Targeting Lysine Specific Demethylase 1A (LSD1) with small molecule inhibitors and Proteolysis Targeting Chimeras (PROTACs)

The study of proteins associated with epigenetics is an ever expanding field of study. Lysine Specific Demethylase 1A (LSD1), existing within the co-repressor to the transcription factor REST (CoREST) complex, is an epigenetic protein responsible for the demethylation of lysine residues on histone tails, causing both gene activation and repression. Therapeutic agents targeting LSD1 have the potential to treat many diseases, most notably acute myeloid leukaemia and Ewing Sarcoma. This thesis describes a variety of techniques to target LSD1 within the CoREST complex. Herein, a variety of small molecule inhibitors, heterobifunctional molecules and dual peptidic inhibitors have been synthesised with the purpose of probing LSD1.

A variety of functionalised inhibitors have been synthesised in a structure-activity-relationship (SAR) study to investigate the 3D space surrounding an established non-competitive LSD1 inhibitor SP2509. An SP2509 analogue, functionalised with a polyethylene glycol (PEG) linker, was found to possess an IC50 of 159 nM against LSD1 in the CoREST complex in vitro. Overall, a number of novel SP2509 analogues have been discovered with submicromolar IC50 values for LSD1 in the CoREST complex. Heterobifunctional molecules, including Proteolysis Targeting Chimeras (PROTACs) and a biotinylated conjugate, were designed and synthesised based on the PEG functionalised inhibitor discovered in the SAR study. The PROTAC library, employing CRBN and VHL ligands with alkyl and PEG linker functionalities, were successfully synthesised and tested against HCT116 cell line. These preliminary studies showed low levels of degradation; 49 % degradation with a VHL recruiting PROTAC and 28 % degradation with a CRBN recruiting PROTAC. Furthermore, a small peptide study was conducted, whereby a variety of dual functionalised HDAC-LSD1 peptides were synthesised. This was in order to investigate the possibly of probing LSD1 alongside its complex partner, histone deacetylase (HDAC) I/II in a simultaneous 1:1 dual inhibition manner. Quantitative NMR was conducted on purified peptides to determine the accurate peptide content.