RESUMO
NVR 3-778 is the first capsid assembly modulator (CAM) that has demonstrated antiviral activity in hepatitis B virus (HBV)-infected patients. NVR 3-778 inhibited the generation of infectious HBV DNA-containing virus particles with a mean antiviral 50% effective concentration (EC50) of 0.40 µM in HepG2.2.15 cells. The antiviral profile of NVR 3-778 indicates pan-genotypic antiviral activity and a lack of cross-resistance with nucleos(t)ide inhibitors of HBV replication. The combination of NVR 3-778 with nucleos(t)ide analogs in vitro resulted in additive or synergistic antiviral activity. Mutations within the hydrophobic pocket at the dimer-dimer interface of the core protein could confer resistance to NVR 3-778, which is consistent with the ability of the compound to bind to core and to induce capsid assembly. By targeting core, NVR 3-778 inhibits pregenomic RNA encapsidation, viral replication, and the production of HBV DNA- and HBV RNA-containing particles. NVR 3-778 also inhibited de novo infection and viral replication in primary human hepatocytes with EC50 values of 0.81 µM against HBV DNA and between 3.7 and 4.8 µM against the production of HBV antigens and intracellular HBV RNA. NVR 3-778 showed favorable pharmacokinetics and safety in animal species, allowing serum levels in excess of 100 µM to be achieved in mice and, thus, enabling efficacy studies in vivo The overall preclinical profile of NVR 3-778 predicts antiviral activity in vivo and supports its further evaluation for safety, pharmacokinetics, and antiviral activity in HBV-infected patients.
Assuntos
Antivirais/farmacologia , Benzamidas/farmacologia , Capsídeo/efeitos dos fármacos , DNA Viral/antagonistas & inibidores , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Piperidinas/farmacologia , RNA Viral/antagonistas & inibidores , Animais , Antígenos Virais/genética , Antígenos Virais/metabolismo , Antivirais/sangue , Antivirais/química , Antivirais/farmacocinética , Benzamidas/sangue , Benzamidas/química , Benzamidas/farmacocinética , Capsídeo/química , Capsídeo/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Células Hep G2 , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Piperidinas/sangue , Piperidinas/química , Piperidinas/farmacocinética , Cultura Primária de Células , RNA Viral/genética , RNA Viral/metabolismo , Proteínas do Core Viral/antagonistas & inibidores , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
This report describes the development, optimization, and implementation of a cell-based assay for high-throughput screening (HTS) to identify inhibitors to hepatitis C virus (HCV) replication. The assay is based on a HCV subgenomic RNA replicon that expresses beta-lactamase as a reporter for viral replication in enhanced Huh-7 cells. The drug targets in this assay are viral and cellular enzymes required for HCV replication, which are monitored by fluorescence resonance energy transfer using cell-permeable CCF4-AM as a beta-lactamase substrate. Digital image processing was used to visualize cells that harbor viral RNA and to optimize key assay development parameters such as transfection and culturing conditions to obtain a cell line which produced a robust assay window. Formatting the assay for compound screening was problematic due to small signal-to-background ratio and reduced potency to known HCV inhibitors. These technical difficulties were solved by using clavulanic acid, an irreversible inhibitor of beta-lactamase, to eliminate residual beta-lactamase activity after HCV replication was terminated, thus resulting in an improved assay window. HTS was carried out in 384-well microplate format, and the signal-to-background ratio and Z factor for the assay plates during the screen were approximately 13-fold and 0.5, respectively.