Novel Approaches for Targeted Protein Degradation of Histone Deacetylases: Beyond Conventional E3 Ligase Recruitment

Abstract

Targeted protein degradation (TPD) has emerged as an alternative strategy in drug discovery, offering a catalytic, event-driven mechanism to eradicate disease-relevant proteins. Unlike conventional occupancy-driven inhibitors that transiently block activity, degraders induce the removal of the protein of interest (POI), enabling prolonged pharmacological effects, thereby overcoming resistance and addressing proteins previously considered undruggable. Proteolysis targeting chimeras (PROTACs) are the most common modality in TPD, consisting of an E3 ligase recruiter and a POI ligand to induce proximity for subsequent degradation of the POI. <br/> In recent years, this approach was applied to target histone deacetylases (HDACs) for degradation. These epigenetic regulators remove acetyl groups from histones and other substrates. HDACs are key drivers in cancer progression, with overexpression linked to poor prognosis. Although various HDAC ligands have been used in HDAC PROTACs, successful applications have so far been limited to the three well-characterized E3 ligases: cereblon, von Hippel–Lindau, and inhibitor of apoptosis protein. <br/> To expand the repertoire of E3 ligases for HDAC degradation and harness understudied ligases, two projects of this thesis investigated feminization-1 homolog B (FEM1B) and DDB1–CUL4–associated factor 11 (DCAF11) as alternative E3 ligases. Two mini libraries of PROTACs were prepared mainly by solid-phase synthesis. Both sets exhibited notable selectivity profiles: despite that the PROTACs did utilize a non-selective HDAC ligand, FEM1B recruitment enabled selective HDAC1-3 degradation, while DCAF11 recruitment led to unselective degradation of all tested isoforms. For FEM1B-based HDAC degradation, short spacers were beneficial, whereas long spacers were most efficient for DCAF11-based degradation. HDAC degradation was accompanied by antiproliferative effects in MM.1S cells. These initial results were validated in further cell lines, followed by the evaluation of apoptosis induction and cell cycle arrest. Mechanistical studies confirmed ubiquitin-proteasome system dependency. In addition, bystander degradation of HDAC-containing multiprotein complex members was also observed. <br/> A third study introduced hydrophobic tagging as an alternative strategy to PROTACs for HDAC degradation. By mimicking unfolded proteins, hydrophobic tags exploit the quality control machinery for POI degradation. Therefore, different HDAC ligands were fused to two types of hydrophobic tags, with and without spacers. While engaging HDAC1, 2, and 6, the hydrophobic-tagged HDAC ligands degraded only HDAC1 and 2. This degradation was at least partially proteasome-dependent, consistent with the hydrophobic tagging mechanism. However, the exact degradation mechanism remains unclear. Besides degradation, the most effective compounds also exhibited antiproliferative effects and induced apoptosis. <br/> In conclusion, these investigations expand the degradation toolbox for HDACs by establishing FEM1B and DCAF11 as alternative E3 ligases for PROTAC design and validate hydrophobic tagging as an alternative degradation modality. Importantly, all degraders are based on non-selective HDAC ligands yet achieved distinct isoform selectivity degradation profiles. These findings underscore the importance of selecting an appropriate degradation strategy for precise and effective HDAC degradation.