DSpace Community:

Molecular  recognition  from  atomic  interactions:   insights  into  drug  discovery

Mon, 07 May 2012 23:00:00 GMT

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.

Expression, purification and characterisation of recombinant chromatin assembly factor 1

Tue, 08 Jan 2013 00:00:00 GMT

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.

MitoInteractome: Mitochondrial protein interactome database, and its application in 'aging network' analysis

Thu, 03 Dec 2009 00:00:00 GMT

Title: MitoInteractome: Mitochondrial protein interactome database, and its application in 'aging network' analysis Abstract: Abstract Background Mitochondria play a vital role in the energy production and apoptotic process of eukaryotic cells. Proteins in the mitochondria are encoded by nuclear and mitochondrial genes. Owing to a large increase in the number of identified mitochondrial protein sequences and completed mitochondrial genomes, it has become necessary to provide a web-based database of mitochondrial protein information. Results We present 'MitoInteractome', a consolidated web-based portal containing a wealth of information on predicted protein-protein interactions, physico-chemical properties, polymorphism, and diseases related to the mitochondrial proteome. MitoInteractome contains 6,549 protein sequences which were extracted from the following databases: SwissProt, MitoP, MitoProteome, HPRD and Gene Ontology database. The first general mitochondrial interactome has been constructed based on the concept of 'homologous interaction' using PSIMAP (Protein Structural Interactome MAP) and PEIMAP (Protein Experimental Interactome MAP). Using the above mentioned methods, protein-protein interactions were predicted for 74 species. The mitochondrial protein interaction data of humans was used to construct a network for the aging process. Analysis of the 'aging network' gave us vital insights into the interactions among proteins that influence the aging process. Conclusion MitoInteractome is a comprehensive database that would (1) aid in increasing our understanding of the molecular functions and interaction networks of mitochondrial proteins, (2) help in identifying new target proteins for experimental research using predicted protein-protein interaction information, and (3) help in identifying biomarkers for diagnosis and new molecular targets for drug development related to mitochondria. MitoInteractome is available at http://mitointeractome.kobic.kr/. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.

The genetic control of growth rate: a systems biology study in yeast.

Fri, 13 Jan 2012 00:00:00 GMT

Title: The genetic control of growth rate: a systems biology study in yeast. Authors: Pir, Pinar; Gutteridge, Alex; Wu, Jian; Rash, Bharat; Kell, Douglas B; Zhang, Nianshu; Oliver, Stephen G Abstract: Abstract Background Control of growth rate is mediated by tight regulation mechanisms in all free-living organisms since long-term survival depends on adaptation to diverse environmental conditions. The yeast, Saccharomyces cerevisiae, when growing under nutrient-limited conditions, controls its growth rate via both nutrient-specific and nutrient-independent gene sets. At slow growth rates, at least, it has been found that the expression of the genes that exert significant control over growth rate (high flux control or HFC genes) is not necessarily regulated by growth rate itself. It has not been determined whether the set of HFC genes is the same at all growth rates or whether it is the same in conditions of nutrient limitation or excess. Results HFC genes were identified in competition experiments in which a population of hemizygous diploid yeast deletants were grown at, or close to, the maximum specific growth rate in either nutrient-limiting or nutrient-sufficient conditions. A hemizygous mutant is one in which one of any pair of homologous genes is deleted in a diploid, These HFC genes divided into two classes: a haploinsufficient (HI) set, where the hemizygous mutants grow slower than the wild type, and a haploproficient (HP) set, which comprises hemizygotes that grow faster than the wild type. The HI set was found to be enriched for genes involved in the processes of gene expression, while the HP set was enriched for genes concerned with the cell cycle and genome integrity. Conclusion A subset of growth-regulated genes have HFC characteristics when grown in conditions where there are few, or no, external constraints on the rate of growth that cells may attain. This subset is enriched for genes that participate in the processes of gene expression, itself (i.e. transcription and translation). The fact that haploproficiency is exhibited by mutants grown at the previously determined maximum rate implies that the control of growth rate in this simple eukaryote represents a trade-off between the selective advantages of rapid growth and the need to maintain the integrity of the genome. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.

Molecular characterization and evolutionary plasticity of protein-protein interfaces

Fri, 01 Jan 2010 00:00:00 GMT

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'.

A metabolomic strategy defines the regulation of lipid content and global metabolism by Delta-9 desaturases in Caenorhabditis elegans

Fri, 20 Jan 2012 00:00:00 GMT

Title: A metabolomic strategy defines the regulation of lipid content and global metabolism by Delta-9 desaturases in Caenorhabditis elegans Authors: Castro, Cecilia; Sar, Funda; Shaw, W. Robert; Mishima, Masanori; Miska, Eric A; Griffin, Julian L Abstract: Abstract Background Caenorhabditis elegans provides a genetically tractable model organism to investigate the network of genes involved in fat metabolism and how regulation is perturbed to produce the complex phenotype of obesity. C. elegans possess the full range of desaturases, including the Δ9 desaturases expressed by fat-5, fat-6 and fat-7. They regulate the biosynthesis of monounsaturated fatty acids, used for the synthesis of lipids including phospholipids, triglycerides and cholesteryl esters. Results Liquid chromatography mass spectrometry (LC-MS), gas chromatography mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy were used to define the metabolome of all the possible knock-outs for the Δ9 desaturases, including for the first time intact lipids. Despite the genes having similar enzymatic roles, excellent discrimination was achievable for all single and viable double mutants highlighting the distinctive roles of fat-6 and fat-7, both expressing steroyl-CoA desaturases. The metabolomic changes extend to aqueous metabolites demonstrating the influence Δ9 desaturases have on regulating global metabolism and highlighting how comprehensive metabolomics is more discriminatory than classically used dyes for fat staining. Conclusions The propagation of metabolic changes across the network of metabolism demonstrates that modification of the Δ9 desaturases places C.elegans into a catabolic state compared with wildtype controls. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.

Genome-wide assessment of the carriers involved in the cellular uptake of drugs: a model system in yeast.

Sun, 23 Oct 2011 23:00:00 GMT

Title: Genome-wide assessment of the carriers involved in the cellular uptake of drugs: a model system in yeast. Authors: Lanthaler, Karin; Bilsland, Elizabeth; Dobson, Paul D; Moss, Harry J; Pir, Pinar; Kell, Douglas B; Oliver, Stephen G Abstract: Abstract Background The uptake of drugs into cells has traditionally been considered to be predominantly via passive diffusion through the bilayer portion of the cell membrane. The recent recognition that drug uptake is mostly carrier-mediated raises the question of which drugs use which carriers. Results To answer this, we have constructed a chemical genomics platform built upon the yeast gene deletion collection, using competition experiments in batch fermenters and robotic automation of cytotoxicity screens, including protection by 'natural' substrates. Using these, we tested 26 different drugs and identified the carriers required for 18 of the drugs to gain entry into yeast cells. Conclusions As well as providing a useful platform technology, these results further substantiate the notion that the cellular uptake of pharmaceutical drugs normally occurs via carrier-mediated transport and indicates that establishing the identity and tissue distribution of such carriers should be a major consideration in the design of safe and effective drugs. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.

How yeast re-programmes its transcriptional profile in response to different nutrient impulses

Sat, 24 Sep 2011 23:00:00 GMT

Title: How yeast re-programmes its transcriptional profile in response to different nutrient impulses Authors: Dikicioglu, Duygu; Karabekmez, Erkan; Rash, Bharat; Pir, Pinar; Kirdar, Betul; Oliver, Stephen G Abstract: Abstract Background A microorganism is able to adapt to changes in its physicochemical or nutritional environment and this is crucial for its survival. The yeast, Saccharomyces cerevisiae, has developed mechanisms to respond to such environmental changes in a rapid and effective manner; such responses may demand a widespread re-programming of gene activity. The dynamics of the re-organization of the cellular activities of S. cerevisiae in response to the sudden and transient removal of either carbon or nitrogen limitation has been studied by following both the short- and long-term changes in yeast's transcriptomic profiles. Results The study, which spans timescales from seconds to hours, has revealed the hierarchy of metabolic and genetic regulatory switches that allow yeast to adapt to, and recover from, a pulse of a previously limiting nutrient. At the transcriptome level, a glucose impulse evoked significant changes in the expression of genes concerned with glycolysis, carboxylic acid metabolism, oxidative phosphorylation, and nucleic acid and sulphur metabolism. In ammonium-limited cultures, an ammonium impulse resulted in the significant changes in the expression of genes involved in nitrogen metabolism and ion transport. Although both perturbations evoked significant changes in the expression of genes involved in the machinery and process of protein synthesis, the transcriptomic response was delayed and less complex in the case of an ammonium impulse. Analysis of the regulatory events by two different system-level, network-based approaches provided further information about dynamic organization of yeast cells as a response to a nutritional change. Conclusions The study provided important information on the temporal organization of transcriptomic organization and underlying regulatory events as a response to both carbon and nitrogen impulse. It has also revealed the importance of a long-term dynamic analysis of the response to the relaxation of a nutritional limitation to understand the molecular basis of the cells' dynamic behaviour. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.

Comprehensive, atomic-level characterization of structurally characterized protein-protein interactions: the PICCOLO database.

Thu, 28 Jul 2011 23:00:00 GMT

Title: Comprehensive, atomic-level characterization of structurally characterized protein-protein interactions: the PICCOLO database. Authors: Bickerton, George R; Higueruelo, Alicia P; Blundell, Tom L Abstract: Abstract Background Structural studies are increasingly providing huge amounts of information on multi-protein assemblies. Although a complete understanding of cellular processes will be dependent on an explicit characterization of the intermolecular interactions that underlie these assemblies and mediate molecular recognition, these are not well described by standard representations. Results Here we present PICCOLO, a comprehensive relational database capturing the details of structurally characterized protein-protein interactions. Interactions are described at the level of interacting pairs of atoms, residues and polypeptide chains, with the physico-chemical nature of the interactions being characterized. Distance and angle terms are used to distinguish 12 different interaction types, including van der Waals contacts, hydrogen bonds and hydrophobic contacts. The explicit aim of PICCOLO is to underpin large-scale analyses of the properties of protein-protein interfaces. This is exemplified by an analysis of residue propensity and interface contact preferences derived from a much larger data set than previously reported. However, PICCOLO also supports detailed inspection of particular systems of interest. Conclusions The current PICCOLO database comprises more than 260 million interacting atom pairs from 38,202 protein complexes. A web interface for the database is available at http://www-cryst.bioc.cam.ac.uk/piccolo. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.

The contrasting roles of PPARdelta and PPARgamma in regulating the metabolic switch between oxidation and storage of fats in white adipose tissue

Wed, 10 Aug 2011 23:00:00 GMT

Title: The contrasting roles of PPARdelta and PPARgamma in regulating the metabolic switch between oxidation and storage of fats in white adipose tissue Authors: Roberts, Lee D; Murray, Andrew J; Menassa, David; Ashmore, Tom; Nicholls, Andrew W; Griffin, Julian L Abstract: Abstract Background The nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and peroxisome proliferator-activated receptor δ (PPARδ) play central roles in regulating metabolism in adipose tissue, as well as being targets for the treatment of insulin resistance. While the role of PPARγ in regulating insulin sensitivity has been well defined, research into PPARδ has been limited until recently due to a scarcity of selective PPARδ agonists. Results The metabolic effects of PPARγ and PPARδ activation have been examined in vivo in white adipose tissue from ob/ob mice and in vitro in cultured 3T3-L1 adipocytes using 1H nuclear magnetic resonance spectroscopy and mass spectrometry metabolomics to understand the receptors' contrasting roles. These steady state measurements were supplemented with 13C-stable isotope substrate labeling to assess fluxes, in addition to respirometry and transcriptomic microarray analysis. The metabolic effects of the receptors were readily distinguished, with PPARγ activation characterized by increased fat storage, synthesis and elongation, while PPARδ activation caused increased fatty acid β-oxidation, tricarboxylic acid cycle rate and oxidation of extracellular branch chain amino acids. Stimulated glycolysis and increased fatty acid desaturation were common pathways for the agonists. Conclusions PPARγ and PPARδ restore insulin sensitivity through varying mechanisms. PPARδ activation increases total oxidative metabolism in white adipose tissue, a tissue not traditionally thought of as oxidative. However, the increased metabolism of branch chain amino acids may provide a mechanism for muscle atrophy, which has been linked to activation of this nuclear receptor. PPARδ has a role as an anti-obesity target and as an anti-diabetic, and hence may target both the cause and consequences of dyslipidemia. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.

Atomic resolution structure of EhpR: phenazine resistance in Enterobacter agglomerans Eh1087 follows principles of bleomycin / mitomycin C resistance in other bacteria

Tue, 16 Aug 2011 23:00:00 GMT

Title: Atomic resolution structure of EhpR: phenazine resistance in Enterobacter agglomerans Eh1087 follows principles of bleomycin / mitomycin C resistance in other bacteria Authors: Yu, Shen; Vit, Allegra; Devenish, Sean; Mahanty, H Khris; Itzen, Aymelt; Goody, Roger S; Blankenfeldt, Wulf Abstract: Abstract Background The phenazines are redox-active secondary metabolites that a large number of bacterial strains produce and excrete into the environment. They possess antibiotic activity owing to the fact that they can reduce molecular oxygen to toxic reactive oxygen species. In order to take advantage of this activity, phenazine producers need to protect themselves against phenazine toxicity. Whereas it is believed that phenazine-producing pseudomonads possess highly active superoxide dismutases and catalases, it has recently been found that the plant-colonizing bacterium Enterobacter agglomerans expresses a small gene ehpR to render itself resistant towards D-alanyl-griseoluteic acid, the phenazine antibiotic produced by this strain. Results To understand the resistance mechanism installed by EhpR we have determined its crystal structure in the apo form at 2.15 Å resolution and in complex with griseoluteic acid at 1.01 Å, respectively. While EhpR shares a common fold with glyoxalase-I/bleomycin resistance proteins, the ligand binding site does not contain residues that some related proteins employ to chemically alter their substrates. Binding of the antibiotic is mediated by π-stacking interactions of the aromatic moiety with the side chains of aromatic amino acids and by a few polar interactions. The dissociation constant KD between EhpR and griseoluteic acid was quantified as 244 ± 45 μM by microscale thermophoresis measurements. Conclusions The data accumulated here suggest that EhpR confers resistance by binding D-alanyl-griseoluteic acid and acting as a chaperone involved in exporting the antibiotic rather than by altering it chemically. It is tempting to speculate that EhpR acts in concert with EhpJ, a transport protein of the major facilitator superfamily that is also encoded in the phenazine biosynthesis operon of E. agglomerans. The low affinity of EhpR for griseoluteic acid may be required for its physiological function. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.

Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles

Thu, 09 Jun 2011 23:00:00 GMT

Title: Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles Authors: O'Brien, John A; Lummis, Sarah C R Abstract: Abstract Background Biolistic transfection is proving an increasingly popular method of incorporating DNA or RNA into cells that are difficult to transfect using traditional methods. The technique routinely uses 'microparticles', which are ~1 μm diameter projectiles, fired into tissues using pressurised gas. These microparticles are efficient at delivering DNA into cells, but cannot efficiently transfect small cells and may cause significant tissue damage, thus limiting their potential usefulness. Here we describe the use of 40 nm diameter projectiles - nanoparticles - in biolistic transfections to determine if they are a suitable alternative to microparticles. Results Examination of transfection efficiencies in HEK293 cells, using a range of conditions including different DNA concentrations and different preparation procedures, reveals similar behaviour of microparticles and nanoparticles. The use of nanoparticles, however, resulted in ~30% fewer damaged HEK293 cells following transfection. Biolistic transfection of mouse ear tissue revealed similar depth penetration for the two types of particles, and also showed that < 10% of nuclei were damaged in nanoparticle-transfected samples, compared to > 20% in microparticle-transfected samples. Visualising details of small cellular structures was also considerably enhanced when using nanoparticles. Conclusions We conclude that nanoparticles are as efficient for biolistic transfection as microparticles, and are more appropriate for use in small cells, when examining cellular structures and/or where tissue damage is a problem. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.

Probing the druggability of the Notch1 ankyrin domain using a fragment-based approach

Tue, 08 Feb 2011 00:00:00 GMT

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.

Genome-wide dynamics of a bacterial response to antibiotics that target the cell envelope

Tue, 10 May 2011 23:00:00 GMT

Title: Genome-wide dynamics of a bacterial response to antibiotics that target the cell envelope Authors: Hesketh, Andrew; Hill, Chris; Mokhtar, Jehan; Novotna, Gabriela; Tran, Ngat; Bibb, Mervyn; Hong, Hee-Jeon Abstract: AbstractBackgroundA decline in the discovery of new antibacterial drugs, coupled with a persistent rise in the occurrence of drug-resistant bacteria, has highlighted antibiotics as a diminishing resource. The future development of new drugs with novel antibacterial activities requires a detailed understanding of adaptive responses to existing compounds. This study uses Streptomyces coelicolor A3(2) as a model system to determine the genome-wide transcriptional response following exposure to three antibiotics (vancomycin, moenomycin A and bacitracin) that target distinct stages of cell wall biosynthesis. ResultsA generalised response to all three antibiotics was identified that involves activation of transcription of the cell envelope stress sigma factor E, together with elements of the stringent response, and of the heat, osmotic and oxidative stress regulons. Attenuation of this system by deletion of genes encoding the osmotic stress sigma factor B or the ppGpp synthetase RelA reduced resistance to both vancomycin and bacitracin. Many antibiotic-specific transcriptional changes were identified, representing cellular processes potentially important for tolerance to each antibiotic. Sensitivity studies using mutants constructed on the basis of the transcriptome profiling confirmed a role for several such genes in antibiotic resistance, validating the usefulness of the approach. ConclusionsAntibiotic inhibition of bacterial cell wall biosynthesis induces both common and compound-specific transcriptional responses. Both can be exploited to increase antibiotic susceptibility. Regulatory networks known to govern responses to environmental and nutritional stresses are also at the core of the common antibiotic response, and likely help cells survive until any specific resistance mechanisms are fully functional. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.

Comparative evaluation of linear and exponential amplification techniques for expression profiling at the single-cell level

Tue, 07 Mar 2006 00:00:00 GMT

Title: Comparative evaluation of linear and exponential amplification techniques for expression profiling at the single-cell level Authors: Subkhankulova, Tatiana; Livesey, Frederick J Abstract: Abstract Background Single-cell microarray expression profiling requires 108-109-fold amplification of the picogram amounts of total RNA typically found in eukaryotic cells. Several methods for RNA amplification are in general use, but little consideration has been given to the comparative analysis of those methods in terms of the overall validity of the data generated when amplifying from single-cell amounts of RNA, rather than their empirical performance in single studies. Results We tested the performance of three methods for amplifying single-cell amounts of RNA under ideal conditions: T7-based in vitro transcription; switching mechanism at 5' end of RNA template (SMART) PCR amplification; and global PCR amplification. All methods introduced amplification-dependent noise when mRNA was amplified 108-fold, compared with data from unamplified cDNA. PCR-amplified cDNA demonstrated the smallest number of differences between two parallel replicate samples and the best correlation between independent amplifications from the same cell type, with SMART outperforming global PCR amplification. SMART had the highest true-positive rate and the lowest false-positive rate when comparing expression between two different cell types, but had the lowest absolute discovery rate of all three methods. Direct comparison of the performance of SMART and global PCR amplification on single-cell amounts of total RNA and on single neural stem cells confirmed these findings. Conclusion Under the conditions tested, PCR amplification was more reliable than linear amplification for detecting true expression differences between samples. SMART amplification had a higher true-positive rate than global amplification, but at the expense of a considerably lower absolute discovery rate and a systematic compression of observed expression ratios. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.

Application of phage display to high throughput antibody generation and characterisation

Thu, 29 Nov 2007 00:00:00 GMT

Title: Application of phage display to high throughput antibody generation and characterisation Authors: Schofield, Darren J; Pope, Anthony R; Clementel, Veronica; Buckell, Jenny; Chapple, Susan D J; Clarke, Kay F; Conquer, Jennie S; Crofts, Anna M; Crowther, Sandra R E; Dyson, Michael R; Flack, Gillian; Griffin, Gareth J; Hooks, Yvette; Howat, William J; Kolb-Kokocinski, Anja; Kunze, Susan; Martin, Cecile D; Maslen, Gareth L; Mitchell, Joanne N; O'Sullivan, Maureen; Perera, Rajika L; Roake, Wendy; Shadbolt, S Paul; Vincent, Karen J; Warford, Anthony; Wlson, Wendy E; Xie, Jane; Young, Joyce L; McCafferty, John Abstract: Abstract We have created a high quality phage display library containing over 1010 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry assay. Furthermore, positive staining by immunohistochemistry on tissue microarrays was found for 37% (143/381) of antibodies. Thus, we have demonstrated the potential of and illuminated the issues associated with genome-wide monoclonal antibody generation. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.

Application of the comprehensive set of heterozygous yeast deletion mutants to elucidate the molecular basis of cellular chromium toxicity

Tue, 18 Dec 2007 00:00:00 GMT

Title: Application of the comprehensive set of heterozygous yeast deletion mutants to elucidate the molecular basis of cellular chromium toxicity Authors: Holland, Sara; Lodwig, Emma; Sideri, Theodora; Reader, Tom; Clarke, Ian; Gkargkas, Konstantinos; Hoyle, David C; Delneri, Daniela; Oliver, Stephen G; Avery, Simon V Abstract: Abstract Background The serious biological consequences of metal toxicity are well documented, but the key modes of action of most metals are unknown. To help unravel molecular mechanisms underlying the action of chromium, a metal of major toxicological importance, we grew over 6,000 heterozygous yeast mutants in competition in the presence of chromium. Microarray-based screens of these heterozygotes are truly genome-wide as they include both essential and non-essential genes. Results The screening data indicated that proteasomal (protein degradation) activity is crucial for cellular chromium (Cr) resistance. Further investigations showed that Cr causes the accumulation of insoluble and toxic protein aggregates, which predominantly arise from proteins synthesised during Cr exposure. A protein-synthesis defect provoked by Cr was identified as mRNA mistranslation, which was oxygen-dependent. Moreover, Cr exhibited synergistic toxicity with a ribosome-targeting drug (paromomycin) that is known to act via mistranslation, while manipulation of translational accuracy modulated Cr toxicity. Conclusion The datasets from the heterozygote screen represent an important public resource that may be exploited to discover the toxic mechanisms of chromium. That potential was validated here with the demonstration that mRNA mistranslation is a primary cause of cellular Cr toxicity. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.

Increased hepatic oxidative metabolism distinguishes the action of Peroxisome proliferator-activated receptor delta from Peroxisome proliferator-activated receptor gamma in the ob/ob mouse

Mon, 07 Dec 2009 00:00:00 GMT

Title: Increased hepatic oxidative metabolism distinguishes the action of Peroxisome proliferator-activated receptor delta from Peroxisome proliferator-activated receptor gamma in the ob/ob mouse Authors: Roberts, Lee D; Hassall, David G; Winegar, Deborah A; Haselden, John N; Nicholls, Andrew W; Griffin, Julian L Abstract: Abstract Background The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors and members of the nuclear receptor superfamily. The PPAR family consists of three members: PPARα, PPARγ, and PPARδ. PPARδ controls the transcription of genes involved in multiple physiological pathways, including cellular differentiation, lipid metabolism and energy homeostasis. The receptor is expressed almost ubiquitously, with high expression in liver and skeletal muscle. Although the physiological ligands of PPARδ remain undefined, a number of high affinity synthetic ligands have been developed for the receptor as a therapeutic target for type 2 diabetes mellitus, dyslipidemia and the metabolic syndrome. Methods In this study, the metabolic role of PPARδ activation has been investigated in liver, skeletal muscle, blood serum and white adipose tissue from ob/ob mice using a high affinity synthetic ligand and contrasted with PPARγ activation. To maximize the analytical coverage of the metabolome, 1H-nuclear magnetic resonance (1H-NMR) spectroscopy, gas chromatography-mass spectrometry (GC-MS) and ultra performance liquid chromatography-mass spectrometry (UPLC-MS) were used to examine metabolites from tissue extracts. Results Analysis by multivariate statistics demonstrated that PPARδ activation profoundly affected glycolysis, gluconeogenesis, the TCA cycle and linoleic acid and α-linolenic acid essential fatty acid pathways. Conclusions Although activation of both PPARδ and PPARγ lead to increased insulin sensitivity and glucose tolerance, PPARδ activation was functionally distinct from PPARγ activation, and was characterized by increased hepatic and peripheral fatty acid oxidative metabolism, demonstrating the distinctive catabolic role of this receptor compared with PPARγ.

PEDRo: A database for storing, searching and disseminating experimental proteomics data

Thu, 16 Sep 2004 23:00:00 GMT

Title: PEDRo: A database for storing, searching and disseminating experimental proteomics data Authors: Garwood, Kevin; McLaughlin, Thomas; Garwood, Chris; Joens, Scott; Morrison, Norman; Taylor, Christopher F; Carroll, Kathleen; Evans, Caroline; Whetton, Anthony D; Hart, Sarah; Stead, David; Yin, Zhikang; Brown, Alistair J P; Hesketh, Andrew; Chater, Keith; Hansson, Lena; Mewissen, Muriel; Ghazal, Peter; Howard, Julie; Lilley, Kathryn S; Gaskell, Simon J; Brass, Andy; Hubbard, Simon J; Oliver, Stephen G; Paton, Norman W Abstract: Abstract Background Proteomics is rapidly evolving into a high-throughput technology, in which substantial and systematic studies are conducted on samples from a wide range of physiological, developmental, or pathological conditions. Reference maps from 2D gels are widely circulated. However, there is, as yet, no formally accepted standard representation to support the sharing of proteomics data, and little systematic dissemination of comprehensive proteomic data sets. Results This paper describes the design, implementation and use of a Proteome Experimental Data Repository (PEDRo), which makes comprehensive proteomics data sets available for browsing, searching and downloading. It is also serves to extend the debate on the level of detail at which proteomics data should be captured, the sorts of facilities that should be provided by proteome data management systems, and the techniques by which such facilities can be made available. Conclusions The PEDRo database provides access to a collection of comprehensive descriptions of experimental data sets in proteomics. Not only are these data sets interesting in and of themselves, they also provide a useful early validation of the PEDRo data model, which has served as a starting point for the ongoing standardisation activity through the Proteome Standards Initiative of the Human Proteome Organisation. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.

Activation of microglial NADPH oxidase is synergistic with glial iNOS expression in inducing neuronal death: a dual-key mechanism of inflammatory neurodegeneration

Sun, 11 Sep 2005 23:00:00 GMT

Title: Activation of microglial NADPH oxidase is synergistic with glial iNOS expression in inducing neuronal death: a dual-key mechanism of inflammatory neurodegeneration Authors: Mander, Palwinder K; Brown, Guy C Abstract: Abstract Background Inflammation-activated glia are seen in many CNS pathologies and may kill neurons through the release of cytotoxic mediators, such as nitric oxide from inducible NO synthase (iNOS), and possibly superoxide from NADPH oxidase (NOX). We set out to determine the relative role of these species in inducing neuronal death, and to test the dual-key hypothesis that the production of both species simultaneously is required for significant neuronal death. Methods Primary co-cultures of cerebellar granule neurons and glia from rats were used to investigate the effect of NO (from iNOS, following lipopolysaccharide (LPS) and/or cytokine addition) or superoxide/hydrogen peroxide (from NOX, following phorbol 12-myristate 13-acetate (PMA), ATP analogue (BzATP), interleukin-1β (IL-1β) or arachidonic acid (AA) addition) on neuronal survival. Results Induction of glial iNOS caused little neuronal death. Similarly, activation of NOX alone resulted in little or no neuronal death. However, if NOX was activated (by PMA or BzATP) in the presence of iNOS (induced by LPS and interferon-γ) then substantial delayed neuronal death occurred over 48 hours, which was prevented by inhibitors of iNOS (1400W), NOX (apocynin) or a peroxynitrite decomposer (FeTPPS). Neurons and glia were also found to stain positive for nitrotyrosine (a putative marker of peroxynitrite) only when both iNOS and NOX were simultaneously active. If NOX was activated by weak stimulators (IL-1β, AA or the fibrillogenic prion peptide PrP106-126) in the presence of iNOS, it caused microglial proliferation and delayed neurodegeneration over 6 days, which was prevented by iNOS or NOX inhibitors, a peroxynitrite decomposer or a NMDA-receptor antagonist (MK-801). Conclusion These results suggest a dual-key mechanism, whereby glial iNOS or microglial NOX activation alone is relatively benign, but if activated simultaneously are synergistic in killing neurons, through generating peroxynitrite. This mechanism may mediate inflammatory neurodegeneration in response to cytokines, bacteria, ATP, arachidonate and pathological prions, in which case neurons may be protected by iNOS or NOX inhibitors, or scavengers of NO, superoxide or peroxynitrite. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.