Despite advances in antiretroviral therapy, HIV-1 infection remains a global health challenge with no cure. One promising therapeutic target is the HIV-1 accessory protein Nef, which downregulates key immune receptor proteins to enable immune evasion. Specifically, Nef downregulates MHC-I by hijacking the Clathrin adaptor protein AP1, which impairs antigen presentation and allows infected cells to escape surveillance by the cytotoxic T lymphocytes (CTLs). In addition, Nef downregulates CD4 through interaction with Clathrin AP2, which allows infected cell to evade antibody-dependent cellular cytotoxicity (ADCC). Inhibiting these Nef functions has the potential to restore immune surveillance mechanisms, which may target and clear infected cells possibly leading to a cure. To detect such inhibitors, we have developed two fluorescence polarization (FP) assays, one monitoring the binding of the MHC-I cytoplasmic tail to the Nef-AP1 complex and one monitoring the binding of the CD4 cytoplasmic tail to the Nef-AP2 complex. Both assays have been optimized to have high signal-to-noise ratios, great tolerance to detergent and DMSO, and robust Z’-factors (∼0.6) indicating suitability for high-throughput screening. Using these assays, we performed a preliminary screening against a medium-sized compound library and successfully identified hits. Some hits were subsequently validated by concentration-response studies using the FP assays as well as by other orthogonal assays. Cellular tests of the hits are now underway. We plan to use these assays, and our established workflow, to screen more small molecule libraries to identify potent Nef inhibitors. Our ultimate goal is to develop novel Nef inhibitors into real-world antiretrovirals, which may contribute to the HIV-1 cure strategies.
Taklifi et al. (Sun,) studied this question.