Developing Proteolysis-Targeting Chimeras (PROTACs) to Individual Class-I Histone Deacetylase Complexes

Epigenetics is the study of how histones – the packaging of DNA – and their modifications affect gene transcription and the overall cell. Histone deacetylase (HDAC) enzymes are a large family of epigenetic modulators vital for transcriptional regulation. HDACs 1, 2 and 3 perform this role via multi-protein complexes such as the CoREST complex, which consists of HDAC 1 or 2, LSD1 and RCOR1, 2 or 3. This complex is the only HDAC complex that includes LSD1 (Lysine-Specific Demethylase 1), so is the only complex to contain both deacetylase and demethylase activity.

This thesis describes work to design Proteolysis-Targeting Chimeras (PROTACs) – heterobifunctional molecules linking a ligand of a protein of interest to an E3 ligase ligand that can hijack a cell’s ubiquitin-proteasome pathway to degrade that protein – to selectively target and degrade LSD1 using ligands based on tranylcypromine (TCP). Two functionalised TCP-based ligands were designed, synthesised and used to construct a library of nine PROTACs with two different E3 ligase ligands and several different linkers. While no PROTACs described here caused any degradation of LSD1 or changes in histone methylation, four of them as well as the two functionalised inhibitors were found to inhibit LSD1 at a sub-micromolar level, with several below 100 nM, proving that they engage and inhibit LSD1.

Furthermore, LSD1-dTAG cells, incorporating an FKBP12F36V tag onto LSD1 in order to degrade it selectively in situ with dTAGV-1, were used for further investigations. It was shown that the dTAGV-1 fully degrades the LSD1 fusion protein by 24 hours. This effect was used to investigate the differences between inhibition and degradation of LSD1 by RNA-sequencing. It was found that degradation had a much more pronounced effect on the cells than inhibition, with suggestion that a cooperative mechanism between the demethylase and deacetylase activities of the CoREST complex is more important than demethylation alone.