RESUMO
Research toward a next-generation HCV NS5A inhibitor has identified fluorobenzimidazole analogs that demonstrate potent, broad-genotype in vitro activity against HCV genotypes 1-6 replicons as well as HCV NS5A variants that are orders of magnitude less susceptible to inhibition by first-generation NS5A inhibitors in comparison to wild-type replicons. The fluorobenzimidazole inhibitors have improved pharmacokinetic properties in comparison to non-fluorinated benzimidazole analogs. Discovery of these inhibitors was facilitated by exploring SAR in a structurally simplified inhibitor series.
Assuntos
Antivirais/química , Antivirais/farmacologia , Benzimidazóis/química , Benzimidazóis/farmacologia , Hepacivirus/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/farmacocinética , Benzimidazóis/farmacocinética , Cães , Genótipo , Halogenação , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C/tratamento farmacológico , Humanos , Camundongos , Ratos , Replicon/efeitos dos fármacos , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Ombitasvir (ABT-267) is a hepatitis C virus (HCV) NS5A inhibitor with picomolar potency, pan-genotypic activity, and 50% effective concentrations (EC50s) of 0.82 to 19.3 pM against HCV genotypes 1 to 5 and 366 pM against genotype 6a. Ombitasvir retained these levels of potency against a panel of 69 genotype 1 to 6 chimeric replicons containing the NS5A gene derived from HCV-infected patients, despite the existence of natural sequence diversity within NS5A. In vitro resistance selection identified variants that conferred resistance to ombitasvir in the HCV NS5A gene at amino acid positions 28, 30, 31, 58, and 93 in genotypes 1 to 6. Ombitasvir was evaluated in vivo in a 3-day monotherapy study in 12 HCV genotype 1-infected patients at 5, 25, 50, or 200 mg dosed once daily. All patients in the study were HCV genotype 1a infected and were without preexisting resistant variants at baseline as determined by clonal sequencing. Decreases in HCV RNA up to 3.1 log10 IU/ml were observed. Resistance-associated variants at position 28, 30, or 93 in NS5A were detected in patient samples 48 hours after the first dose. Clonal sequencing analysis indicated that wild-type virus was largely suppressed by ombitasvir during 3-day monotherapy, and at doses higher than 5 mg, resistant variant M28V was also suppressed. Ombitasvir was well tolerated at all doses, and there were no serious or severe adverse events. These data support clinical development of ombitasvir in combination with inhibitors targeting HCV NS3/4A protease (ABT-450 with ritonavir) and HCV NS5B polymerase (ABT-333, dasabuvir) for the treatment of chronic HCV genotype 1 infection. (Study M12-116 is registered at ClinicalTrials.gov under registration no. NCT01181427.).
Assuntos
Anilidas/uso terapêutico , Antivirais/uso terapêutico , Carbamatos/uso terapêutico , Hepatite C/tratamento farmacológico , Linhagem Celular , Farmacorresistência Viral , Hepacivirus/efeitos dos fármacos , Humanos , Prolina , ValinaRESUMO
The Rho family GTPase Cdc42 is a critical regulator of cellular polarization from yeast to man. An analysis of its function in T cell activation is therefore of interest. This analysis poses two substantial challenges, similar to the analysis of many other critical T cell signaling intermediates. First, Cdc42 is required for development and cell survival, necessitating short-term manipulation of its activity. Second, Cdc42 is likely involved in multiple signaling pathways, requiring approaches to distinguish multiple roles. To address these challenges, we first determined and quantified spatio-temporal patterns of Cdc42 activity using live cell video fluorescence microscopy. This generates hypotheses at which times and locations Cdc42 might play possibly distinct roles. Second and as the focus of this manuscript, we employed protein transduction to manipulate Cdc42 activity for the generation of causality. Protein transduction allows such manipulation to be short-term, quantitative, and with multiple reagents. Here, we characterize uptake, retention, and subcellular distribution of protein transduction reagents. We describe how a more quantitative single cell analysis of Cdc42 activity provides superior distinction between experimental conditions. And we show how we have used dose responses of the protein transduction reagents to minimize side effects while retaining efficacy. We suggest that our strategy is an important complement to more established techniques to study protein function in primary T cells, in particular in the investigation of signaling intermediates that are essential for cell survival and regulate multiple aspects of T cell activation.
Assuntos
Proteínas/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Transporte Proteico/genética , Transporte Proteico/imunologia , Proteínas/metabolismo , Transdução Genética/métodosRESUMO
We describe here N-phenylpyrrolidine-based inhibitors of HCV NS5A with excellent potency, metabolic stability, and pharmacokinetics. Compounds with 2S,5S stereochemistry at the pyrrolidine ring provided improved genotype 1 (GT1) potency compared to the 2R,5R analogues. Furthermore, the attachment of substituents at the 4-position of the central N-phenyl group resulted in compounds with improved potency. Substitution with tert-butyl, as in compound 38 (ABT-267), provided compounds with low-picomolar EC50 values and superior pharmacokinetics. It was discovered that compound 38 was a pan-genotypic HCV inhibitor, with an EC50 range of 1.7-19.3 pM against GT1a, -1b, -2a, -2b, -3a, -4a, and -5a and 366 pM against GT6a. Compound 38 decreased HCV RNA up to 3.10 log10 IU/mL during 3-day monotherapy in treatment-naive HCV GT1-infected subjects and is currently in phase 3 clinical trials in combination with an NS3 protease inhibitor with ritonavir (r) (ABT-450/r) and an NS5B non-nucleoside polymerase inhibitor (ABT-333), with and without ribavirin.
Assuntos
Anilidas/farmacologia , Antivirais/farmacologia , Carbamatos/farmacologia , Genótipo , Hepacivirus/efeitos dos fármacos , Sulfonamidas/farmacologia , Uracila/análogos & derivados , Proteínas não Estruturais Virais/antagonistas & inibidores , 2-Naftilamina , Anilidas/química , Anilidas/farmacocinética , Animais , Antivirais/química , Antivirais/farmacocinética , Disponibilidade Biológica , Carbamatos/química , Carbamatos/farmacocinética , Linhagem Celular , Descoberta de Drogas , Hepacivirus/enzimologia , Humanos , Prolina , Ratos , Sulfonamidas/química , Sulfonamidas/farmacocinética , Uracila/química , Uracila/farmacocinética , Uracila/farmacologia , ValinaRESUMO
T cell polarization toward and within the cellular interface with an APC is critical for effective T cell activation. The Rho family GTPase Cdc42 is a central regulator of cellular polarization. Using live-cell imaging, we characterized the spatiotemporal patterns of Cdc42 activity and their physiological regulation. Using three independent means of experimental manipulation of Cdc42 activity, we established that Cdc42 is a critical regulator of T cell actin dynamics, TCR clustering, and cell cycle entry. Using quantification of three-dimensional data, we could relate distinct spatiotemporal patterns of Cdc42 activity to specific elements of T cell activation. This result suggests that Cdc42 activity in specific locations at specific times is most critical for its function in T cell activation.