RESUMO
The farnesoid X receptor (FXR) may play a crucial role in a number of metabolic diseases and, as such, could potentially serve as a target for the development of therapeutics as a treatment for those diseases. Previous work has described GW4064 as an FXR agonist with an interesting activity profile. This manuscript will describe the synthesis of novel analogs of GW4064 and the activity profile of those analogs.
Assuntos
Isoxazóis/química , Isoxazóis/farmacologia , Oxazolidinonas/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Avaliação Pré-Clínica de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Humanos , Modelos Moleculares , Estrutura Molecular , Oxazolidinonas/química , Relação Estrutura-AtividadeRESUMO
Two novel series of spirocyclic piperidine analogs appended to a pyrazolo[1,5-a]pyridine core were designed, synthesized and evaluated for their anti-HCV activity. A series of piperidine ketals afforded dispiro 6p which showed excellent in vitro anti-HCV activities (EC50 of 1.5nM and 1.2nM against genotype 1a and 1b replicons, respectively). A series of piperidine oxazolidinones afforded 27c which showed EC50's of 10.9nM and 6.1nM against 1a and 1b replicons, respectively. Both compounds 6p and 27c bound directly to non-structural NS4B protein in vitro (IC50's=10.2 and 30.4nM, respectively) and exhibited reduced potency in replicons containing resistance mutations encoding changes in the NS4B protein.
Assuntos
Antivirais/química , Antivirais/farmacologia , Hepacivirus/fisiologia , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Antivirais/síntese química , Desenho de Fármacos , Hepacivirus/efeitos dos fármacos , Hepacivirus/metabolismo , Humanos , Terapia de Alvo Molecular , Compostos de Espiro/síntese químicaRESUMO
GSK2485852 (referred to here as GSK5852) is a hepatitis C virus (HCV) NS5B polymerase inhibitor with 50% effective concentrations (EC50s) in the low nanomolar range in the genotype 1 and 2 subgenomic replicon system as well as the infectious HCV cell culture system. We have characterized the antiviral activity of GSK5852 using chimeric replicon systems with NS5B genes from additional genotypes as well as NS5B sequences from clinical isolates of patients infected with HCV of genotypes 1a and 1b. The inhibitory activity of GSK5852 remained unchanged in these intergenotypic and intragenotypic replicon systems. GSK5852 furthermore displays an excellent resistance profile and shows a <5-fold potency loss across the clinically important NS5B resistance mutations P495L, M423T, C316Y, and Y448H. Testing of a diverse mutant panel also revealed a lack of cross-resistance against known resistance mutations in other viral proteins. Data from both the newer 454 sequencing method and traditional population sequencing showed a pattern of mutations arising in the NS5B RNA-dependent RNA polymerase in replicon cells exposed to GSK5852. GSK5852 was more potent than HCV-796, an earlier inhibitor in this class, and showed greater reductions in HCV RNA during long-term treatment of replicons. GSK5852 is similar to HCV-796 in its activity against multiple genotypes, but its superior resistance profile suggests that it could be an attractive component of an all-oral regimen for treating HCV.
Assuntos
Antivirais/farmacologia , Ácidos Borônicos/farmacologia , Farmacorresistência Viral/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Replicon/efeitos dos fármacos , Sulfonamidas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Benzofuranos/farmacologia , Linhagem Celular , Farmacorresistência Viral/genética , Ensaios Enzimáticos , Genótipo , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Hepatite C Crônica/virologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cinética , Testes de Sensibilidade Microbiana , Tipagem Molecular , Mutação , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
We have synthesized and evaluated a series of novel HCV NS3 protease inhibitors with various P4 capping groups, which include urea, carbamate, methoxy-carboxamide, cyclic carbamate and amide, pyruvic amide, oxamate, oxalamide and cyanoguanidine. Most of these compounds are remarkably potent, exhibiting single-digit to sub-nanomolar activity in the enzyme assay and cell-based replicon assay. Selected compounds were also evaluated in the protease-inhibitor-resistant mutant transient replicon assay, and they were found to show quite different potency profiles against a panel of HCV protease-inhibitor-resistant mutants.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Amidas/química , Animais , Antivirais/química , Carbamatos/química , Relação Dose-Resposta a Droga , Farmacorresistência Viral/genética , Guanidinas/química , Hepacivirus/enzimologia , Hepacivirus/genética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ácido Oxâmico/química , Ratos , Inibidores de Serina Proteinase/química , Relação Estrutura-Atividade , Ureia/química , Proteínas não Estruturais Virais/metabolismoRESUMO
To further explore the optimum placement of the acid moiety in conformationally constrained analogs of GW 4064 1a, a series of stilbene replacements were prepared. The benzothiophene 1f and the indole 1g display the optimal orientation of the carboxylate for enhanced FXR agonist potency.
Assuntos
Isoxazóis/química , Isoxazóis/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Estilbenos/química , Estilbenos/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Conformação Molecular , Dados de Sequência Molecular , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
We have synthesized and evaluated a new series of acyclic P4-benzoxaborole-based HCV NS3 protease inhibitors. Structure-activity relationships were investigated, leading to the identification of compounds 5g and 17 with low nanomolar potency in the enzymatic and cell-based replicon assay. The linker-truncated compound 5j was found to exhibit improved absorption and oral bioavailability in rats, suggesting that further reduction of molecular weight and polar surface area could result in improved drug-like properties of this novel series.
Assuntos
Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Inibidores de Proteases/química , Inibidores de Proteases/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
To improve on the drug properties of GSK8062 1b, a series of heteroaryl bicyclic naphthalene replacements were prepared. The quinoline 1c was an equipotent FXR agonist with improved drug developability parameters relative to 1b. In addition, analog 1c lowered body weight gain and serum glucose in a DIO mouse model of diabetes.
Assuntos
Isoxazóis/química , Naftalenos/química , Quinolinas/química , Receptores Citoplasmáticos e Nucleares/agonistas , Animais , Sítios de Ligação , Glicemia/metabolismo , Cristalografia por Raios X , Diabetes Mellitus Experimental/metabolismo , Cães , Transferência Ressonante de Energia de Fluorescência , Humanos , Isoxazóis/síntese química , Isoxazóis/farmacocinética , Ligantes , Camundongos , Conformação Molecular , Estrutura Terciária de Proteína , Quinolinas/síntese química , Quinolinas/farmacocinética , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Aumento de Peso/efeitos dos fármacosRESUMO
HCV NS3/4A serine protease is essential for the replication of the HCV virus and has been a clinically validated target. A series of HCV NS3/4A protease inhibitors containing a novel acylsulfamoyl benzoxaborole moiety at the P1' region was synthesized and evaluated. The resulting P1-P3 and P2-P4 macrocyclic inhibitors exhibited sub-nanomolar potency in the enzymatic assay and low nanomolar activity in the cell-based replicon assay. The in vivo PK evaluations of selected compounds are also described.
Assuntos
Compostos de Boro/química , Hepacivirus/enzimologia , Inibidores de Proteases/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Animais , Compostos de Boro/síntese química , Compostos de Boro/farmacocinética , Domínio Catalítico , Hepacivirus/efeitos dos fármacos , Masculino , Modelos Moleculares , Inibidores de Proteases/química , Inibidores de Proteases/farmacocinética , Ratos , Ratos Sprague-Dawley , Replicação Viral/efeitos dos fármacosRESUMO
A novel series of P2-P4 macrocyclic HCV NS3/4A protease inhibitors with α-amino cyclic boronates as warheads at the P1 site was designed and synthesized. When compared to their linear analogs, these macrocyclic inhibitors exhibited a remarkable improvement in cell-based replicon activities, with compounds 9a and 9e reaching sub-micromolar potency in replicon assay. The SAR around α-amino cyclic boronates clearly established the influence of ring size, chirality and of the substitution pattern. Furthermore, X-ray structure of the co-crystal of inhibitor 9a and NS3 protease revealed that Ser-139 in the enzyme active site traps boron in the warhead region of 9a, thus establishing its mode of action.
Assuntos
Compostos de Boro/química , Ácidos Borônicos/química , Compostos Macrocíclicos/química , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Sítios de Ligação , Compostos de Boro/síntese química , Compostos de Boro/farmacologia , Domínio Catalítico , Cristalografia por Raios X , Hepacivirus/efeitos dos fármacos , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/farmacologia , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismoRESUMO
Starting from the known FXR agonist GW 4064 1a, a series of alternately 3,5-substituted isoxazoles was prepared. Several of these analogs were potent full FXR agonists. A subset of this series, with a tether between the isoxazole ring and the 3-position aryl substituent, were equipotent FXR agonists to GW 4064 1a, with the 2,6-dimethyl phenol analog 1t having greater FRET FXR potency than GW 4064 1a.
Assuntos
Isoxazóis/síntese química , Receptores Citoplasmáticos e Nucleares/agonistas , Animais , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Isoxazóis/química , Isoxazóis/farmacologia , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-AtividadeRESUMO
Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Replacement of the metabolically labile stilbene with either benzothiophene or naphthalene rings led to the identification of potent full agonists 2a and 2g.
Assuntos
Isoxazóis/química , Naftalenos/química , Receptores Citoplasmáticos e Nucleares/agonistas , Tiofenos/química , Animais , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Humanos , Isoxazóis/farmacologia , Naftalenos/farmacocinética , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-Atividade , Tiofenos/farmacocinéticaRESUMO
Antagonizing the action of the human nuclear xenobiotic receptor pregnane X receptor (PXR) may have important clinical implications in preventing drug-drug interactions and improving therapeutic efficacy. We provide evidence that a naturally occurring phytoestrogen, coumestrol, is an antagonist of the nuclear receptor PXR (NR1I2). In transient transfection assays, coumestrol was able to suppress the agonist effects of SR12813 on human PXR activity. PXR activity was assessed and correlated with effects on the metabolism of the anesthetic tribromoethanol and on gene expression in primary human hepatocytes. We found that coumestrol was able to suppress the effects of PXR agonists on the expression of the known PXR target genes, CYP3A4 and CYP2B6, in primary human hepatocytes as well as inhibit metabolism of tribromoethanol in humanized PXR mice. Coumestrol at concentrations above 1.0 microm competed in scintillation proximity assays with a labeled PXR agonist for binding to the ligand-binding cavity. However, mammalian two-hybrid assays and transient transcription data using ligand-binding-cavity mutant forms of PXR show that coumestrol also antagonizes coregulator recruitment. This effect is likely by binding to a surface outside the ligand-binding pocket. Taken together, these data imply that there are antagonist binding site(s) for coumestrol on the surface of PXR. These studies provide the basis for development of novel small molecule inhibitors of PXR with the ultimate goal of clinical applications toward preventing drug-drug interactions.
Assuntos
Cumestrol/farmacologia , Fitoestrógenos/farmacologia , Receptores de Esteroides/antagonistas & inibidores , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Linhagem Celular , Células Cultivadas , Receptor Constitutivo de Androstano , Cumestrol/química , Cumestrol/metabolismo , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Etanol/análogos & derivados , Etanol/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Imuno-Histoquímica , Espectrometria de Massas , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Coativador 1 de Receptor Nuclear , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Fitoestrógenos/química , Fitoestrógenos/metabolismo , Receptor de Pregnano X , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Development of an efficient and scalable synthesis of 6-formylindolo[3,2-b]carbazole (FICZ), a naturally-occurring aryl hydrocarbon receptor (AhR) ligand, allowed its biological and physical properties to be studied. FICZ was shown to be the most potent among a series of 6-substituted indolo[3,2-b]carbazoles for activation of AhR in cells. Photostability studies of FICZ revealed a non-enzymatic mechanism for its conversion to a biologically active quinone. These results further support the hypothesis that FICZ is a light-dependent hormone that links sun exposure to regulation of biological pathways in peripheral tissues.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carbazóis/síntese química , Receptores de Hidrocarboneto Arílico/metabolismo , Carbazóis/química , Carbazóis/farmacologia , Linhagem Celular , Estabilidade de Medicamentos , Humanos , Ligantes , Estrutura Molecular , Processos FotoquímicosRESUMO
We previously described the discovery of GSK5852 (1), a non-nucleoside polymerase (NS5B) inhibitor of hepatitis C virus (HCV), in which an N-benzyl boronic acid was essential for potent antiviral activity. Unfortunately, facile benzylic oxidation resulted in a short plasma half-life (5 h) in human volunteers, and a backup program was initiated to remove metabolic liabilities associated with 1. Herein, we describe second-generation NS5B inhibitors including GSK8175 (49), a sulfonamide- N-benzoxaborole analog with low in vivo clearance across preclinical species and broad-spectrum activity against HCV replicons. An X-ray structure of NS5B protein cocrystallized with 49 revealed unique protein-inhibitor interactions mediated by an extensive network of ordered water molecules and the first evidence of boronate complex formation within the binding pocket. In clinical studies, 49 displayed a 60-63 h half-life and a robust decrease in viral RNA levels in HCV-infected patients, thereby validating our hypothesis that reducing benzylic oxidation would improve human pharmacokinetics and lower efficacious doses relative to 1.
Assuntos
Antivirais/farmacologia , Ácidos Borônicos/farmacologia , Desenho de Fármacos , Hepacivirus/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Animais , Antivirais/química , Antivirais/farmacocinética , Ácidos Borônicos/química , Ácidos Borônicos/farmacocinética , Cristalografia por Raios X , Cães , Meia-Vida , Humanos , Macaca fascicularis , Camundongos , Estrutura Molecular , Inibidores da Síntese de Ácido Nucleico/química , Inibidores da Síntese de Ácido Nucleico/farmacocinética , RatosRESUMO
Starting from the known FXR agonist GW 4064 1a, a series of stilbene replacements were prepared. The 6-substituted 1-naphthoic acid 1b was an equipotent FXR agonist with improved developability parameters relative to 1a. Analog 1b also reduced the severity of cholestasis in the ANIT acute cholestatic rat model.
Assuntos
Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/farmacologia , Colestase/tratamento farmacológico , Proteínas de Ligação a DNA/agonistas , Isoxazóis/síntese química , Isoxazóis/farmacologia , Naftalenos/síntese química , Naftalenos/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/agonistas , Administração Oral , Animais , Ácidos Carboxílicos/química , Colestase/metabolismo , Colestase/patologia , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Modelos Animais de Doenças , Cães , Mucosa Gástrica/metabolismo , Haplorrinos , Isoxazóis/química , Camundongos , Conformação Molecular , Estrutura Molecular , Naftalenos/química , Ratos , Receptores Citoplasmáticos e Nucleares/química , Estômago/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Transcrição/químicaRESUMO
Tapinarof (GSK2894512) is a naturally derived topical treatment with demonstrated efficacy for patients with psoriasis and atopic dermatitis, although the biologic target and mechanism of action had been unknown. We demonstrate that the anti-inflammatory properties of tapinarof are mediated through activation of the aryl hydrocarbon receptor (AhR). We show that tapinarof binds and activates AhR in multiple cell types, including cells of the target tissue-human skin. In addition, tapinarof moderates proinflammatory cytokine expression in stimulated peripheral blood CD4+ T cells and ex vivo human skin, and impacts barrier gene expression in primary human keratinocytes; both of these processes are likely to be downstream of AhR activation based on current evidence. That the anti-inflammatory properties of tapinarof derive from AhR agonism is conclusively demonstrated using the mouse model of imiquimod-induced psoriasiform skin lesions. Topical treatment of AhR-sufficient mice with tapinarof leads to compound-driven reductions in erythema, epidermal thickening, and tissue cytokine levels. In contrast, tapinarof has no impact on imiquimod-induced skin inflammation in AhR-deficient mice. In summary, these studies identify tapinarof as an AhR agonist and confirm that its efficacy is dependent on AhR.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Dermatite Atópica/tratamento farmacológico , Inflamação/tratamento farmacológico , Psoríase/tratamento farmacológico , Receptores de Hidrocarboneto Arílico/agonistas , Resorcinóis/administração & dosagem , Estilbenos/administração & dosagem , Administração Tópica , Animais , Células Cultivadas , Citocinas/metabolismo , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Modelos Animais de Doenças , Humanos , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Psoríase/metabolismo , Psoríase/patologia , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologiaRESUMO
HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs) are part of the combination therapy currently used to treat HIV infection. The features of a new NNRTI drug for HIV treatment must include selective potent activity against both wild-type virus as well as against mutant virus that have been selected by use of current antiretroviral treatment regimens. Based on analogy with known HIV-1 NNRTI inhibitors and modeling studies utilizing the X-ray crystal structure of inhibitors bound in the HIV-1 RT, a series of substituted 2-quinolones was synthesized and evaluated as HIV-1 inhibitors.
Assuntos
Fármacos Anti-HIV/síntese química , Farmacorresistência Viral , Transcriptase Reversa do HIV/química , Quinolonas/síntese química , Inibidores da Transcriptase Reversa/síntese química , Alcinos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Benzoxazinas , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Ciclopropanos , Desenho de Fármacos , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , Modelos Moleculares , Estrutura Molecular , Mutação , Oxazinas/química , Quinolonas/química , Quinolonas/farmacologia , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Relação Estrutura-AtividadeRESUMO
GW4511, GW4751, and GW3011 showed IC50 values < or =2 nM against wild type HIV-1 and <10 nM against 16 mutants. They were particularly potent against NNRTI-resistant viruses containing Y181C-, K103N-, and K103N-based double mutations, which account for a significant proportion of the clinical failure of the three currently marketed NNRTIs. The antiviral data together with the favorable pharmacokinetic data of GW4511 suggested that these benzophenones possess attributes of a new NNRTI drug candidate.
Assuntos
Fármacos Anti-HIV/síntese química , Benzofenonas/síntese química , Transcriptase Reversa do HIV/antagonistas & inibidores , Inibidores da Transcriptase Reversa/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Benzofenonas/química , Benzofenonas/farmacologia , Linhagem Celular , Cristalografia por Raios X , Farmacorresistência Viral , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/genética , Humanos , Concentração Inibidora 50 , Mutação , Ligação Proteica , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Relação Estrutura-AtividadeRESUMO
Hepatitis C virus (HCV) assembles many host cellular proteins into unique membranous replication structures as a prerequisite for viral replication, and PI4KIIIα is an essential component of these replication organelles. RNA interference of PI4KIIIα results in a breakdown of this replication complex and cessation of HCV replication in Huh-7 cells. PI4KIIIα is a lipid kinase that interacts with the HCV nonstructural 5A protein (NS5A) and enriches the HCV replication complex with its product, phosphoinositol 4-phosphate (PI4P). Elevated levels of PI4P at the endoplasmic reticulum have been linked to HCV infection in the liver of HCV infected patients. We investigated if small molecule inhibitors of PI4KIIIα could inhibit HCV replication in vitro. The synthesis and structure-activity relationships associated with the biological inhibition of PI4KIIIα and HCV replication are described. These efforts led directly to identification of quinazolinone 28 that displays high selectivity for PI4KIIIα and potently inhibits HCV replication in vitro.
Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Animais , Antivirais/química , Descoberta de Drogas , Inibidores Enzimáticos/química , Hepacivirus/enzimologia , Hepacivirus/fisiologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Ratos , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
We describe the preclinical development and in vivo efficacy of a novel chemical series that inhibits hepatitis C virus replication via direct interaction with the viral nonstructural protein 4B (NS4B). Significant potency improvements were realized through isosteric modifications to our initial lead 1a. The temptation to improve antiviral activity while compromising physicochemical properties was tempered by the judicial use of ligand efficiency indices during lead optimization. In this manner, compound 1a was transformed into (+)-28a which possessed an improved antiviral profile with no increase in molecular weight and only a modest elevation in lipophilicity. Additionally, we employed a chimeric "humanized" mouse model of HCV infection to demonstrate for the first time that a small molecule with high in vitro affinity for NS4B can inhibit viral replication in vivo. This successful proof-of-concept study suggests that drugs targeting NS4B may represent a viable treatment option for curing HCV infection.