http://www.dspace.cam.ac.uk:80/handle/1810/219483 Wed, 22 May 2013 16:10:47 GMT 2013-05-22T16:10:47Z http://www.dspace.cam.ac.uk:80/handle/1810/244380 Title: Molecular
 recognition
 from
 atomic 
interactions:

 insights 
into 
drug 
discovery
 Authors: Higueruelo, Alicia Perez Abstract: The failure of the pharmaceutical industry to increase the delivery of new drugs into the market is driving a re-assessment of practices and methods in drug discovery and development. In particular alternative strategies are being pursued to find therapeutics that are more selective, including small molecules that target protein-protein interactions. However, success depends on improving our understanding of the recognition of small molecules by interfaces in order to develop better methods for maximising their affinity and selectivity, whilst trying to confer an appropriate therapeutic profile. This thesis starts with the description of the creation of TIMBAL, a database that holds small molecules disrupting protein-protein interactions. The thesis then focuses on the analysis of these molecules and their interactions in a medicinal chemistry and structural biology context. TIMBAL molecules are profiled against other sets of molecules (drugs, drug-like and screening compounds) in terms of molecular properties. Using the structural databases in the Blundell group, the atomic detail of the interaction patterns of TIMBAL molecules with their protein targets are compared with other molecules interacting with proteins, comprising natural molecules, small peptides, synthetic small molecules (including drug-like and drugs) and other proteins. The structural features and composition of the binding sites of these complexes are also analysed. Keeping in mind that current drug candidates are somewhat too lipophilic to succeed, these interaction profiles are defined in terms of polar and apolar contacts, with the aim of migrating natural patterns into the design of new therapeutics. Mon, 07 May 2012 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/244380 2012-05-07T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/244243 Title: Expression, purification and characterisation of recombinant chromatin assembly factor 1 Authors: Royle, Nikki Abstract: Chromatin Assembly Factor 1 (CAF-1) is the only known replication dependant histone chaperone, responsible for the deposition of the histone H3/H4 tetramer onto DNA. Found in all eukaryotes, CAF-1 consists of three subunits, p150, p60 and p48. Since its identification work on CAF-1 has mainly focused on in vivo studies due to the lack of a reliable method to produce large quantities of recombinant protein for biochemical studies. Herein the cloning, production and purification of the three subunits of recombinant CAF-1 is described. The proteins were expressed as complexes and individually in insect cells and Escherichia coli, optimised protocols are described for maximum protein recovery and purity. Constructs of p150 and p60 were also produced and used to analyse the binding regions and modes of both the p48 and p60 proteins to p150. It is shown that the two smaller subunits of CAF-1 do not interact in the absence of p150 and that the p150 subunit of CAF-1 acts as a scaffold for assembly of the complex, binding directly to both p48 and p60. The stoichiometry of the CAF-1 complex was also investigated and a basis for further work, including structural studies, discussed. Tue, 08 Jan 2013 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/244243 2013-01-08T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/242014 Title: Molecular characterization and evolutionary plasticity of protein-protein interfaces Authors: Bickerton, George Richard James Abstract: Abstract The sequencing of the human genome provides the parts list for understanding cellular processes. However, as 70% of eukaryotic genes work through multi-protein systems, it is only through detailed study of the interactions of these components, that a more complete, systems-level understanding can be gained. This thesis is centred on the establishment of PICCOLO - a comprehensive database of structurally characterized protein interactions. In generating the resource, issues of interface definition, quaternary structure, data redundancy, structural environment and interaction type are addressed. The resource enables a variety of analyses to be performed concerning interface properties including residue propensity, hydropathy, polarity, interface size, sequence entropy and residue contact preference. PICCOLO has been applied to probing the patterns of substitutions that are accepted in protein interfaces across evolution, and whether these patterns are distinguishable from those seen in other structural environments. The derivation of a high-quality set of multiple structural alignments in the form of the database TOCCATA, a prerequisite for such analysis, is described, as well as procedures to derive environment-specific substitution tables. The Blundell group has contributed a series of methods to predict the likely effect of non-synonymous Single Nucleotide Polymorphisms (nsSNPs) on protein stability, function and interactions in order to triage the large volumes of data created from high-throughput genetic screening studies, enabling prioritization of those nsSNPs most likely to be phenotypically detrimental. PICCOLO's contribution to these predictions is described. Historically there has been little focus on protein-protein interactions as drug targets for small-molecule therapeutics. However, alanine-scanning mutagenesis studies have revealed that only a subset of residues contribute the greater part of free energy to binding - so-called "hot-spots". Molecular characterization of hot-spots performed using PICCOLO, probes the molecular basis underlying this important phenomenon leading to the possibility of predictive methods to identify hot-spots 'in silico'. Fri, 01 Jan 2010 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/242014 2010-01-01T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/238434 Title: Probing the druggability of the Notch1 ankyrin domain using a fragment-based approach Authors: Abdel-Rahman, Noha Abstract: Notch signalling is a highly conserved pathway that is important in the developmental processes that control cell differentiation and cell fates. This canonical pathway involves binding of a transmembrane ligand in one cell to the extraceullular domain of a transmembrane Notch receptor in an adjacent cell. Ligand binding triggers two sequential proteolytic cleavages that shed a Notch intracellular domain (NICD). This is followed by translocation of NICD to the nucleus where it interacts with a transcription factor CSL and forms an activated Notch transcription complex, which induces the transcription of Notch target genes. Abnormal expression or mutations in the different components of the pathway are associated with a number of diseases and cancers. An enhanced activity of Notch signalling resulting from a mutation in the extracellular domain is implicated in the progression of T-acute lymphoblastic leukaemia (T-ALL). Several therapeutic agents have been developed to target the Notch signalling pathway such as, γ-secretase inhibitors, antibodies targeting different regions of the Notch receptor and recently a synthetic stapled peptide, which was found to inhibit the formation of the transcription complex. The current inhibitors have their own disadvantages including lack of selectivity, cost of goods and delivery to the target. Thus, a more selective approach to target downstream proteinprotein interactions by small molecules would provide an attractive approach to the design of new therapeutic agents that target this pathway. Here I report a fragment-based approach to target the ankyrin domain, a historically known but challenging, often-considered “undruggable” target. In this dissertation I describe the application of various biophysical and computational approaches to find, characterise and design compounds. The initial screening of a commercial fragment-library exploited a fluorescent-based thermal shift assay that identified 36 fragment hits. Some of the fragments were kinetically characterised by Surface Plasmon Resonance (SPR) and their affinities were found to be in the millimolar range. Several attempts at soaking vii and co-crystallising the fragments in the ankyrin domain crystal resulted in only two successful crystal structures that clearly define the positions of the fragments and their interactions with the ankyrin domain. One fragment binds to a pre-defined hotspot residue at the interface between the ankyrin domain and CSL. The other fragment is located at the interface between the ankyrin domain and Mastermind (MAML). The structural and kinetic data assisted the design of larger compounds with more extensive interactions using drug design software such as SPROUT and a docking program (GOLD). However, the optimised fragments did not show much improvement in affinity underlying the difficulty of flat protein-protein interface. The results reported here show the first structures of small molecules binding to the ankyrin domain of Notch1 receptor. Tue, 08 Feb 2011 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/238434 2011-02-08T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/228702 Title: Structural and biochemical studies of the yeast linker histone, Hho1p Authors: Osmotherly, Lara May Abstract: The basic unit of eukaryotic chromatin is the nucleosome core, which contains 147 base pairs of DNA wrapped around an octamer of core histone proteins. Linker histones bind through their globular domain at the nucleosome dyad and to internucleosomal DNA through their C-terminal basic tail. The Saccharomyces cerevisiae linker histone homologue, Hho1p, contains two domains, GI and GII, that have sequence similarity to the globular domain of the canonical linker histone H1. The individual domains of Hho1p differ in their structural and functional properties, for example in 10 mM sodium phosphate GI is folded while GII exists as two species: folded and “unfolded”. In Chapter 2 the structure of the second globular domain of Hho1p, GII, is further investigated. NMR studies indicate residual structure in the “unfolded” form of GII, especially at the start of helices I and III. Chapter 3 considers the structural roles of Hho1p within chromatin. Semi-quantitativeWestern blotting is used to measure the abundance of Hho1p relative to nucleosomes in yeast. Analysis of reconstituted nucleosome arrays containing NGIL (Hho1p with the second globular domain removed) are indistinguishable from those containing full-length Hho1p, in gel-based assays and by analytical ultracentrifugation, suggesting the GII domain may not have a major role in chromatin compaction. Chapter 4 focuses on the interaction of Hho1p with chromatin proteins. Chemical cross-linking and gel filtration indicate that Hho1p does not interact significantly with the putative HMGB1 homologues Hmo1p and Nhp6ap in vitro. Hho1p and Htz1p, the yeast histone H2A.Z subtype, do not appear to interact directly in co-immunoprecipitation and chemical cross-linking assays, while chromatin immunoprecipitation studies show no evidence of colocalisation across the ADH2 and PHO5 genes. Hho1p and Sir2p cross-link in solution, but purification difficulties precluded further investigation. The effect of phosphorylation on the interaction of Hho1p and related truncation proteins with DNA and chromatin are investigated in Chapter 5. Phosphorylation reduces their affinity for linear DNA, but has different effects on the binding to four-way junction DNA for Hho1p and NGIL, compared with LGII (the linker region and GII domain of Hho1p). Phosphorylation has no obvious effect on the affinity of these proteins for chromatin in sucrose gradient centrifugation assays. NMR spectroscopy studies show that the linker region is mostly unstructured, with a short region showing some α-helical character. Phosphorylation of the linker domain changes its structural character. Description: The first part of Figure 2.6, sections a-f, in separate PDF. Tue, 16 Nov 2010 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/228702 2010-11-16T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/226857 Title: Molecular pharmacology of an insect GABA receptor Authors: McGonigle, Ian Vincent Abstract: Cys-loop receptors are ligand-gated ion channels that are involved in fast synaptic neurotransmission in the central and peripheral nervous system. The Cys-loop receptor RDL (‘resistant to dieldrin’) is a GABA-gated chloride channel from Drosophila melanogaster and is a major target site for insecticides. The aim of this dissertation was to characterise RDL receptors with particular focus on the agonist binding site. To assess the potency of a range of GABA analogues on RDL receptors, I expressed receptors in Xenopus oocytes and used voltage-clamp electrophysiology to detect receptor responses. I carried out computational modelling of these analogues to determine the dipole separation distances and atomic charges. Computational calculations and functional experiments revealed that agonists require a charged ammonium and an anionic centre, with the most potent agonists having a dipole separation distance of ~5 Å. I made a homology model of the extracellular domain of RDL and docked the active analogues into the putative binding site. I then conducted mutagenesis studies to test the accuracy of this model. Functional data from mutagenesis studies broadly support the location of GABA within this model. This model may be useful for further structure−activity studies and rational drug design. Natural compounds from the traditional Chinese medicine ‘Ginkgo biloba’ (ginkgolide A, ginkgolide B and bilobalide) have potent insecticidal properties and are similar in structure to picrotoxin. I tested the effect of these compounds on RDL receptor function using voltage-clamp electrophysiology. All compounds were found to inhibit RDL receptor function. I probed the binding site of these compounds using site-directed mutagenesis and electrophysiology. Mutations to the 2'A and 6'T channel-lining (M2) residues greatly reduced the potency of these compounds. I then made a homology model of the transmembrane domain of RDL and docked these compounds into the channel. Compounds docked into the channel pore close to the 2' and 6' channel-lining residues and H-bonding interactions were detected at these locations. Ginkgolides are therefore antagonists of RDL receptors, binding in the channel close to the 2' and 6' residues and this may be the mechanism underlying their potent insecticidal properties. The 5-HT3 receptor is a member of the Cys-loop receptor family and shows homology to RDL receptors. To explore different techniques for studying Cys-loop receptor function I assessed the functionality of two brain derived transcripts of the 5-HT3B subunit (Br1 and Br2) using single-channel electrophysiology and a fluorometric assay. Receptors containing Br1 were found to have a conductance identical to the 5-HT3B subunit whilst Br2 receptors were found not to be expressed. This finding has implications for 5-HT3 brain signalling, in which Br1 may play an important role. In conclusion, work here has described how agonists bind to and activate RDL GABA receptors and I have identified a candidate mechanism for the potent insecticidal properties of Ginkgo biloba extracts. I have also confirmed that 5-HT3 receptor brain transcript Br1 forms functional channels with similar properties to the 5-HT3B subunit. Mon, 11 Oct 2010 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/226857 2010-10-11T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/226743 Title: Defining the metabolic effect of peroxisome proliferator-activated receptor δ activation Authors: Roberts, Lee D Abstract: Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that function as ligand activated transcription factors. There are three identified isotypes: PPAR alpha, PPAR gamma and PPAR delta, together controlling the expression of genes involved in inflammation, cell differentiation, proliferation, lipid and carbohydrate metabolism and energy homeostasis. The PPARs are potential targets for the treatment of dyslipidaemia, type II diabetes mellitus and the metabolic syndrome. This thesis uses a multi-platform metabolomics approach, 13C-isotope substrate flux analysis, respirometry and transcriptomics to determine the role PPAR delta and PPAR gamma play in metabolic control both in adipose tissue and systemically. To achieve this, the metabolic phenotype of the 3T3-L1 adipocyte cell line was defined to generate a metabolically phenotyped in vitro model of adipose tissue. The importance of fatty acid alpha-oxidation in the differentiation of adipocytes was emphasised The effects of PPAR delta and PPAR gamma activation in white adipose tissue from the ob/ob mouse model of insulin resistance, and in the phenotyped 3T3-L1 adipocyte model, were investigated. PPAR delta activation was distinguished by oxidative catabolism of fatty acids and citric acid cycle intermediates. Conversely, PPAR gamma activation was identified by the sequestration of lipids into adipose tissue. Moreover, to address the systemic influence of PPAR activation, with a focus on the Cori cycle and the interactions of the liver and skeletal muscle, the metabolic changes that occur in these tissues following PPAR delta and PPAR gamma activation in the ob/ob mouse were examined. PPAR delta activation was characterised by the mobilisation and release of triacylglycerols (TAGs) into circulation as an energy source for peripheral tissues whereas PPAR gamma activation was defined by a reduction and sequestration of circulating TAGs. This thesis has better characterised the role of the PPARs as master regulators of metabolism and emphasised their potential as therapeutic targets for metabolic diseases of global importance. Mon, 07 Jun 2010 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/226743 2010-06-07T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/224844 Title: On the origins of enzyme inhibitor selectivity and promiscuity: a case study of protein kinase binding to staurosporine Authors: Tanramluk, Duangrudee Abstract: Protein kinases are important regulatory enzymes in signal transduction and in cell regulation. Understanding inhibition mechanisms of kinases is important for the further development of new therapies for cancer and inflammatory diseases. I have developed a statistical approach based on the Mantel test to find the relationship between the shapes of ATP binding sites and their affinities for inhibitors. My shape-based dendrogram shows clustering of the kinases based on similarity in shape. I investigate the pocket in terms of conservation of surrounding amino acids and atoms in order to identify the key determinants of ligand binding. I find that the most conserved regions are the main chain atoms in the hinge region and I show that the tetrahydropyran ring of staurosporine causes induced-fit of the glycine rich loop. I apply multiple linear regression to select distances measured between the distinctive parts of residues which correlate with the binding constants. This method allows me to understand the importance of the size of the gatekeeper residue and the closure between the first glycine of the GXGXXG motif and the aspartate of the DFG loop, which act together to promote tight binding to staurosporine. I also find that the greater the number of hydrogen bonds made by the kinase around the methylamine group of staurosporine, the tighter the binding to staurosporine. The website I have developed allows a better understanding of cross reactivity and may be useful for narrowing down the options for a synthetic strategy to design kinase inhibitors. Description: The determinants of protein kinase inhibitor selectivity Tue, 09 Feb 2010 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/224844 2010-02-09T00:00:00Z