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Title: Design of small-molecule inhibitors of sulfatase 2 and ERK5
Author: Reuillon, Tristan Denis Constantin Rene
ISNI:       0000 0004 7960 9977
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2015
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Modern targeted cancer therapeutics are directed against components of cell signalling pathways, responsible for driving tumour progression. In this thesis, chemical tools and inhibitors of two enzymes involved in cell signalling, namely sulfatase 2 (Sulf-2) and extracellular signal-regulated kinase 5 (ERK5), have been investigated. Sulfatase 2 is a heparan sulfate (HS) processing enzyme, which has been implicated in the progression of several cancers including hepatocellular carcinoma (HCC). In HCC patients, high Sulf-2 mRNA expression correlates with a poor prognosis. The first published small-molecule Sulf-2 inhibitors were monosaccharide glucosamine derivatives, bearing a sulfamate at the O6-position. The most potent analogue from this series, was 5, having a reported IC50 against Sulf-2 of 130 μM. Although 5 was not sufficiently potent for use in target validation studies, its discovery provided encouragement that viable Sulf-2 inhibition was possible with low molecular weight compounds. In the absence of a published crystal structure and of a biological assay suitable for a highthroughput screening campaign, the structure of the endogenous substrate was considered as a potential starting point for identification of probe compounds, for use in target validation studies. Biphenyl A and biphenyl ether B sulfamates were designed in an attempt to identify a non-saccharide scaffold, which could mimic the spatial arrangements of groups believed to be important for binding of the endogenous substrate to Sulf-2. Access to these targets was facilitated by the development of a sulfamate protecting group strategy, which enabled a more flexible approach to the synthesis of phenolic O-sulfamates. v Preliminary sulfatase inhibition data have been generated, indicating that biphenyl 162 and biphenyl ether sulfamates 197 exhibited better potency against Sulf-2 than monosaccharide glucosamine 5 in our assay format. Extracellular signal-regulated kinase 5 (ERK5) is a member of the protein kinase superfamily, which plays an essential role in the transduction of extracellular signals to intracellular effectors. Activation of the ERK5 signalling pathway is associated with cell survival, proliferation, and differentiation, and ERK5 over-expression has been implicated in tumour development. An ERK5 inhibitor, XMD8-92 218, which was reported in the literature, displayed selectivity for ERK5 when tested against a panel of 402 kinases in an ATP-site competition binding assay (ERK5 IC50 = 300 nM) and showed reasonable activity in HeLa cells (GI50 = 1.5 μM). This compound also inhibited the growth of two human tumour xenograft models (HeLa cells and Lewis lung cells), highlighting that ERK5 is a valid target for anti-cancer therapy. A pyrrole carboxamide series of ERK5 inhibitors was optimised from lead compounds such as 318, which had good absorption in the Caco-2 assay but suffered from low solubility and medium clearance in mouse liver microsomes in vitro, and translated to poor oral bioavailability in vivo. The objective of the optimisation studies was to improve ERK5 inhibitory activity, both in the biochemical and cellular assays, and improve pharmacokinetic parameters to deliver compounds suitable for in vivo efficacy studies. Three areas were identified for investigation i.e. alkylation of the amide nitrogen; replacement of the meta-chloro on the aroyl ring; and substitution on the heteroaromatic amide ring at the ortho and para positions. vi Docking of compounds into a recently published co-crystal structure of an analogue of XMD8-92 bound to ERK5 has provided information on the possible binding mode of the pyrrole carboxamide series and was used to guide the design of inhibitors (Figure 1). Extension of the carboxamide heteroaromatic amine at the para position, with aliphatic and cyclic aliphatic side-chains bearing a basic centre, resulted in significantly improved ERK5 inhibitory activity (IC50 < 30 nM) as exemplified by 406, which achieved a 3-fold improvement in cellular inhibitory activity, and combined excellent microsomal stability with high solubility. However all the compounds with a basic centre suffered from a high efflux ratio and low membrane permeability in the Caco-2 assay, and as a result, had poor in vivo oral bioavailability in mouse PK studies. The work presented in this thesis has extended the SARs around the pyrrole carboxamide core but did not allow to deliver a new in vivo tool.
Supervisor: Not available Sponsor: Cancer Research UK ; Medical Research Council ; Astex Pharmaceuticals
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available