Small Molecule Inhibitors of Nef-Mediated Immune Evasion

About This Grant

PROJECT SUMMARY: The currently available antiretrovirals can effectively control HIV-1 replication and thus prevent disease pro- gression to AIDS. Such antiretroviral treatment (ART), however, cannot eradicate the infection. Latently infect- ed cells persist through ART and, if ART is stopped, will lead to rebound of viral replications within weeks. In- fected individuals therefore need to take medications for the entire life span and consequently suffer from drug resistance as well as severe side effects including cardiovascular complications, metabolic disorders, and neu- rocognitive damage. Novel antiretrovirals that could eliminate HIV-1 infection and cure the disease is thus high- ly desired. It is believed that HIV cure could be possibly achieved through empowering host immune cells to recognize and kill HIV-1-infected cells. One significant barrier here, however, is that HIV-1 has evolved mech- anisms that enable infected cells to evade host immunity. A key player here is the HIV-1 Nef protein. Nef is a master regulator of HIV-1 immune evasion. By downregulating MHC-I from the surface of the host cell, Nef dis- rupts host cell’s antigen presentation, which then allows the infected cell to evade immune surveillance by host cytotoxic T lymphocytes (CTLs). Nef also downregulates CD4 from the cell surface; this benefits the infected cell in several ways including evading killing by natural killer (NK) cells through antibody-dependent cellular cytotoxicity (ADCC). Inhibition of these Nef functions should revitalize both CTLs and NK cells, empowering them to find and kill HIV-1 infected cells. This project is aimed at developing such inhibitors and thus overcome the barrier of viral immune evasion to facilitate the cure. Four in vitro assays have been developed and opti- mized for screening small molecule libraries to find inhibitors. Two assays, orthogonal to each other, are both capable of identifying inhibitors that can block Nef’s activity on MHC-I and CTLs (having two orthogonal assays here allows cross-validation of hits to ensure discovery of true inhibitors). Another two assays, also orthogonal to each other, are capable of finding inhibitors that can block Nef’s activity on CD4 and ADCC. These assays have been tested in a proof-of-concept screen against a medium-sized compound library. All four assays per- formed excellently, indicating their suitability for high-throughput screening. A hit compound has been found from this modest screen and was subsequently validated through concentration-dependent studies in vitro. Building on these promising results, we now propose to carry out—using our developed assays and the estab- lished workflow—large-scale library screens to identify inhibitors against the two targeted Nef functions (Aim 1). We will then use both in vitro and cell-based assays to characterize inhibitors and identify those with out- standing potency and efficacy (Aim 2). We will also solve high-resolution structures of inhibitor-Nef complexes and then use the structural information obtained to guide chemical derivatization of lead compounds (Aim 3). Upon completion of the project, we will obtain potent inhibitors of Nef-mediated immune evasion, ready for fur- ther development into real-world antiretrovirals to facilitate HIV cure.