Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc Investigations on small molecule inhibitors targeting the histone H3K4 tri-methyllysine binding PHD-finger of JmjC histone demethylases Bhaskar Bhushan a,b,e, Alexandre Erdmann a,e, Yijia Zhang a, Roman Belle a, Catrine Johannson a,c, ⇑ Udo Oppermann c, Richard J. Hopkinson a,d, Christopher J. Schofield a, Akane Kawamura a,b, a Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, United Kingdom b Radcliffe Department of Medicine, Division of Cardiovascular Medicine, University of Oxford, The Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN, United Kingdom c Botnar Research Centre, NIHR Oxford Biomedical Research Unit, University of Oxford, Oxford, United Kingdom article info abstract Article history: Plant homeodomain (PHD) finger containing proteins are important epigenetic regulators and are of Received 15 February 2018 interest as potential drug targets. Inspired by the amiodarone derivatives reported to inhibit the PHD Revised 10 March 2018 finger 3 of KDM5A (KDM5A(PHD3)), a set of compounds were synthesised. Amiodarone and its Accepted 18 March 2018 derivatives were observed to weakly disrupt the interactions of a histone H3K4me3 peptide with Available online xxxx KDM5A(PHD3). Selected amiodarone derivatives inhibited catalysis of KDM5A, but in a PHD-finger independent manner. Amiodarone derivatives also bind to H3K4me3-binding PHD-fingers from the Keywords: KDM7 subfamily. Further work is required to develop potent and selective PHD finger inhibitors. Epigenetics Ó 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http:// PHD-finger inhibitor Plant Homeodomain creativecommons.org/licenses/by/4.0/). JmjC-KDMs Histone demethylases Contents 1. Introduction . ....................................................................................................... 00 2. Results. ....................................................................................................... 00 2.1. Synthesis of AMI derivatives . .......................................................................... 00 2.2. Development of KDM5A(PHD3) AlphaScreen binding assay . ....................................................... 00 2.3. Inhibition of KDM5A demethylation activity by AMI derivatives . ....................................................... 00 2.4. Inhibition of H3K4me3-binding PHD-fingers of other JmjC-KDMs by AMI derivatives . .................................... 00 2.5. Inhibition of binding and catalytic activity of KDM7A/B(PHD-JmjC) by AMI derivatives . .................................... 00 3. Discussion. ....................................................................................................... 00 Acknowledgments. ........................................................................................ 00 Funding. ....................................................................................................... 00 A. Supplementary data . .................................................................................... 00 References . ....................................................................................................... 00 1. Introduction genetic marks’ are recognised and maintained by a diverse set of regulatory proteins and enzymes.1 The maintenance of these Most human genes are subject to epigenetic regulation, includ- marks is vital for the functioning and maintenance of cells, and ing by the post-translational modifications of histones. These ‘epi- their dysregulation is linked to multiple diseases, including cancer, cardiovascular disease, and developmental disorders.2–5 ⇑ Corresponding author. Plant homeodomain (PHD) fingers are C4HC3 type zinc-finger E-mail address: [email protected] (A. Kawamura). binding domains present in many chromatin-modifying pro- d Current address: Leicester Institute of Structural and Chemical Biology and teins.6,7 These small 50–10033 residue domains bind to histones Department of Chemistry, University of Leicester, Leicester LE1 7RH, United Kingdom. e These authors contributed equally to this work. to enable the localisation of enzyme(s) to specific targets and https://doi.org/10.1016/j.bmc.2018.03.030 0968-0896/Ó 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Please cite this article in press as: Bhushan B., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.03.030 2 B. Bhushan et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx Fig. 1. PHD-finger domains associated with the JmjC-KDMs. A) Phylogenetic tree of the PHD-finger domains in human JmjC-KDM family proteins. Branch lengths are indicated as a cladogram, and recognized histone marks in green. B) Domain architectures of selected JmjC-KDMs with PHD-fingers. promote the recruitment of transcription factors or chromatin- retardation, and manifests reduced H3K4 demethylase activity.13 associated protein complexes.7 While the roles of many PHD- Overall, these results identify their PHD-finger domains to be fingers are unclear, some PHD-fingers recognise specific histone crucial to the KDM5 function(s) (both catalytic and non-catalytic), modifications, including non-methylated or methylated lysines and in addition to JmjC-targeting,22,23 suggest they are interesting (e.g., histone H3 at K4 and K9), arginines (e.g., H3R2me1/me2), targets for oncology. and acetylated lysines (e.g., H3K14).7,8 PHD-fingers can also func- By contrast to the catalytic domains of epigenetic proteins, (e.g., tion as allosteric modulators of the activities of associated DNA methyltransferases, histone deacetylases, histone methyl- enzymes. Mutations, deletions or chromosomal translocations of transferases, demethylases and bromodomains),24,25 chemical PHD-finger encoding genes are linked to a range of diseases, tools for PHD-fingers are lacking and progress towards inhibitor including cancer, immunodeficiency and neurological disorders;6,7 development has been limited.26,27 PHD-finger inhibitors will be thus PHD-fingers are important epigenetic regulators. useful in exploring their biological functions and therapeutic Histone modifying enzymes, such as in the Jumonji-C (JmjC) potential. domain-containing histone lysine demethylases (JmjC-KDMs), In 2012, Wagner et al. identified amiodarone (AMI), an antiar- sometimes contain multiple PHD-fingers (Fig. 1).9 The KDM5 sub- rhythmic drug, as an inhibitor of KDM5A(PHD3).26 Analogues of family of JmjC-KDMs (KDM5A-D) catalyses demethylation of the AMI (WAG-003, WAG-005) were reported to inhibit the binding transcriptionally activating tri- and di-methylated lysine-4 mark of KDM5A(PHD3) to H3K4me3 with IC50 values of 30 ± 14 mM on histone H3 (H3K4me3/2), and is generally associated with and 41 ± 16 mM, respectively, on the basis of a HaloTag-based pep- transcriptional repression.10–13 The KDM5s are associated with tide displacement assay, and supported by fluorescence polarisa- development and progression of multiple cancers,11,14 and can tion assay results.26 While WAG-003 also inhibited other mediate cancer cell drug tolerance and maintain tumour-initiating H3K4me3 binding domains (PHD in RAG and double tudor domain cells.15,16 KDM5A/B have three PHD-fingers (PHD1-3, numbered (DTD) in KDM4A), it showed modest selectivity over other tested sequentially from the N-terminus), whereas KDM5C/D have two. PHD-fingers and Tudor domains (AIRE PHD1, BHC80 PHD, UHRF1 The roles of KDM5 PHD-fingers are partially characterised: (TDD)). However, the mode of action of AMI derivatives and their KDM5A/B(PHD3) binds to H3K4me3, with decreasing affinity for potential effect on KDM5A catalytic activity was unclear. lower methylation states,17 whereas KDM5A/B(PHD1) recognizes We describe the synthesis of a series of AMI derivatives and H3K4me017 (demethylation product), and is implicated in structure-activity-relationship (SAR) studies on their KDM5A ‘allosteric’ activation of KDM5 catalysis.18 It is proposed that (PHD3) binding and H3K4me3 demethylation catalysis by KDM5A PHD3 of KDM5A/B directs the JmjC domain to the H3K4me3 site; (Fig. 2). The results reveal that, while AMI and its derivatives bind PHD1 binds to the demethylated product H3K4me0 and activates weakly to PHD-fingers of KDM5A and other PHD-fingers within the the JmjC domain through a positive-feedback mechanism. This is JmjC-KDMs, they also inhibit the demethylation activity in a PHD- thought to propagate the transcriptionally inactive state of chro- finger independent manner, suggesting AMI derivatives can act via matin by K4me3 removal along the H3K4me3-enriched promot- more than one binding mode. ers.17,18 KDM5A(PHD3) is implicated in acute myeloid leukemia (AML) and forms a fusion protein with nucleoporin protein 98 2. Results (NUP98), a common translocation partner.19 This fused KDM5A (PHD3):NUP98 found in AML patients directs the ‘‘oncoprotein” 2.1. Synthesis of AMI derivatives to H3K4me3 promoter sites, inducing aberrant active transcription 20 leading to AML, as shown in cellular and animal models. PHD3 The AMI derivatives were synthesised as in Scheme 1.To mutations that disrupt H3K4me3 binding inhibit leukaemic trans- explore the importance of alkylation at the 20-position of the ben- 20 - formation. In ER breast cancers, KDM5A promotes progression zofuran