Design, Synthesis and Evaluation of Novel Small Molecule Inhibitors of the Histone Methyltransferase DOT1L and Ubiquitination Facilitator Keap1 Sophie S. Spurr A thesis submitted for the award of doctor of philosophy School of Pharmacy, University College London 2017 Declaration I, Sophie S. Spurr confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Sophie S. Spurr i Abstract This thesis details the design, synthesis and evaluation of novel small molecule inhibitors of the histone methyltransferase DOT1L and the ubiquitination facilitator Keap1. The thesis is in two parts as outlined below. Part 1: The first part of this thesis details efforts towards identification of novel small molecule inhibitors of DOT1L, a histone methyltransferase which has been implicated in the development and proliferation of mixed lineage leukaemias (MLL). This work aims to optimise the drug-like properties of published DOT1L inhibitors while retaining potency, through further exploration of the nucleobase template. Structure-activity relationships (SARs) identified polar substituents and small heterocycles as favourable replacements for the halogen in 5-ITC, a small molecule inhibitor of DOT1L. Alternative nucleobase templates also demonstrated comparable DOT1L inhibition. To demonstrate proof of concept, a polar nitrile substituent was translated into the inhibitor Br-SAH as a direct replacement of the bromide. Activity was retained and a crystal structure obtained which demonstrated the nitrile occupied the same hydrophobic pocket. This work also demonstrated the use of a nitrile as a non-traditional replacement for heavy halogen atoms. Part 2: The second part of this thesis details identification of novel inhibitors of the Keap1-Nrf2 protein-protein interactions (PPI) using an approach based on kinetic target-guided synthesis (kTGS). Keap1 is a dimeric cytoplasmic protein that mediates the ubiquitination of Nrf2, a transcription factor that acts as a regulator of cellular antioxidant responses. Disruption of the PPI between Keap1 and Nrf2 has been shown to have a therapeutic benefit in diseases associated with oxidative stress and inflammation as well as providing a potential route to chemopreventative agents for cancer. Biased kTGS was applied as proof of concept. A biased ligand was designed and screened against a focused library of azides. The 1,3-dipolar cycloaddition products formed in the presence of the Keap1 Kelch domain were evaluated and validated through chemical synthesis and screening. A novel triazole structure was identified with improved activity over the initial biased fragment thus demonstrating kTGS as a valid approach for identifying novel inhibitors of the Keap1-Nrf2 PPI interaction. ii Impact statement This thesis contributes to the academic area of research of drug discovery, particularly in relation to the development of novel small molecule inhibitors of the histone methyltransferase DOT1L and the ubiquitination facilitator Keap1. The findings from this body of work could ultimately be beneficial to public health. Inhibition of DOT1L has been shown to have a therapeutic benefit in treatment of paediatric patients harbouring MLL-rearranged leukaemia. The aim of this research was to discover new inhibitors of DOT1L activity with improved pharmacokinetic properties. A strategy was adopted to identify novel functional groups which could exploit the hydrophobic cavity adjacent to the adenosine nucleobase. This work led to the identification of a nanomolar DOT1L inhibitor, CN-SAH, with an improved drug-like template. This substitution can be translated to other published inhibitor scaffolds, with the effect of improving the pharmacokinetic properties. Disruption of the PPI between Keap1 and Nrf2 has been shown to have a therapeutic benefit in diseases associated with oxidative stress and inflammation as well as providing a potential route to chemopreventative agents for cancer. The aim of this research was to discover novel small molecule inhibitors of the Keap1-Nrf2 PPI using a fragment-based drug discovery approach, kTGS. This work led to the identification of a novel triazole structure demonstrating kTGS as a valid approach for identifying novel inhibitors of the Keap1- Nrf2 PPI interaction. The work undertaken provides a contribution to knowledge through publication in Bioorganic Medicinal Chemistry Letters (impact factor 2.973, 2014). It is anticipated the results gained toward inhibition of Keap1 will provide future publications. iii Acknowledgments First, I am very grateful to Professor Paul Fish for the opportunity to undertake this PhD. I would like to thank all my supervisors, Professor Paul Fish, Dr Geoff Wells and Dr Jess Healy for their fantastic support and guidance. I would like to acknowledge the encouragement and camaraderie I received from everyone – past and present – in the Fish, Wells and Healy groups. I would particularly like to thank Dr Carrie-Anne Molyneaux, Dr Niall Igoe and Dr Elliott Bayle for their support. I am also thankful to my collaborators at the Structural Genomics Consortium, particularly Dr Matthieu Schapira for his helpful advice. I am also grateful to my funding body, the UCL Drug Discovery PhD studentship programme. I would like to thank Dr Jamie Baker for his support at the start of my PhD during the 6- month project rotation. I would like to mention Dr Rosemary Huckvale, Dr Samantha Gibson and Dr Rachel Lanigan-Mills for their ‘liquid encouragement’ (although I think my liver has suffered from our prosecco days!). Last but not least, I would like to thank my family and my friends, particularly Tom Gockelen-Kozlowski, Richard Oliver, Tom Skelton and Neil Thompson, for their unending support through the tears, merriment, presentation practise, pub trips and endless viewings of Murder She Wrote episodes. Finally, I give a particular mention to my partner, Dr Andrew Wright for being by my side. iv Abbreviations AAC azide alkyne cycloaddition AChE acetylcholinesterase AcOH acetic acid AD Alzheimer’s disease ALL acute lymphoblastic leukaemia AML acute myeloid leukaemia ARE antioxidant response element BCL-2 B-cell lymphoma 2 BcL-XL B-cell lymphoma-extra large BSA Bovine serum albumin BTB broad complex, tram-track and bric-a-brac bZIP basic leucine zipper CAAC Cu catalysed AAC CARMI coactivator associated arginine methyltransferase 1 cLogP calculated LogP CNC cap'n'collar COPD chronic obstructive pulmonary disease CTR C-terminal region DBCL diffuse large B cell lymphomas DBPS Dulbecco’s phosphate buffer solution DCC dynamic combinatorial chemistry DCL dynamic combinatorial library DOT1L disruptor of telomeric silencing 1-like DSF differential scanning fluorimetry v E. coli Escherichia coli EMT epithelial-mesenchymal transition EtOAc ethyl acetate EtOH ethanol EZH1 enhancer of zeste homolog 1 FBDD fragment-based drug discovery FDA food and drug administration FITC fluorescein isothiocyanate FP fluorescence polarisation GCL glutamate-cysteine ligase GSH glutathione GST glutathione S-transferase GWAS genome-wide analysis Halo halogen H-bonds hydrogen bonds HMDS hexamethyldisilazane HMDT histone demethyltransferase HMPA hexamethylphosphoramide HMT histone methyltransferase HO-1 heme-oxygenase-1 HTS high throughput screen IBD inflammatory bowel disease IC50 half maximal inhibitory concentration in-situ in situation IPTG isopropyl β-D-1-thiogalactopyranoside IVR intervening region vi Keap1 Kelch-like ECH-associated protein 1 kTGS kinetic target-guided synthesis LB Luria-Bertani LE ligand efficiency LipE ligand lipophilic efficiency LogP log of lipophilicity mCPBA meta-chloroperbenzoic acid MOA mechanism of action MLL mixed lineage leukaemia MLPCN molecular libraries probe production centres network mol% mole percent Mtase methyltransferase NAD(P)H nicotinamide amine dinucleotide phosphate Neh Nrf2-ECH homology NMSF National Mass Spectrometry Facility NQO1 NAD(P)H quinone oxidoreductase 1 Nrf2 nuclear factor erythroid 2-related factor 2 NTR N-terminal region OA osteoarthritis PA product amplification PAGE polyacrylamide gel electrophoresis PD Parkinson's disease PDB protein databank PEG polyethylene glycol PKMT protein lysine methyltransferase PMT protein methyltransferase vii PPI protein-protein interaction PRMT protein arginine methyltransferase PSA polar surface area PTM post-translational modification qPCR quantitative polymerase chain reaction RAAC Ru catalysed AAC RNS reactive nitrogen species ROS reactive oxygen species SAH S-adenosylhomocysteine SAM S-adenosylmethionine SBDD structure-based drug design SDS sodium dodecyl sulfate SET su(var)3–9, enhancer-of-zeste, trithorax SGC Structural Genomics Consortium SIM single ion monitoring SPR surface plasmon resonance TAT transactivating transcriptional activator tBHQ tert-Butylhydroquinone TBTA 2,2,2-trichloroacetimidate TF triazole formation TGS target-guided synthesis Tm protein melt point Tris tris(hydroxymethyl)aminomethane Ub ubquitination viii Contents Declaration .................................................................................................................... i Abstract ........................................................................................................................ ii Impact statement .......................................................................................................... iii Acknowledgments .......................................................................................................