A Mechanism Based Probe for Visualising Chromatinmodifying Enzyme Lysine-Specific Histone Demethylase 1

Lysine-specific demethylase 1 (LSD1), the first histone demethylase to be identified, catalyses specifically the demethylation of the mono and dimethyl groups of histone 3 (H3) lysine 4 (K4), and its dysregulation is thought to contribute to the development of cancer. GlaxoSmithKline (GSK) and Oryzon Genomics (ORY) have submitted numerous N-alkylated phenylcyclopropylamine (PCPA) molecules to phase II clinical trials against several different cancers. Eight probes have been designed and synthesised with alkyne and azide tags from PCPA. Their inhibitory values have been investigated towards Monoamine Oxidase (MAO) and LSD1, showing over two times the increase in selectivity towards LSD1. Probe 1 has been subject to cell treatment and its ability to inhibit LSD1 confirmed using NTERA2 cells. Furthermore, these synthesised probes are conjugated to a peptide to successfully guide the probes into the cell. The addition of the peptide causes an increase in the inhibitory values towards LSD1 by on average seven fold. Chiral separation was undertaken on probe 7 to explore the potential inhibition differences of the two enantiomers. Single Crystal X-Ray Diffraction analysis and 1H NMR Nuclear Overhauser effect (nOe) confirmed the chiral separation, with inhibitory data showing (1 R – 2 S)-probe 7 is a more potent inhibitor of LSD1 than its enantiomer. In addition to this, N-alkylation of probe 4 achieved a successfully increase of potency towards LSD1 over PCPA. The mechanistic inhibition pathway for PCPA inhibiting LSD1 is currently unknown. Here, DFT is used on cluster models of Flavin Adenine Dinucleotide (FAD) and the active site of LSD1 (135 atoms and 218 atoms) to investigate the mechanistic inhibition pathway for PCPA inhibiting the FAD cofactor. The calculated energy of the potential rate determining step was 45.3 kcal mol-1 which is 23.5 kcal mol-1 greater than the experimental activation energy for the inhibition of LSD1.