BioPROTAC-Mediated Degradation of HIV-1 Rev - Project Summary In 2023, approximately 40 million people globally, including 1.2 million Americans, were living with HIV- 1, which causes acquired immunodeficiency syndrome (AIDS). The current standard care involves daily small-molecule antiviral drugs, which reduce viral load and the probability of transmission but do not cure the infection. Adherence to treatment is challenging due to side effects and complex regimens, leading to drug resistance and treatment failure. One way to combat the emergence of drug resistance is to develop novel anti-HIV-1 agents against alternative targets. Since small-molecule anti-HIV-1 drugs are generally limited to targeting viral enzymes that possess hydrophobic binding pockets, we propose to target critical, non-enzymatic HIV-1 proteins with nanobodies. In this project, we aim to develop protein-based proteolysis targeting chimeras (bioPROTACs) to degrade HIV-1 Rev protein, which is critical for the HIV-1 lifecycle but does not contain any hydrophobic pocket for small molecules to bind. This approach is enabled by the recent development of a powerful intracellular protein delivery platform, membrane translocation domains (MTDs), in one of our laboratories. Specific Aim 1 is to generate and biochemically characterize a small family of Rev-degrading bioPROTACs by recombinantly fusing MTD4 (one of the most active MTDs), a previously reported Rev-binding nanobody (Nb190), and several commonly used E3 ubiquitin ligases. Specific Aim 2 is to test and validate the bioPROTACs from Aim 1 for the targeted degradation of HIV-1 Rev in live cells. The development of a bioPROTAC strategy targeting Rev or other essential viral proteins for HIV-1 therapy presents several compelling advantages in the fight against HIV. This approach opens a novel pathway for targeting viral proteins that are difficult to inhibit using traditional small molecules. By leveraging PROTACs, which harness the cell’s own machinery to degrade the target protein, the likelihood of resistance arising from viral escape mutants—common in conventional inhibition-based therapies—is significantly reduced. PROTACs can also be designed for high specificity, minimizing off-target effects and toxicities often associated with small-molecule drugs. Furthermore, combining PROTACs with traditional antiretroviral therapy could create a multifaceted strategy, enhancing long-term viral suppression.
