RESUMO
Daclatasvir (DCV) is a first-in-class hepatitis C virus (HCV) nonstructural 5A replication complex inhibitor (NS5A RCI) that is clinically effective in interferon-free combinations with direct-acting antivirals (DAAs) targeting alternate HCV proteins. Recently, we reported NS5A RCI combinations that enhance HCV inhibitory potential in vitro, defining a new class of HCV inhibitors termed NS5A synergists (J. Sun, D. R. O'Boyle II, R. A. Fridell, D. R. Langley, C. Wang, S. Roberts, P. Nower, B. M. Johnson F. Moulin, M. J. Nophsker, Y. Wang, M. Liu, K. Rigat, Y. Tu, P. Hewawasam, J. Kadow, N. A. Meanwell, M. Cockett, J. A. Lemm, M. Kramer, M. Belema, and M. Gao, Nature 527:245-248, 2015, doi:10.1038/nature15711). To extend the characterization of NS5A synergists, we tested new combinations of DCV and NS5A synergists against genotype (gt) 1 to 6 replicons and gt 1a, 2a, and 3a viruses. The kinetics of inhibition in HCV-infected cells treated with DCV, an NS5A synergist (NS5A-Syn), or a combination of DCV and NS5A-Syn were distinctive. Similar to activity observed clinically, DCV caused a multilog drop in HCV, followed by rebound due to the emergence of resistance. DCV-NS5A-Syn combinations were highly efficient at clearing cells of viruses, in line with the trend seen in replicon studies. The retreatment of resistant viruses that emerged using DCV monotherapy with DCV-NS5A-Syn resulted in a multilog drop and rebound in HCV similar to the initial decline and rebound observed with DCV alone on wild-type (WT) virus. A triple combination of DCV, NS5A-Syn, and a DAA targeting the NS3 or NS5B protein cleared the cells of viruses that are highly resistant to DCV. Our data support the observation that the cooperative interaction of DCV and NS5A-Syn potentiates both the genotype coverage and resistance barrier of DCV, offering an additional DAA option for combination therapy and tools for explorations of NS5A function.
Assuntos
Antivirais/farmacologia , Compostos de Bifenilo/farmacologia , Hepacivirus/efeitos dos fármacos , Imidazóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Carbamatos , Linhagem Celular , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Simulação de Acoplamento Molecular , Pirrolidinas , Replicon/efeitos dos fármacos , Replicon/genética , Valina/análogos & derivadosRESUMO
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 fármacosRESUMO
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 fármacosRESUMO
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/genéticaRESUMO
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/genéticaRESUMO
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 & derivadosRESUMO
The exceptional in vitro potency of the hepatitis C virus (HCV) NS5A inhibitor BMS-790052 has translated into an in vivo effect in proof-of-concept clinical trials. Although the 50% effective concentration (EC(50)) of the initial lead, the thiazolidinone BMS-824, was ~10 nM in the replicon assay, it underwent transformation to other inhibitory species after incubation in cell culture medium. The biological profile of BMS-824, including the EC(50), the drug concentration required to reduce cell growth by 50% (CC(50)), and the resistance profile, however, remained unchanged, triggering an investigation to identify the biologically active species. High-performance liquid chromatography (HPLC) biogram fractionation of a sample of BMS-824 incubated in medium revealed that the most active fractions could readily be separated from the parental compound and retained the biological profile of BMS-824. From mass spectral and nuclear magnetic resonance data, the active species was determined to be a dimer of BMS-824 derived from an intermolecular radical-mediated reaction of the parent compound. Based upon an analysis of the structural elements of the dimer deemed necessary for anti-HCV activity, the stilbene derivative BMS-346 was synthesized. This compound exhibited excellent anti-HCV activity and showed a resistance profile similar to that of BMS-824, with changes in compound sensitivity mapped to the N terminus of NS5A. The N terminus of NS5A has been crystallized as a dimer, complementing the symmetry of BMS-346 and allowing a potential mode of inhibition of NS5A to be discussed. Identification of the stable, active pharmacophore associated with these NS5A inhibitors provided the foundation for the design of more potent inhibitors with broad genotype inhibition. This culminated in the identification of BMS-790052, a compound that preserves the symmetry discovered with BMS-346.
Assuntos
Alanina/análogos & derivados , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Imidazóis/farmacologia , Tiazolidinas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Alanina/química , Alanina/farmacologia , Antivirais/química , Carbamatos , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Vírus da Diarreia Viral Bovina/crescimento & desenvolvimento , Descoberta de Drogas , Farmacorresistência Viral/genética , Hepacivirus/fisiologia , Humanos , Imidazóis/química , Espectrometria de Massas , Ressonância Magnética Nuclear Biomolecular , Prolina/análogos & derivados , Prolina/química , Prolina/farmacologia , Pirrolidinas , Estilbenos/química , Estilbenos/farmacologia , Tiazolidinas/química , Valina/análogos & derivados , Replicação Viral/efeitos dos fármacosRESUMO
Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multi-functional protein that is expressed in basally phosphorylated (p56) and in hyperphosphorylated (p58) forms. NS5A phosphorylation has been implicated in regulating multiple aspects of HCV replication. We recently reported the identification of a class of compounds that potently inhibit HCV RNA replication by targeting NS5A. Although the precise mechanism of inhibition of these compounds is not well understood, one activity that has been described is their ability to block expression of the hyperphosphorylated form of NS5A. Here, we report that an NS5A inhibitor impaired hyperphosphorylation without affecting basal phosphorylation at the C-terminal region of NS5A. This inhibitor activity did not require NS5A domains II and III and was distinct from that of a cellular kinase inhibitor that also blocked NS5A hyperphosphorylation, results that are consistent with an inhibitor-binding site within the N-terminal region of NS5A. In addition, we observed that an NS5A inhibitor promoted the accumulation of an HCV polyprotein intermediate, suggesting that inhibitor binding to NS5A may occur prior to the completion of polyprotein processing. Finally, we observed that NS5A p56 and p58 separated into different membrane fractions during discontinuous sucrose gradient centrifugation, consistent with these NS5A phosphoforms performing distinct replication functions. The p58 localization pattern was disrupted by an NS5A inhibitor. Collectively, our results suggest that NS5A inhibitors probably impact several aspects of HCV expression and regulation. These findings may help to explain the exceptional potency of this class of HCV replication complex inhibitors.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Fosforilação , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismoRESUMO
Using a cell-based replicon screen, we identified a class of compounds with a thiazolidinone core structure as inhibitors of hepatitis C virus (HCV) replication. The concentration of one such compound, BMS-824, that resulted in a 50% inhibition of HCV replicon replication was approximately 5 nM, with a therapeutic index of >10,000. The compound showed good specificity for HCV, as it was not active against several other RNA and DNA viruses. Replicon cells resistant to BMS-824 were isolated, and mutations were identified. A combination of amino acid substitutions of leucine to valine at residue 31 (L31V) and glutamine to leucine at residue 54 (Q54L) in NS5A conferred resistance to this chemotype, as did a single substitution of tyrosine to histidine at amino acid 93 (Y93H) in NS5A. To further explore the region(s) of NS5A involved in inhibitor sensitivity, genotype-specific NS5A inhibitors were used to evaluate a series of genotype 1a/1b hybrid replicons. Our results showed that, consistent with resistance mapping, the inhibitor sensitivity domain also mapped to the N terminus of NS5A, but it could be distinguished from the key resistance sites. In addition, we demonstrated that NS5A inhibitors, as well as an active-site inhibitor that specifically binds NS3 protease, could block the hyperphosphorylation of NS5A, which is believed to play an essential role in the viral life cycle. Clinical proof of concept has recently been achieved with derivatives of these NS5A inhibitors, indicating that small molecules targeting a nontraditional viral protein like NS5A, without any known enzymatic activity, can also have profound antiviral effects on HCV-infected subjects.
Assuntos
Hepacivirus/efeitos dos fármacos , Tiazolidinas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Substituição de Aminoácidos , Antivirais/química , Antivirais/farmacologia , Domínio Catalítico , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Genótipo , Humanos , Fosforilação/efeitos dos fármacos , Replicon/efeitos dos fármacos , Tiazolidinas/uso terapêutico , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
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 ElectrosprayRESUMO
A medicinal chemistry campaign that was conducted to address a potential genotoxic liability associated with an aniline-derived scaffold in a series of HCV NS5A inhibitors with dual GT-1a/-1b inhibitory activity is described. Anilides 3b and 3c were used as vehicles to explore structural modifications that retained antiviral potency while removing the potential for metabolism-based unmasking of the embedded aniline. This effort resulted in the discovery of a highly potent biarylimidazole chemotype that established a potency benchmark in replicon assays, particularly toward HCV GT-1a, a strain with significant clinical importance. Securing potent GT-1a activity in a chemotype class lacking overt structural liabilities was a critical milestone in the effort to realize the full clinical potential of targeting the HCV NS5A protein.
Assuntos
Antivirais/farmacologia , Genótipo , Hepacivirus/efeitos dos fármacos , Imidazóis/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Replicon/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/química , Antivirais/farmacocinética , Cristalografia por Raios X , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Hepacivirus/enzimologia , Hepacivirus/genética , Hepacivirus/fisiologia , Imidazóis/química , Imidazóis/farmacocinética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
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 & derivadosRESUMO
The hepatitis C virus NS5A protein is an established and clinically validated target for antiviral intervention by small molecules. Characterizations are presented of compounds identified as potent inhibitors of HCV replication to provide insight into structural elements that interact with the NS5A protein. UV-activated cross linking and affinity isolation was performed with one series to probe the physical interaction between the inhibitors and the NS5A protein expressed in HCV replicon cells. Resistance mapping with the second series was used to determine the functional impact of specific inhibitor subdomains on the interaction with NS5A. The data provide evidence for a direct high-affinity interaction between these inhibitors and the NS5A protein, with the interaction dependent on inhibitor stereochemistry. The functional data supports a model of inhibition that implicates inhibitor binding by covalently combining distinct pharmacophores across an NS5A dimer interface to achieve maximal inhibition of HCV replication.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Farmacorresistência Viral , Humanos , Ligação ProteicaRESUMO
The iminothiazolidinone BMS-858 (2) was identified as a specific inhibitor of HCV replication in a genotype 1b replicon assay via a high-throughput screening campaign. A more potent analogue, BMS-824 (18), was used in resistance mapping studies, which revealed that inhibitory activity was related to disrupting the function of the HCV nonstructural protein 5A. Despite the development of coherent and interpretable SAR, it was subsequently discovered that in DMSO 18 underwent an oxidation and structural rearrangement to afford the thiohydantoin 47, a compound with reduced HCV inhibitory activity. However, HPLC bioassay fractionation studies performed after incubation of 18 in assay media led to the identification of fractions containing a dimeric species 48 that exhibited potent antiviral activity. Excision of the key elements hypothesized to be responsible for antiviral activity based on SAR observations reduced 48 to a simplified, symmetrical, pharmacophore realized most effectively with the stilbene 55, a compound that demonstrated potent inhibition of HCV in a genotype 1b replicon with an EC50 = 86 pM.
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.