Development of A Fluorescence Polarization Assay for High- Throughput Screening of Inhibitors against HIV-1 Nef-Mediated CD4 Downregulation

Abstract

Abstract Therapeutic inhibition of the viral protein Nef is an intriguing direction of antiretroviral drug discovery as 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 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 this 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 ensure the proper formation of the CD4-binding pocket, and a fluorophore labeled CD4 cytoplasmic tail binds competently to the Nef-AP2 complex. The optimized assay has a good signal-to-noise ratio, excellent tolerance of DMSO and detergent, and the ability to detect competitive inhibition, making it suitable for high-throughput screening.