Epigenetics have within the last decade evolved into an exciting new strategy to target diseases
linked to changes in the transcriptome of a cell. Both DNA methylation and posttranslational
modifications of histone proteins are important regulators of gene expression, and aberrant
regulation of histone- and DNA-modifying enzymes can lead to the development of diseases
such as cancer. The histone demethylases of the KDM4 family have been implicated in a wide
range of diseases, and are hence important drug targets. KDM4s belong to the bigger family of
2-OG oxygenases, an enzyme class sharing high sequence homology along their active site.
Since most of the known inhibitors target the active site of KDM4 in a substrate- or cofactordependent
manner, they often suffer from off-target effects on other 2-OG oxygenases.
In this thesis, novel strategies to target KDM4C were explored. To discover new small
molecule scaffolds targeting KDM4 enzymes, a heterocyclic ring library was screened against
KDM4C. Two 4-hydroxypyrazoles were identified as inhibitors of KDM4C, and represent an
interesting novel scaffold for inhibitor development. Secondly, a phage display screening against
KDM4C was performed prior to this thesis to identify peptides interacting with the surface of
the enzyme in a substrate and cofactor-independent manner. Two cyclic peptides were identified
as binders of KDM4C (peptide 1: ACKWMDDGYCGGG-CONH2, peptide 2: ACYTRNMNQC
GGG-CONH2). During this thesis, these peptides were developed into inhibitors of KDM4C and
their mode of inhibition was evaluated in vitro. The regions on KDM4C affected by peptide
binding were identified and characterized by hydrogen/deuterium exchange mass spectrometry
(HDX-MS). Substrate-competition experiments indicated that the peptides do not target
KDM4C through competition with the histone peptide substrate, but possibly in a cooperative
manner. This hypothesis is supported by the results of the HDX-MS analysis that indicated two
distinct binding sites for the peptides on the N- and C-terminal site of KDM4C, remote from the
active site.