A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated CD4 downregulation.

Karimian Shamsabadi, Mohammad; Jia, Xiaofei

Karimian Shamsabadi, Mohammad; Jia, Xiaofei.

Afiliación

Karimian Shamsabadi M; Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, Dartmouth, Massachusetts, USA; The Biomedical Engineering and Biotechnology Program, University of Massachusetts Dartmouth, Dartmouth, Massachusetts, USA.
Jia X; Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, Dartmouth, Massachusetts, USA; The Biomedical Engineering and Biotechnology Program, University of Massachusetts Dartmouth, Dartmouth, Massachusetts, USA. Electronic address: xjia@umassd.edu.

J Biol Chem ; 300(8): 107528, 2024 Aug.

Article en En | MEDLINE | ID: mdl-38960038

ABSTRACT

ABSTRACT

Therapeutic inhibition of the viral protein Nef is an intriguing direction of antiretroviral drug discovery-it may revitalize immune mechanisms to target, and potentially clear, HIV-1-infected cells. Of the many cellular functions of Nef, the most conserved is the downregulation of surface CD4, which takes place through Nef hijacking the clathrin adaptor protein complex 2 (AP2)-dependent endocytosis. Our recent crystal structure has unraveled the molecular details of the CD4-Nef-AP2 interaction. Guided by the new structural knowledge, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef's activity on CD4. In our assay, AP2 is included along with Nef to facilitate the proper formation of the CD4-binding pocket and a fluorescently labeled CD4 cytoplasmic tail binds competently to the Nef-AP2 complex generating the desired polarization signal. The optimized assay has a good signal-to-noise ratio, excellent tolerance of dimethylsulfoxide and detergent, and the ability to detect competitive binding at the targeted Nef pocket, making it suitable for high-throughput screening.

Asunto(s)

Antígenos CD4; Regulación hacia Abajo; Polarización de Fluorescencia; VIH-1; Ensayos Analíticos de Alto Rendimiento; Productos del Gen nef del Virus de la Inmunodeficiencia Humana; Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo; Productos del Gen nef del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores; Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química; Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética; Antígenos CD4/metabolismo; Antígenos CD4/química; Humanos; Polarización de Fluorescencia/métodos; VIH-1/metabolismo; VIH-1/efectos de los fármacos; Ensayos Analíticos de Alto Rendimiento/métodos; Regulación hacia Abajo/efectos de los fármacos; Complejo 2 de Proteína Adaptadora/metabolismo; Fármacos Anti-VIH/farmacología; Fármacos Anti-VIH/química; Unión Proteica

Palabras clave

CD4; HIV; Nef; antiretroviral; fluorescence polarization; high-throughput screening

Texto completo

Añadir a Mi BVS

Imprimir

XML

PubMed Links

Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Antígenos CD4 / Regulación hacia Abajo / VIH-1 / Productos del Gen nef del Virus de la Inmunodeficiencia Humana / Ensayos Analíticos de Alto Rendimiento / Polarización de Fluorescencia Límite: Humans Idioma: En Revista: J Biol Chem Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos

Texto completo

Añadir a Mi BVS

Imprimir

XML

PubMed Links

Buscar en Google