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De La Roche M.,University of Cambridge | Rutherford T.J.,University of Cambridge | Gupta D.,University of Cambridge | Veprintsev D.B.,University of Cambridge | And 4 more authors.
Nature Communications

Wnt/β-catenin signalling controls development and tissue homeostasis. Moreover, activated β-catenin can be oncogenic and, notably, drives colorectal cancer. Inhibiting oncogenic β-catenin has proven a formidable challenge. Here we design a screen for small-molecule inhibitors of β-catenin's binding to its cofactor BCL9, and discover five related natural compounds, including carnosic acid from rosemary, which attenuates transcriptional β-catenin outputs in colorectal cancer cells. Evidence from NMR and analytical ultracentrifugation demonstrates that the carnosic acid response requires an intrinsically labile α-helix (H1) amino-terminally abutting the BCL9-binding site in β-catenin. Similarly, in colorectal cancer cells with hyperactive β-catenin signalling, carnosic acid targets predominantly the transcriptionally active ('oncogenic') form of β-catenin for proteasomal degradation in an H1-dependent manner. Hence, H1 is an 'Achilles' Heel' of β-catenin, which can be exploited for destabilization of oncogenic β-catenin by small molecules, providing proof-of-principle for a new strategy for developing direct inhibitors of oncogenic β-catenin. © 2012 Macmillan Publishers Limited. All rights reserved. Source

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro 174(4.59)-Met 180(4.66)) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln 174(4.60) and Phe 178(4.64) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu 1 and Trp 3 of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe 178(4.64) of the receptor and Trp 3 of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu 175(4.61), Ile 177(4.63), and Met 180(4.66) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe 177(4.63) and Phe 178(4.64) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Source

Smith H.,University of Dundee | Liu X.-Y.,Nanyang Technological University | Dai L.,Nanyang Technological University | Goh E.T.H.,University of Dundee | And 12 more authors.
Biochemical Journal

Mammalian Pellino isoforms are phosphorylated by IRAK (interleukin receptor associated kinase) 1/IRAK4 in vitro, converting them into active E3 ubiquitin ligases. In the present paper we report a striking enhancement in both transcription of the gene encoding Pellino 1 and Pellino 1 protein expression when murine BMDMs (bone-marrow-derived macrophages) are stimulated with LPS (lipopolysaccharide) or poly(I:C). This induction occurs via a TRIF [TIR (Toll/interleukin-1 receptor)-domain-containing adaptor-inducing interferon-β]-dependent IRAK-independent pathway and is prevented by inhibition of the IKK [IκB(inhibitor of nuclear factor κB) kinase]-related protein kinases, TBK1 {TANK [TRAF (tumour-necrosis-factor- receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1} and IKK?. Pellino 1 is not induced in IRF3 (interferon regulatory factor 3) -/- BMDMs, and its induction is only reduced slightly in type 1 interferon receptor -/- BMDMs, identifying Pellino 1 as a new IRF3-dependent gene. We also identify Pellino 1 in a two-hybrid screen using IKKε as bait, and show that IKKε/TBK1 activate Pellino 1 in vitro by phosphorylating Ser 76, Thr 288 and Ser 293. Moreover, we show that the E3 ligase activity of endogenous Pellino 1 is activated in LPS- or poly(I:C)-stimulated macrophages. This occurs more rapidly than the increase in Pellino 1 mRNA and protein expression, is prevented by the inhibition of IKKε/TBK1 and is reversed by phosphatase treatment. Thus IKKε/TBK1mediate the activation of Pellino 1's E3 ligase activity, as well as inducing the transcription of its gene and protein expression in response to TLR3 and TLR4 agonists. © The Authors Journal compilation © 2011 Biochemical Society. Source

Leeper A.D.,University of Edinburgh | Farrell J.,MRC Technology | Dixon Michael J.,University of Edinburgh | Wedden S.E.,MRC Technology | And 2 more authors.
Journal of Visualized Experiments

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account. We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies. We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines. © 2011 Journal of Visualized Experiments. Source

Miller T.C.R.,University of Cambridge | Rutherford T.J.,University of Cambridge | Birchall K.,MRC Technology | Chugh J.,MRC Technology | And 2 more authors.
ACS Chemical Biology

The Pygo-BCL9 complex is a chromatin reader, facilitating β-catenin-mediated oncogenesis, and is thus emerging as a potential therapeutic target for cancer. Its function relies on two ligand-binding surfaces of Pygos PHD finger that anchor the histone H3 tail methylated at lysine 4 (H3K4me) with assistance from the BCL9 HD1 domain. Here, we report the first use of fragment-based screening by NMR to identify small molecules that block protein-protein interactions by a PHD finger. This led to the discovery of a set of benzothiazoles that bind to a cleft emanating from the PHD-HD1 interface, as defined by X-ray crystallography. Furthermore, we discovered a benzimidazole that docks into the H3K4me specificity pocket and displaces the native H3K4me peptide from the PHD finger. Our study demonstrates the ligandability of the Pygo-BCL9 complex and uncovers a privileged scaffold as a template for future development of lead inhibitors of oncogenesis. © 2014 American Chemical Society. Source

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