RESUMEN
Lassa fever virus (LASV) is endemic in West Africa and causes severe hemorrhagic fever and sensorineural hearing loss. We identified a small molecule inhibitor of LASV and used it to analyze the mechanism of entry. Using a photo-reactive analog that retains antiviral activity as a probe, we identified the inhibitor target as lysosome-associated membrane protein 1 (LAMP1), a host factor that binds to the LASV glycoprotein (GP) during infection. We found that LAMP1 binding to LASV GP is cholesterol-dependent, and that the inhibitor blocks infection by competing with cholesterol in LAMP1. Mutational analysis of a docking-based model identified a putative inhibitor binding site in the cholesterol-binding pocket within the LAMP1 domain that binds GP. These findings identify a critical role for cholesterol in LASV entry and a potential target for therapeutic intervention.
Asunto(s)
Colesterol/metabolismo , Virus Lassa/fisiología , Virus Lassa/patogenicidad , Proteínas de Membrana de los Lisosomas/fisiología , Receptores Virales/fisiología , Adamantano/análogos & derivados , Adamantano/química , Adamantano/farmacología , Animales , Antivirales/química , Antivirales/farmacología , Línea Celular , Chlorocebus aethiops , Células HEK293 , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Humanos , Fiebre de Lassa/etiología , Virus Lassa/efectos de los fármacos , Proteínas de Membrana de los Lisosomas/antagonistas & inhibidores , Proteínas de Membrana de los Lisosomas/genética , Modelos Moleculares , Mutación , Estabilidad Proteica , Estructura Terciaria de Proteína , Receptores Virales/antagonistas & inhibidores , Receptores Virales/genética , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/fisiología , Internalización del Virus/efectos de los fármacosRESUMEN
Previous studies identified an adamantane dipeptide piperazine 3.47 that inhibits Ebola virus (EBOV) infection by targeting the essential receptor Niemann-Pick C1 (NPC1). The physicochemical properties of 3.47 limit its potential for testing in vivo. Optimization by improving potency, reducing hydrophobicity, and replacing labile moieties identified 3.47 derivatives with improved in vitro ADME properties that are also highly active against EBOV infection, including when tested in the presence of 50% normal human serum (NHS). In addition, 3A4 was identified as the major cytochrome P450 isoform that metabolizes these compounds, and accordingly, mouse microsome stability was significantly improved when tested in the presence of the CYP3A4 inhibitor ritonavir that is approved for clinical use as a booster of anti-HIV drugs. Oral administration of the EBOV inhibitors with ritonavir resulted in a pharmacokinetic profile that supports a b.i.d. dosing regimen for efficacy studies in mice.
