RESUMEN
While new direct-acting antiviral agents for the treatment of chronic hepatitis C virus (HCV) infection have been approved, there is a continued need for novel antiviral agents that act on new targets and can be used in combination with current therapies to enhance efficacy and to restrict the emergence of drug-resistant viral variants. To this end, we have identified a novel class of small molecules, exemplified by PTC725, that target the nonstructural protein 4B (NS4B). PTC725 inhibited HCV 1b (Con1) replicons with a 50% effective concentration (EC50) of 1.7 nM and an EC90 of 9.6 nM and demonstrated a >1,000-fold selectivity window with respect to cytotoxicity. The compounds were fully active against HCV replicon mutants that are resistant to inhibitors of NS3 protease and NS5B polymerase. Replicons selected for resistance to PTC725 harbored amino acid substitutions F98L/C and V105M in NS4B. Anti-replicon activity of PTC725 was additive to synergistic in combination with alpha interferon or with inhibitors of HCV protease and polymerase. Immunofluorescence microscopy demonstrated that neither the HCV inhibitors nor the F98C substitution altered the subcellular localization of NS4B or NS5A in replicon cells. Oral dosing of PTC725 showed a favorable pharmacokinetic profile with high liver and plasma exposure in mice and rats. Modeling of dosing regimens in humans indicates that a once-per-day or twice-per-day oral dosing regimen is feasible. Overall, the preclinical data support the development of PTC725 for use in the treatment of chronic HCV infection.
Asunto(s)
Antivirales/metabolismo , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Indoles/farmacología , Sulfonamidas/farmacología , Proteínas no Estructurales Virales/metabolismo , Sustitución de Aminoácidos , Animales , Antivirales/farmacocinética , Línea Celular Tumoral , Farmacorresistencia Viral/genética , Sinergismo Farmacológico , Humanos , Indoles/metabolismo , Indoles/farmacocinética , Interferón-alfa/farmacología , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Ratas , Ratas Sprague-Dawley , Sulfonamidas/metabolismo , Sulfonamidas/farmacocinética , Proteínas no Estructurales Virales/genética , Replicación Viral/efectos de los fármacosRESUMEN
Truncated human coronavirus HCoV-229E spike glycoproteins containing amino acids 407 to 547 bound to purified, soluble virus receptor, human aminopeptidase N (hAPN). Soluble hAPN neutralized the infectivity of HCoV-229E virions at 37 degrees C, but not 4 degrees C. Binding of hAPN may therefore trigger conformational changes in the viral spike protein at 37 degrees C that facilitate virus entry.
Asunto(s)
Antígenos CD13/fisiología , Coronavirus Humano 229E/fisiología , Coronavirus Humano 229E/patogenicidad , Receptores Virales/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Antígenos CD13/química , Antígenos CD13/genética , Línea Celular , Coronavirus Humano 229E/genética , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Receptores de Coronavirus , Receptores Virales/química , Receptores Virales/genética , Eliminación de Secuencia , Solubilidad , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/fisiologíaRESUMEN
A competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of turkey coronavirus (TCV) antibodies. The cELISA utilized a recombinant baculovirus (Autographa californica nuclear polyhedrosis virus)-expressed TCV nucleocapsid (N) protein and biotin-labeled TCV N protein-specific monoclonal antibody. Sensitivity and specificity of the cELISA for detection of TCV antibodies were determined by comparison with the indirect fluorescent antibody test (IFAT) with 1269 reference, experimentally derived, and field-origin sera. Sera with discordant cELISA and IFAT results were further evaluated by western immunoblot analyses. The cELISA detected antibodies specific for TCV and infectious bronchitis virus, a closely related coronavirus, but did not detect antibodies specific for other avian viruses. A high degree of concordance was observed between the cELISA and IFAT; sensitivity and specificity of the cELISA relative to IFAT were 92.9% and 96.2%, respectively. Western immunoblot analyses provided additional evidence of cELISA specificity. The findings indicate that the cELISA is a rapid, sensitive, and specific serologic test for detection of TCV antibodies in turkeys.
