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.
