármacos , Replicon/efeitos dos fármacos , Valina/análogos & derivados , Proteínas não Estruturais Virais/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
14.
Bioorg Med Chem Lett ; 22(19): 6063-6, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-22959243

RESUMO

In a previous disclosure,(1) we reported the dimerization of an iminothiazolidinone to form 1, a contributor to the observed inhibition of HCV genotype 1b replicon activity. The dimer was isolated via bioassay-guided fractionation experiments and shown to be a potent inhibitor of genotype 1b HCV replication for which resistance mapped to the NS5A protein. The elements responsible for governing HCV inhibitory activity were successfully captured in the structurally simplified stilbene prolinamide 2. We describe herein the early SAR and profiling associated with stilbene prolinamides that culminated in the identification of analogs with PK properties sufficient to warrant continued commitment to this chemotype. These studies represent the key initial steps toward the discovery of daclatasvir (BMS-790052), a compound that has demonstrated clinical proof-of-concept for inhibiting the NS5A replication complex in the treatment of HCV infection.


Assuntos
Antivirais/farmacologia , Imidazóis/farmacologia , Prolina/análogos & derivados , Estilbenos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Antivirais/síntese química , Antivirais/química , Carbamatos , Relação Dose-Resposta a Droga , Imidazóis/síntese química , Imidazóis/química , Estrutura Molecular , Prolina/síntese química , Prolina/química , Prolina/farmacologia , Pirrolidinas , Estilbenos/síntese química , Estilbenos/química , Relação Estrutura-Atividade , Valina/análogos & derivados
15.
Antimicrob Agents Chemother ; 54(9): 3641-50, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20585111

RESUMO

BMS-790052 is the most potent hepatitis C virus (HCV) inhibitor reported to date, with 50% effective concentrations (EC(50)s) of < or = 50 pM against genotype 1 replicons. This exceptional potency translated to rapid viral load declines in a phase I clinical study. By targeting NS5A, BMS-790052 is distinct from most HCV inhibitors in clinical evaluation. As an initial step toward correlating in vitro and in vivo resistances, multiple cell lines and selective pressures were used to identify BMS-790052-resistant variants in genotype 1 replicons. Similarities and differences were observed between genotypes 1a and 1b. For genotype 1b, L31F/V, P32L, and Y93H/N were identified as primary resistance mutations. L23F, R30Q, and P58S acted as secondary resistance substitutions, enhancing the resistance of primary mutations but themselves not conferring resistance. For genotype 1a, more sites of resistance were identified, and substitutions at these sites (M28T, Q30E/H/R, L31M/V, P32L, and Y93C/H/N) conferred higher levels of resistance. For both subtypes, combining two resistance mutations markedly decreased inhibitor susceptibility. Selection studies with a 1b/1a hybrid replicon highlighted the importance of the NS5A N-terminal region in determining genotype-specific inhibitor responses. As single mutations, Q30E and Y93N in genotype 1a conferred the highest levels of resistance. For genotype 1b, BMS-790052 retained subnanomolar potency against all variants with single amino acid substitutions, suggesting that multiple mutations will likely be required for significant in vivo resistance in this genetic background. Importantly, BMS-790052-resistant variants remained fully sensitive to alpha interferon and small-molecule inhibitors of HCV protease and polymerase.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral/genética , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Imidazóis/farmacologia , Carbamatos , Linhagem Celular Tumoral , Genótipo , Humanos , Mutação/genética , Fenótipo , Pirrolidinas , Valina/análogos & derivados
16.
J Med Chem ; 57(5): 1976-94, 2014 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-23573957

RESUMO

A series of symmetrical E-stilbene prolinamides that originated from the library-synthesized lead 3 was studied with respect to HCV genotype 1a (G-1a) and genotype 1b (G-1b) replicon inhibition and selectivity against BVDV and cytotoxicity. SAR emerging from an examination of the prolinamide cap region revealed 11 to be a selective HCV NS5A inhibitor exhibiting submicromolar potency against both G-1a and G-1b replicons. Additional structural refinements resulted in the identification of 30 as a potent, dual G-1a/1b HCV NS5A inhibitor.


Assuntos
Antivirais/farmacologia , Genótipo , Hepacivirus/efeitos dos fármacos , Inibidores de Proteases/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Replicon/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Antivirais/química , Hepacivirus/genética , Hepacivirus/fisiologia , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Inibidores de Proteases/química , Espectrometria de Massas por Ionização por Electrospray
17.
J Virol Methods ; 193(1): 68-76, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23684844

RESUMO

A 96-well based replicon elimination and colony formation assay is presented for comparing the resistance barrier of the hepatitis C virus (HCV) NS5A replication complex inhibitor daclatasvir (DCV, BMS-790052) on three HCV genotypes (gts) in a proof of concept experimental protocol. The 96-well assay format provides both individual colony as well as population characterization and is readily applicable to other HCV direct-acting antiviral agents (DAAs). The assay provides an assessment of HCV replication levels over a 5log10 range by measuring a luciferase reporter resident in the HCV replicons. Individual colony status can be measured with a separate and compatible resazurin assay to assess relative host cell fitness following inhibitor treatments. The methods employed are non-toxic and leave intact isolatable colonies that can be used for phenotyping and genotyping. The utility of the assay is demonstrated by the identification and isolation of resistant variants as well as in the ranking of the relative resistance barrier for the replication complex inhibitor DCV for gts 1a, 1b and 2a. The format provides a quantitative ranking based upon luciferase activity and has the ability to monitor DAA resistance development over time for large numbers of compounds.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Hepacivirus/efeitos dos fármacos , Imidazóis/farmacologia , Virologia/métodos , Replicação Viral/efeitos dos fármacos , Carbamatos , Linhagem Celular , Genótipo , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Hepacivirus/fisiologia , Humanos , Luciferases/análise , Testes de Sensibilidade Microbiana , Pirrolidinas , Coloração e Rotulagem/métodos , Valina/análogos & derivados
18.
Antimicrob Agents Chemother ; 49(4): 1346-53, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15793110

RESUMO

The hepatitis C virus (HCV) replicon is a unique system for the development of a high-throughput screen (HTS), since the analysis of inhibitors requires the quantification of a decrease in a steady-state level of HCV RNA. HCV replicon replication is dependent on host cell factors, and any toxic effects may have a significant impact on HCV replicon replication. Therefore, determining the antiviral specificity of compounds presents a challenge for the identification of specific HCV inhibitors. Here we report the development of an HCV/bovine viral diarrhea virus (BVDV) dual replicon assay suitable for HTS to address these issues. The HCV reporter enzyme is the endogenous NS3 protease contained within the HCV genome, while the BVDV reporter enzyme is a luciferase enzyme engineered into the BVDV genome. The HTS uses a mixture of HCV and BVDV replicon cell lines placed in the same well of a 96-well plate and isolated in the same cell backgrounds (Huh-7). The format consists of three separate but compatible assays: the first quantitates the amount of cytotoxicity based upon the conversion of Alamar blue dye via cellular enzymes, while the second indirectly quantitates HCV replicon replication through measurement of the amount of NS3 protease activity present. The final assay measures the amount of luciferase activity present from the BVDV replicon cells, as an indicator of the specificity of the test compounds. This HCV/BVDV dual replicon assay provides a reliable format to determine the potency and specificity of HCV replicon inhibitors.


Assuntos
Antivirais/farmacologia , Vírus da Diarreia Viral Bovina/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Replicon/efeitos dos fármacos , Animais , Bovinos , Linhagem Celular , Linhagem Celular Tumoral , Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/fisiologia , Transferência Ressonante de Energia de Fluorescência , Hepacivirus/genética , Hepacivirus/fisiologia , Humanos , Luciferases/genética , Luciferases/metabolismo , Oxazinas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Xantenos/metabolismo
19.
Antimicrob Agents Chemother ; 46(12): 3817-22, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12435682

RESUMO

We investigated the mechanism of imipenem resistance in Enterobacter aerogenes strain 810, a clinical isolate from the United States for which the imipenem MIC was 16 micro g/ml and the meropenem MIC was 8 micro g/ml. An imipenem-susceptible revertant, strain 810-REV, was obtained after multiple passages of the strain on nonselective media. For the revertant, the imipenem MIC was /=128 micro g/ml), cefoxitin (>/=32 micro g/ml), and cefotaxime (>/=64 micro g/ml) remained the same. The beta-lactamase and porin profiles of the parent, the revertant, and carbapenem-susceptible type strain E. aerogenes ATCC 13048 were determined. Strains 810 and 810-REV each produced two beta-lactamases with pIs of 8.2 and 5.4. The beta-lactamase activities of the parent and revertant were similar, even after induction with subinhibitory concentrations of imipenem. While 810-REV produced two major outer membrane proteins of 42 and 39 kDa that corresponded to Escherichia coli porins OmpC and OmpF, respectively, the parent strain appeared to produce similar quantities of the 39-kDa protein (OmpF) but decreased amounts of the 42-kDa protein (OmpC). When the parent strain was grown in the presence of imipenem, the 42-kDa protein was not detectable by gel electrophoresis. However, Western blot analysis of the outer membrane proteins of the parent and revertant with polyclonal antisera raised to the OmpC and OmpF analogs of Klebsiella pneumoniae (anti-OmpK36 and anti-OmpK35, respectively) showed that strain 810 expressed only the 42-kDa OmpC analog in the absence of imipenem (the 39-kDa protein was not recognized by the anti-OmpF antisera) and neither the OmpC nor the OmpF analog in the presence of imipenem. The OmpC analog is apparently down-regulated in the presence of imipenem; however, 810-REV expressed both OmpC and OmpF analogs. These data suggest that imipenem resistance in E. aerogenes 810 is primarily associated with the lack of expression of the analogs of the OmpC (42-kDa) and OmpF (39-kDa) outer membrane proteins, which also results in decreased susceptibility to meropenem and cefepime.


Assuntos
Carbapenêmicos/farmacologia , Enterobacter aerogenes/efeitos dos fármacos , Porinas/genética , Farmacorresistência Bacteriana Múltipla , Enterobacter aerogenes/enzimologia , Porinas/isolamento & purificação
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