Abstract
Histone deacetylases (HDACs) play important roles in inflammatory diseases like asthma and chronic obstructive pulmonary disease (COPD). Unravelling of and interfering with the functions of specific isoenzymes contributing to inflammation provides opportunities for drug development. Here we synthesize proteolysis targeting chimeras (PROTACs) for degradation of class I HDACs in which o-aminoanilide-based class I HDAC inhibitors are tethered to the cereblon ligand pomalidomide. One of these PROTACs, denoted HD-TAC7, showed promising degradation effects for HDAC3 with a DC50 value of 0.32μM. In contrast to biochemical evidence using siRNA, HD-TAC7 showed a minimal effect on gene expression in LPS/IFNγ-stimulated RAW 264.7 macrophages. The lack of effect can be attributed to downregulation of the NF-κB subunit p65, which is a known side effect of pomalidomide treatment. Altogether, we describe a novel PROTAC that enables selective downregulation of HDAC3 levels, however we note that concomitant downregulation of the NF-κB subunit p65 can confound the biological outcome.
Highlights
Acetylation of lysine residues of cellular proteins plays a key role in the regulation of cellular signal transduction pathways
The ligands for histone deacetylases (HDACs) 2a and 2b were synthesized starting from Boc-protected ortho-amino aniline or Boc-protected para-fluoro ortho-amino aniline
A condensation reaction was applied to couple intermediate 3a with benzyloxycarbonyl (Cbz) protected amino acid linkers using EDCI and HOBt as reagents to obtain intermediates 4a-4c in yields between 40 and 60%, while intermediate 3b was used to obtain intermediate 4d using the same method with a yield of 56%
Summary
Acetylation of lysine residues of cellular proteins plays a key role in the regulation of cellular signal transduction pathways. We reported anti-inflammatory activity for the HDAC1,2, and 3 selective inhibitor Entinostat both in vitro and in vivo, which is connected to upregulation of the anti-inflammatory cytokine Interleukin-10 (IL10) [10] This could be linked to increased acetylation, nuclear localization and IL-10 promotor binding by the p65 subunit of the NF-kB transcription factor. The HDAC3 selective inhibitor, RGFP966, did not show significant effects on gene expression in RAW 264.7 macrophages [12] This discrepancy between siRNA mediated knockdown and HDAC3 selective inhibition of deacetylase activity suggests that this proteins functions as a scaffold rather than as a catalyst in inflammatory signaling. We report a novel class of class I HDAC directed PROTACs that include o-aminoanilide-based class I HDAC ligands and pomalidomide as ligand for CRBN E3 ligase
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