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Title: Design and synthesis of small-molecule ERK5 inhibitors for anti-cancer therapy
Authors: Martin, Nicholas Charles
Issue Date: 2016
Publisher: Newcastle University
Abstract: Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen activated protein kinase (MAPK) family responsible for activating a number of transcription factors in the cell nucleus. Downstream effects of ERK5 activation have been linked to cell survival, proliferation and differentiation. Overexpression of ERK5 has been observed in a number of tumour cell lines and has been linked with poor prognosis in cancer patients. ERK5 gene knockout studies have proved it plays a role in angiogenesis. Inhibition of ERK5 with XMD8-92 (12) showed correlation with tumour growth. All ERK5 validation experiments to date have lacked a compound with good ERK5 potency, selectivity, cell permeability and metabolic stability. The aim of this project is to prove the role of ERK5 in cancer with a drug-like tool compound through in vivo validation experiments. Small molecule ERK5 inhibitors have been discovered through a high-throughput screen revealing indazole-sulphonamide hit compounds with moderate potency, such as N-(1H-indazol-6-yl)benzenesulfonamide 30 (enzymatic ERK5 IC50 = 4.7 ± 0.04 μM). Hit expansion via library synthesis identified a set of sulphonamide aromatic groups associated with good ERK5 inhibitory activity. Structural optimisation around the sulphonamide linker elucidated a key binding interaction between Lys39 in the ERK5 active site and an S=O group. Removal of the hydrogen bond donor capacity of the sulphonamide yielded compounds with potency in the nM range. Alkylation or removal of heteroatoms in the indazole indicated that compounds form two hydrogen bonds with the hinge region of the kinase active site. Molecular modelling using GOLD predicted the 3- and 4-positions were suitable for substitution. Subsequent synthesis of key bromo intermediate 3 was conducted in a 68% vii yield over five steps allowing for library synthesis of 4-alkyl indazoles, including current lead compound 199 (enzymatic ERK5 IC50 = 12 ± 7 nM). Compound 199 shows favourable drug-like properties (MW = 318, CLogP = 4.7, LE = 0.51, Caco-2 efflux ratio = 0.52) and good ERK5 potency in transfected HEK 293 cells (cellular ERK5 IC50 = 24 nM). However, the compound is rapidly metabolised in mouse liver microsomes (MLM Clint = 378 μl/min/mg) and has a poor oral bioavailability in mice (24%). Compound 199 was also screened for activity against a panel of 456 kinases but unfortunately showed poor selectivity. Metabolite identification conducted with 199 revealed oxidation of the indazole and cyclopropyl group as the major routes of metabolism by CYP enzymes. Unsubstituted 3-, 5-, and 7- positions around the indazole were systematically blocked from metabolism by chlorination, with data indicating the 3-position is most susceptible to oxidation. Improving the LogP of the series was explored by re-optimisation of the sulphonamide aryl group and substitution of the 3-position. It was found that 3-amino Figure 1. A) Key interactions made between compound 199 (cyan) in the ERK5 active site (green); (B) Structure of Compound 199. viii compound 269 had excellent microsomal stability (MLM Clint = 8.3 μl/min/mg) but potency was inferior to 199 (enzymatic ERK5 IC50 = 790 ± 80 nM). The work presented in this thesis describes the synthesis and SARs of over 130 novel, indazole compounds for the inhibition of ERK5. Through SAR development a binding mode has been proposed and a lead compound (199) obtained with excellent ERK5 potency. Major routes of compound metabolism have been identified and methods to impeded metabolism have been demonstrated. The indazole series is in the lead optimisation phase with an aim to provide a tool compound able to critically assess the anti-cancer effect of ERK5 inhibition in vivo.
Description: PhD Thesis
Appears in Collections:Northern Institute for Cancer Research

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