Abstract
The unstructured N-terminal tail of core histones is a hot spot region for additional posttranslational modifications, playing a crucial role in the regulation of transcription. It is not until recently that methylation was considered as an irreversible process. Histone demethylases have been discovered and found to play a critical role in cancer development and progression. KDM4A (lysine-specific demethylase 4A) is an evolutionarily conserved JmjC-domain-containing histone demethylase (JHDM), which demethylates di- and tri methyl lysine 9 and lysine 36 in histone H3 (H3K9me2/3 and H3K36me2/3). H3K9me2 and H3K9me3 are considered as methyl marks of transcriptional repression. KDM4A has been reported to engage in embryonic development, cell cycle, and oncogenesis. In this thesis, N-terminal region containing JmjN and JmjC domain of KDM4A (1―347) was cloned into pET30a expression vector. Purified recombinant protein was demonstrated to possess demethylase activity by MALDI-TOF mass spectrometry analysis and western blot analysis. Screening of inhibitor toward KDM4A and determining of enzyme kinetic parameters were based on a formaldehyde dehydrogenase (FDH) coupled-assay. 29 compounds were selected based on virtual molecular docking analysis, three (NSC 4A, NSC4B and NSC 4C) demonstrated inhibitory effects, with the IC50 = 1.0 ± 0.1 μM and Ki = 1.6 μM for NSC4A and IC50 = 1.5 ± 0.1 μM and Ki = 0.8 μM for NSC 4B. NSC 4A also exhibited a better inhibitory effect than reference inhibitor PD2, suggesting as a putative lead compound for the development of KDM inhibitors with the therapeutic potential.