Investigation of ATV-Based Heterobifunctional Degraders to Combat Growing HIV-1 PR Inhibitor Resistance

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PROJECT SUMMARY/ABSTRACT Heterobifunctional targeted protein degraders (TPDs) offer advantages over traditional occupancy-based inhibitors including a unique catalytic mechanism of action (MOA), greater target selectivity, and a reduced probability for resistance development. TPDs are known to effectively target not only cytosolic proteins, but also nuclear and membrane-bound proteins. This therapeutic modality shows great promise for treating drug-resistant cancers and autoimmune diseases but has seen only limited application in antiviral drug discovery. HIV-1 protease (PR) is essential for proteolytic cleavage of Gag and Gag-Pol polyproteins and, thus, virion maturation and infectivity. Gag-Pol forms dynamic dimers at plasma membrane assembly/budding sites, allowing the embedded precursor PR to dimerize as a prerequisite for auto-processing. We identified precursor PR/Gag-Pol as a promising target for protease inhibitor (PI)-based TPDs. The widely used HIV-1 PI, Atazanavir (ATV), is amenable to conjugation with linkers and ubiquitin E3 ligase recruiting ligands to serve as prototype HIV-1 TPDs. Importantly, ATV inhibits activity of mature PR as well as autoprocessing of precursor PR/Gag-Pol during the assembly and budding processes. The OBJECTIVE of this study is to show proof-of-concept that HIV-1 Gag- Pol assembling on the inner leaflet of the plasma membrane can be targeted for aberrant ubiquitination, degradation, and/or inhibition, thereby impairing HIV infectivity. Importantly, due to TPDs’ established MOA, even low-affinity Gag-Pol/TPD interactions are likely to lead to impaired Gag-Pol function. Thus, we pose the HYPOTHESIS that novel ATV-based TPDs will not only augment the inhibition of ATV-sensitive HIV-1 strains compared to ATV, but also exhibit increased and broader biological activity against drug-resistant variants. The objective of AIM 1 is to design and synthesize ATV-based TPDs built on state-of-the-art computational methods and predictive physicochemical properties currently accepted for in vivo active TPDs. Our approach will feature a modular TPD design to establish the appropriate ATV attachment point, E3 ligase recruiter, and length and composition of linker required for HIV-1 Gag-Pol ubiquitination/proteasomal degradation. The goal of AIM 2 is to provide proof-of-concept that ATV-TPDs exert superior activity versus ATV against wild type and PI-resistant HIV-1 strains. We will systematically screen four series of novel ATV-TPD for activity in vitro, in single-round infectivity, and in multi-round replication assays to identify the most promising ATV-TPD candidates. The latter will be tested in complementary limited-scope mechanistic studies, including comparing ATV-TPDs with analogs bearing inactive E3-ligase ligands, to probe if antiviral activity is consistent with the proposed MOA. The IMPACT will be TPDs targeting HIV-1 Gag-Pol and precursor PR that limit infectivity and replication through a mechanism distinct from occupancy-based HIV-1 PI. This will spur the future development of efficacious regimens against PI-resistant HIV strains with reduced susceptibility for resistance development.