PROJECT SUMMARY / ABSTRACT Small molecules probes are often useful in studying the physiological function of proteins. They also serve as prototype drugs to validate new therapeutic targets. While efforts to develop small molecule inhibitors of viral enzymes have yielded invaluable mechanistic probes and effective drugs (e.g., raltegravir, ritonavir, zidovudine), these successes are outnumbered by the essential viral proteins whose biochemical function(s) are poorly understood and that are considered “undruggable.” In most cases, these proteins are not amenable to conventional structure-based inhibitor design and high-throughput screening. The Nef protein of human immunodeficiency virus 1 (HIV-1) is a prime example. As a multifunctional protein, Nef has been implicated as a critical factor in both viral infectivity and pathogenesis; however, it has been largely recalcitrant to conventional drug discovery approaches. Current Nef inhibitors were identified and optimized largely based on disruption of Nef's interaction with the SH3 domain of hematopoeietic cell kinase (Hck), but it remains unclear whether inhibiting this interaction is sufficient to address all of Nef's biological functions. As an alternative to functional inhibitors, we have begun developing small molecules that act by inducing degradation of the target protein. This is accomplished by synthesis of bifunctional molecules (known as “PROTACs,” “degronimids,” and “degraders”) that contain a ligand specific for the target of interest chemically conjugated via a linker to a ligand for a specific E3 ubiquitin ligase. We propose to apply this strategy to develop small molecules that induce degradation of HIV-1 Nef. To quickly advance this work, we will take advantage of published ligands of Nef as targeting ligands. Candidate Nef degraders will be designed and synthesized using medicinal chemistry and molecular modeling to vary the linkage site on the Nef ligand, the length and composition of the linker, and the E3 ubiquitin ligase ligand. These candidate Nef degraders will be optimized for interaction with Nef, interaction with the E3 ubiquitin ligase, formation of the ternary Nef-degarder-E3 ligase complex, and successful, proteasome-dependent degradation of Nef. Small molecules demonstrated to induce degradation of Nef in cellulo will be tested for antiviral activity against HIV-1 in cell culture. This work builds upon our recent success developing small molecule degraders of the hepatitis C virus NS3- 4A protease. Nef is a particularly attractive target for this approach because there is the potential for degradation- induced pharmacology to be differentiated and superior relative to current inhibitors that exhibit traditional, occupancy-driven pharmacology. Our goals in this exploratory work are to establish proof-of-concept for using small molecule-induced protein degradation to target Nef and to generate lead compounds for the development of Nef degraders that can be used to pro...