TY - BOOK

T1 - Design and Synthesis of Epigenetic Drugs

T2 - Targeting the Histone Demethylase KDM4C

AU - Leurs, Ulrike

PY - 2014/9/27

Y1 - 2014/9/27

N2 - Epigenetics have within the last decade evolved into an exciting new strategy to target diseaseslinked to changes in the transcriptome of a cell. Both DNA methylation and posttranslationalmodifications of histone proteins are important regulators of gene expression, and aberrantregulation of histone- and DNA-modifying enzymes can lead to the development of diseasessuch as cancer. The histone demethylases of the KDM4 family have been implicated in a widerange of diseases, and are hence important drug targets. KDM4s belong to the bigger family of2-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 cofactordependentmanner, 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 smallmolecule scaffolds targeting KDM4 enzymes, a heterocyclic ring library was screened againstKDM4C. Two 4-hydroxypyrazoles were identified as inhibitors of KDM4C, and represent aninteresting novel scaffold for inhibitor development. Secondly, a phage display screening againstKDM4C was performed prior to this thesis to identify peptides interacting with the surface ofthe enzyme in a substrate and cofactor-independent manner. Two cyclic peptides were identifiedas binders of KDM4C (peptide 1: ACKWMDDGYCGGG-CONH2, peptide 2: ACYTRNMNQCGGG-CONH2). During this thesis, these peptides were developed into inhibitors of KDM4C andtheir mode of inhibition was evaluated in vitro. The regions on KDM4C affected by peptidebinding were identified and characterized by hydrogen/deuterium exchange mass spectrometry(HDX-MS). Substrate-competition experiments indicated that the peptides do not targetKDM4C through competition with the histone peptide substrate, but possibly in a cooperativemanner. This hypothesis is supported by the results of the HDX-MS analysis that indicated twodistinct binding sites for the peptides on the N- and C-terminal site of KDM4C, remote from theactive site.

AB - Epigenetics have within the last decade evolved into an exciting new strategy to target diseaseslinked to changes in the transcriptome of a cell. Both DNA methylation and posttranslationalmodifications of histone proteins are important regulators of gene expression, and aberrantregulation of histone- and DNA-modifying enzymes can lead to the development of diseasessuch as cancer. The histone demethylases of the KDM4 family have been implicated in a widerange of diseases, and are hence important drug targets. KDM4s belong to the bigger family of2-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 cofactordependentmanner, 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 smallmolecule scaffolds targeting KDM4 enzymes, a heterocyclic ring library was screened againstKDM4C. Two 4-hydroxypyrazoles were identified as inhibitors of KDM4C, and represent aninteresting novel scaffold for inhibitor development. Secondly, a phage display screening againstKDM4C was performed prior to this thesis to identify peptides interacting with the surface ofthe enzyme in a substrate and cofactor-independent manner. Two cyclic peptides were identifiedas binders of KDM4C (peptide 1: ACKWMDDGYCGGG-CONH2, peptide 2: ACYTRNMNQCGGG-CONH2). During this thesis, these peptides were developed into inhibitors of KDM4C andtheir mode of inhibition was evaluated in vitro. The regions on KDM4C affected by peptidebinding were identified and characterized by hydrogen/deuterium exchange mass spectrometry(HDX-MS). Substrate-competition experiments indicated that the peptides do not targetKDM4C through competition with the histone peptide substrate, but possibly in a cooperativemanner. This hypothesis is supported by the results of the HDX-MS analysis that indicated twodistinct binding sites for the peptides on the N- and C-terminal site of KDM4C, remote from theactive site.

M3 - Ph.D. thesis

BT - Design and Synthesis of Epigenetic Drugs

ER -