LANE, Philip Edward (2021). A Mechanism Based Probe for Visualising Chromatinmodifying Enzyme Lysine-Specific Histone Demethylase 1. Doctoral, Sheffield Hallam University. [Thesis]
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Lane_2021_PhD_MechanismBasedProbe.pdf - Accepted Version
Available under License Creative Commons Attribution Non-commercial No Derivatives.
Lane_2021_PhD_MechanismBasedProbe.pdf - Accepted Version
Available under License Creative Commons Attribution Non-commercial No Derivatives.
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Abstract
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.
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