No abstract

Histone deacetylase 6 (HDAC6) primarily catalyzes the removal of acetyl group from the side chain of acetylated lysine residues in cytoplasmic proteins such as αtubulin and HSP90. HDAC6 is involved in multiple diseaserelevant pathways. Based on the proteolysis targeting chimera strategy, we previously developed the first HDAC6 degrader by tethering a pan-HDAC inhibitor with cereblon (CRBN) E3 ubiquitin ligase ligand. We herein report our new generation of multifunctional HDAC6 degraders by tethering selective HDAC6 inhibitor Nexturastat A with CRBN ligand that can synergize with HDAC6 degradation for the antiproliferation of multiple myeloma (MM). This new class of degraders exhibited improved potency and selectivity for the degradation of HDAC6. After the optimization of the linker length and linking positions, we discovered potent HDAC6 degraders with nanomolar DC 50 and promising antiproliferation activity in multiple myeloma (MM) cells.

Histone deacetylase 6 (HDAC6) is involved in multiple cellular processes such as aggresome formation, protein stability, and cell motility. Numerous HDAC6-selective inhibitors have been developed as cellular chemical tools to elucidate the function of HDAC6. Since HDAC6 has multiple domains that cannot be studied by HDAC6-selective inhibitors, CRISPR-CAS9 and siRNA/shRNA have been employed to elucidate the nonenzymatic functions of HDAC6. However, these genetic methods have many limitations. Proteolysis targeting chimera (PROTAC) is an emerging technology for the development of small molecules that can quickly remove the entire protein in cells. We previously developed multifunctional HDAC6 degraders that can recruit cereblon (CRBN) E3 ubiquitin ligase. These HDAC6 degraders can degrade not only HDAC6 but also neo-substrates of CRBN. They are excellent candidates for the development of anticancer therapeutics, but the multifunctional nature of the CRBN-based HDAC6 degraders has limited their utility as specific chemical probes for the study of HDAC6-related cellular pathways. Herein we report the development of the first cell-permeable HDAC6-selective degraders employing Von Hippel–Lindau (VHL) E3 ubiquitin ligase, which does not have any known neo-substrates. The DC50’s of the most potent compound 3j are 7.1 nM and 4.3 nM in human MM1S and mouse 4935 cell lines, respectively. The D max’s of 3j in these two cell lines are 90% and 57%, respectively.

Proteolysis targeting chimeras (PROTACs) have emerged as useful chemical probes and potential therapeutics by taking advantage of the ubiquitin−proteasome system to degrade intracellular disease-associated proteins. PROTACs are heterobifunctional molecules composed of a target protein ligand, E3 ubiquitin ligase ligand, and a linker between them. The generation of efficient PROTACs requires screening of many parameters, especially the lengths and types of the linkers. We report our proof-ofconcept study using a two-stage strategy to facilitate the development of PROTACs against the estrogen receptor (ER). In stage one, a library of close to 100 PROTACs was synthesized by simply mixing a library of ERα ligands containing a hydrazide functional group at different positions with a preassembled library of E3 ligase ligands bearing different types and lengths of linkers with a terminal aldehyde group in a 1:1 ratio. Cell-based screening occurred without further purification, because the formation of the acylhydrazone linkage is highly efficient and produces water as the only byproduct. Compound A3 was the most potent ER degrader in two ER+ cell lines (DC 50 = ∼ 10 nM, D max = ≥ 95%). Stage two involved transformation to a more stable amide linker to generate a more drug-like molecule. The new compound, AM-A3, showed comparable biological activity (DC 50 = 1.1 nM, D max = 98%) and induced potent antiproliferation (IC 50 = 13.2 nM, I max = 69%) in MCF-7. This proof-of -concept study demonstrates that the two-stage strategy can significantly facilitate the development of PROTACs against ER without the tedious process of making large numbers of PROTACs one by one. It has the potential to be expanded to many other targets.

No abstract