register interest

Dr Brian D Marsden

Research Area: Protein Science and Structural Biology
Technology Exchange: Bioinformatics, Computational biology and Drug discovery
Scientific Themes: Protein Science & Structural Biology and Genetics & Genomics
Keywords: Computational Chemistry, Cheminformatics, Laboratory Informatics Management Systems (LIMS), In silico drug-discovery, Data dissemination and Data Visualisation
Web Links:
iSee - a new way of publishing 3D data.
http://www.thesgc.org/iSee

iSee - a new way of publishing 3D data. http://www.thesgc.org/iSee

Peer-reviewed structural biology data dissemination, the iSee way

Peer-reviewed structural biology data dissemination, the iSee way

Capturing and mining data at the SGC requires a complex and well developed Laboratory Informatics Management System

Capturing and mining data at the SGC requires a complex and well developed Laboratory Informatics ...

The SGC Research Informatics group is responsible the provision of computational resources for all members of the SGC including data management, sample tracking, in silico modelling support plus provision of public access to SGC data.

Our research is focussed upon novel visualisation tools and methodology for structural biology and drug discovery problems.

Name Department Institution Country
Professor Frank von Delft Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Ruben Abagyan The Scripps Research Institute United States
Professor Charlotte Deane University of Oxford United Kingdom
Professor Liz Carpenter Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Dr Alex Bullock Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Dr Opher Gileadi Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Professor Chas Bountra Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Professor Udo CT Oppermann Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Dr Nicola A Burgess-Brown Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Professor Panagis Filippakopoulos Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Associate Professor Wyatt W Yue Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Professor Kim Midwood Kennedy Institute, NDORMS University of Oxford United Kingdom
Aungier S, Cartwright AJ, Schwenzer A, Marshall J, Dyson MR, Slavny P, Parthiban K, Karatt-Vellatt A, Sahbudin I, Culbert E et al. 2019. Response to: 'Potential roles for tenascin in (very) early diagnosis and treatment of rheumatoid arthritis' by Cutolo et al. Ann Rheum Dis, | Read more

Aungier SR, Cartwright AJ, Schwenzer A, Marshall JL, Dyson MR, Slavny P, Parthiban K, Karatt-Vellatt A, Sahbudin I, Culbert E et al. 2019. Targeting early changes in the synovial microenvironment: a new class of immunomodulatory therapy? Ann Rheum Dis, 78 (2), pp. 186-191. | Show Abstract | Read more

OBJECTIVES: Controlled immune responses rely on integrated crosstalk between cells and their microenvironment. We investigated whether targeting proinflammatory signals from the extracellular matrix that persist during pathological inflammation provides a viable strategy to treat rheumatoid arthritis (RA). METHODS: Monoclonal antibodies recognising the fibrinogen-like globe (FBG) of tenascin-C were generated by phage display. Clones that neutralised FBG activation of toll-like receptor 4 (TLR4), without impacting pathogenic TLR4 activation, were epitope mapped by crystallography. Antibodies stained synovial biopsies of patients at different stages of RA development. Antibody efficacy in preventing RA synovial cell cytokine release, and in modulating collagen-induced arthritis in rats, was assessed. RESULTS: Tenascin-C is expressed early in the development of RA, even before disease diagnosis, with higher levels in the joints of people with synovitis who eventually developed RA than in people whose synovitis spontaneously resolved. Anti-FBG antibodies inhibited cytokine release by RA synovial cells and prevented disease progression and tissue destruction during collagen-induced arthritis. CONCLUSIONS: Early changes in the synovial microenvironment contribute to RA progression; blocking proinflammatory signals from the matrix can ameliorate experimental arthritis. These data highlight a new drug class that could offer early, disease-specific immune modulation in RA, without engendering global immune suppression.

Liu L, Damerell DR, Koukouflis L, Tong Y, Marsden BD, Schapira M. 2019. UbiHub: a data hub for the explorers of ubiquitination pathways. Bioinformatics, | Show Abstract | Read more

Motivation: Protein ubiquitination plays a central role in important cellular machineries such as protein degradation or chromatin-mediated signalling. With the recent discovery of the first potent ubiquitin-specific protease inhibitors, and the maturation of PROTACs as promising chemical tools to exploit the ubiquitin-proteasome system, protein target classes associated with ubiquitination pathways are becoming the focus of intense drug discovery efforts. Results: We have developed UbiHub, an online resource that can be used to visualize a diverse array of biological, structural, and chemical data on phylogenetic trees of human protein families involved in ubiquitination signalling, including E3 ligases and deubiquitinases. This interface can inform target prioritization and drug design, and serves as a navigation tool for medicinal chemists, structural and cell biologists exploring ubiquitination pathways. Availability: http://ubihub.thesgc.org.

D'Ascenzio M, Pugh KM, Konietzny R, Berridge G, Tallant C, Hashem S, Monteiro O, Thomas JR, Schirle M, Knapp S et al. 2019. An Activity-Based Probe Targeting Non-Catalytic, Highly Conserved Amino Acid Residues within Bromodomains. Angew Chem Int Ed Engl, 58 (4), pp. 1007-1012. | Show Abstract | Read more

Bromodomain-containing proteins are epigenetic modulators involved in a wide range of cellular processes, from recruitment of transcription factors to pathological disruption of gene regulation and cancer development. Since the druggability of these acetyl-lysine reader domains was established, efforts were made to develop potent and selective inhibitors across the entire family. Here we report the development of a small molecule-based approach to covalently modify recombinant and endogenous bromodomain-containing proteins by targeting a conserved lysine and a tyrosine residue in the variable ZA or BC loops. Moreover, the addition of a reporter tag allowed in-gel visualization and pull-down of the desired bromodomains.

Damerell DR, Strain-Damerell C, Garsot S, Joyce SP, Barrett P, Marsden BD. 2019. SATurn: a modular bioinformatics framework for the design of robust maintainable web-based and standalone applications. Bioinformatics, 35 (2), pp. 349-351. | Show Abstract | Read more

Summary: SATurn is a modular, open-source, bioinformatics platform designed to specifically address the problems of maintenance and longevity commonly associated with the development of simple tools funded by academic research grants. Applications developed in SATurn can be deployed as web-based tools, standalone applications or hybrid tools which have the benefits of both. Within the Structural Genomics Consortium we have utilized SATurn to create a bioinformatics portal which routinely supports a diverse group of scientists including those interested in structural biology, cloning, glycobiology and chemical biology. Availability and implementation: https://github.com/ddamerell53/SATurn. Supplementary information: Supplementary data are available at Bioinformatics online.

Müller S, Ackloo S, Arrowsmith CH, Bauser M, Baryza JL, Blagg J, Böttcher J, Bountra C, Brown PJ, Bunnage ME et al. 2018. Donated chemical probes for open science. Elife, 7 | Show Abstract | Read more

Potent, selective and broadly characterized small molecule modulators of protein function (chemical probes) are powerful research reagents. The pharmaceutical industry has generated many high-quality chemical probes and several of these have been made available to academia. However, probe-associated data and control compounds, such as inactive structurally related molecules and their associated data, are generally not accessible. The lack of data and guidance makes it difficult for researchers to decide which chemical tools to choose. Several pharmaceutical companies (AbbVie, Bayer, Boehringer Ingelheim, Janssen, MSD, Pfizer, and Takeda) have therefore entered into a pre-competitive collaboration to make available a large number of innovative high-quality probes, including all probe-associated data, control compounds and recommendations on use (<ext-link ext-link-type="uri" xlink:href="https://openscienceprobes.sgc-frankfurt.de">https://openscienceprobes.sgc-frankfurt.de</ext-link><ext-link ext-link-type="uri" xlink:href="https://openscienceprobes.sgc-frankfurt.de/">/</ext-link>). Here we describe the chemical tools and target-related knowledge that have been made available, and encourage others to join the project.

Zuliani-Alvarez L, Marzeda AM, Deligne C, Schwenzer A, McCann FE, Marsden BD, Piccinini AM, Midwood KS. 2017. Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers. Nat Commun, 8 (1), pp. 1595. | Show Abstract | Read more

Pattern recognition underpins innate immunity; the accurate identification of danger, including infection, injury, or tumor, is key to an appropriately targeted immune response. Pathogen detection is increasingly well defined mechanistically, but the discrimination of endogenous inflammatory triggers remains unclear. Tenascin-C, a matrix protein induced upon tissue damage and expressed by tumors, activates toll-like receptor 4 (TLR4)-mediated sterile inflammation. Here we map three sites within tenascin-C that directly and cooperatively interact with TLR4. We also identify a conserved inflammatory epitope in related proteins from diverse families, and demonstrate that its presence targets molecules for TLR detection, while its absence enables escape of innate immune surveillance. These data reveal a unique molecular code that defines endogenous proteins as inflammatory stimuli by marking them for recognition by TLRs.

Bradley AR, Echalier A, Fairhead M, Strain-Damerell C, Brennan P, Bullock AN, Burgess-Brown NA, Carpenter EP, Gileadi O, Marsden BD et al. 2017. The SGC beyond structural genomics: redefining the role of 3D structures by coupling genomic stratification with fragment-based discovery. Essays Biochem, 61 (5), pp. 495-503. | Show Abstract | Read more

The ongoing explosion in genomics data has long since outpaced the capacity of conventional biochemical methodology to verify the large number of hypotheses that emerge from the analysis of such data. In contrast, it is still a gold-standard for early phenotypic validation towards small-molecule drug discovery to use probe molecules (or tool compounds), notwithstanding the difficulty and cost of generating them. Rational structure-based approaches to ligand discovery have long promised the efficiencies needed to close this divergence; in practice, however, this promise remains largely unfulfilled, for a host of well-rehearsed reasons and despite the huge technical advances spearheaded by the structural genomics initiatives of the noughties. Therefore the current, fourth funding phase of the Structural Genomics Consortium (SGC), building on its extensive experience in structural biology of novel targets and design of protein inhibitors, seeks to redefine what it means to do structural biology for drug discovery. We developed the concept of a Target Enabling Package (TEP) that provides, through reagents, assays and data, the missing link between genetic disease linkage and the development of usefully potent compounds. There are multiple prongs to the ambition: rigorously assessing targets' genetic disease linkages through crowdsourcing to a network of collaborating experts; establishing a systematic approach to generate the protocols and data that comprise each target's TEP; developing new, X-ray-based fragment technologies for generating high quality chemical matter quickly and cheaply; and exploiting a stringently open access model to build multidisciplinary partnerships throughout academia and industry. By learning how to scale these approaches, the SGC aims to make structures finally serve genomics, as originally intended, and demonstrate how 3D structures systematically allow new modes of druggability to be discovered for whole classes of targets.

Chanalaris A, Doherty C, Marsden BD, Bambridge G, Wren SP, Nagase H, Troeberg L. 2017. Suramin Inhibits Osteoarthritic Cartilage Degradation by Increasing Extracellular Levels of Chondroprotective Tissue Inhibitor of Metalloproteinases 3. Mol Pharmacol, 92 (4), pp. 459-468. | Show Abstract | Read more

Osteoarthritis is a common degenerative joint disease for which no disease-modifying drugs are currently available. Attempts to treat the disease with small molecule inhibitors of the metalloproteinases that degrade the cartilage matrix have been hampered by a lack of specificity. We aimed to inhibit cartilage degradation by augmenting levels of the endogenous metalloproteinase inhibitor, tissue inhibitor of metalloproteinases (TIMP)-3, through blocking its interaction with the endocytic scavenger receptor, low-density lipoprotein receptor-related protein 1 (LRP1). We discovered that suramin (C51H40N6O23S6) bound to TIMP-3 with a KD value of 1.9 ± 0.2 nM and inhibited its endocytosis via LRP1, thus increasing extracellular levels of TIMP-3 and inhibiting cartilage degradation by the TIMP-3 target enzyme, adamalysin-like metalloproteinase with thrombospondin motifs 5. NF279 (8,8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt), a structural analog of suramin, has an increased affinity for TIMP-3 and increased ability to inhibit TIMP-3 endocytosis and protect cartilage. Suramin is thus a promising scaffold for the development of novel therapeutics to increase TIMP-3 levels and inhibit cartilage degradation in osteoarthritis.

Pearce NM, Krojer T, Bradley AR, Collins P, Nowak RP, Talon R, Marsden BD, Kelm S, Shi J, Deane CM, von Delft F. 2017. A multi-crystal method for extracting obscured crystallographic states from conventionally uninterpretable electron density. Nat Commun, 8 (1), pp. 15123. | Show Abstract | Read more

In macromolecular crystallography, the rigorous detection of changed states (for example, ligand binding) is difficult unless signal is strong. Ambiguous ('weak' or 'noisy') density is experimentally common, since molecular states are generally only fractionally present in the crystal. Existing methodologies focus on generating maximally accurate maps whereby minor states become discernible; in practice, such map interpretation is disappointingly subjective, time-consuming and methodologically unsound. Here we report the PanDDA method, which automatically reveals clear electron density for the changed state-even from inaccurate maps-by subtracting a proportion of the confounding 'ground state'; changed states are objectively identified from statistical analysis of density distributions. The method is completely general, implying new best practice for all changed-state studies, including the routine collection of multiple ground-state crystals. More generally, these results demonstrate: the incompleteness of atomic models; that single data sets contain insufficient information to model them fully; and that accuracy requires further map-deconvolution approaches.

Pearce NM, Bradley AR, Krojer T, Marsden BD, Deane CM, von Delft F. 2017. Partial-occupancy binders identified by the Pan-Dataset Density Analysis method offer new chemical opportunities and reveal cryptic binding sites. Struct Dyn, 4 (3), pp. 032104. | Show Abstract | Read more

Crystallographic fragment screening uses low molecular weight compounds to probe the protein surface and although individual protein-fragment interactions are high quality, fragments commonly bind at low occupancy, historically making identification difficult. However, our new Pan-Dataset Density Analysis method readily identifies binders missed by conventional analysis: for fragment screening data of lysine-specific demethylase 4D (KDM4D), the hit rate increased from 0.9% to 10.6%. Previously unidentified fragments reveal multiple binding sites and demonstrate: the versatility of crystallographic fragment screening; that surprisingly large conformational changes are possible in crystals; and that low crystallographic occupancy does not by itself reflect a protein-ligand complex's significance.

Deane CM, Wall ID, Green DVS, Marsden BD, Bradley AR. 2017. WONKA and OOMMPPAA: analysis of protein-ligand interaction data to direct structure-based drug design. Acta Crystallogr D Struct Biol, 73 (Pt 3), pp. 279-285. | Show Abstract | Read more

In this work, two freely available web-based interactive computational tools that facilitate the analysis and interpretation of protein-ligand interaction data are described. Firstly, WONKA, which assists in uncovering interesting and unusual features (for example residue motions) within ensembles of protein-ligand structures and enables the facile sharing of observations between scientists. Secondly, OOMMPPAA, which incorporates protein-ligand activity data with protein-ligand structural data using three-dimensional matched molecular pairs. OOMMPPAA highlights nuanced structure-activity relationships (SAR) and summarizes available protein-ligand activity data in the protein context. In this paper, the background that led to the development of both tools is described. Their implementation is outlined and their utility using in-house Structural Genomics Consortium (SGC) data sets and openly available data from the PDB and ChEMBL is described. Both tools are freely available to use and download at http://wonka.sgc.ox.ac.uk/WONKA/ and http://oommppaa.sgc.ox.ac.uk/OOMMPPAA/.

Krojer T, Talon R, Pearce N, Collins P, Douangamath A, Brandao-Neto J, Dias A, Marsden B, von Delft F. 2017. The XChemExplorer graphical workflow tool for routine or large-scale protein-ligand structure determination. Acta Crystallogr D Struct Biol, 73 (Pt 3), pp. 267-278. | Show Abstract | Read more

XChemExplorer (XCE) is a data-management and workflow tool to support large-scale simultaneous analysis of protein-ligand complexes during structure-based ligand discovery (SBLD). The user interfaces of established crystallographic software packages such as CCP4 [Winn et al. (2011), Acta Cryst. D67, 235-242] or PHENIX [Adams et al. (2010), Acta Cryst. D66, 213-221] have entrenched the paradigm that a `project' is concerned with solving one structure. This does not hold for SBLD, where many almost identical structures need to be solved and analysed quickly in one batch of work. Functionality to track progress and annotate structures is essential. XCE provides an intuitive graphical user interface which guides the user from data processing, initial map calculation, ligand identification and refinement up until data dissemination. It provides multiple entry points depending on the need of each project, enables batch processing of multiple data sets and records metadata, progress and annotations in an SQLite database. XCE is freely available and works on any Linux and Mac OS X system, and the only dependency is to have the latest version of CCP4 installed. The design and usage of this tool are described here, and its usefulness is demonstrated in the context of fragment-screening campaigns at the Diamond Light Source. It is routinely used to analyse projects comprising 1000 data sets or more, and therefore scales well to even very large ligand-design projects.

Cox OB, Krojer T, Collins P, Monteiro O, Talon R, Bradley A, Fedorov O, Amin J, Marsden BD, Spencer J et al. 2016. A poised fragment library enables rapid synthetic expansion yielding the first reported inhibitors of PHIP(2), an atypical bromodomain. Chem Sci, 7 (3), pp. 2322-2330. | Show Abstract | Read more

Research into the chemical biology of bromodomains has been driven by the development of acetyl-lysine mimetics. The ligands are typically anchored by binding to a highly conserved asparagine residue. Atypical bromodomains, for which the asparagine is mutated, have thus far proven elusive targets, including PHIP(2) whose parent protein, PHIP, has been linked to disease progression in diabetes and cancers. The PHIP(2) binding site contains a threonine in place of asparagine, and solution screening have yielded no convincing hits. We have overcome this hurdle by combining the sensitivity of X-ray crystallography, used as the primary fragment screen, with a strategy for rapid follow-up synthesis using a chemically-poised fragment library, which allows hits to be readily modified by parallel chemistry both peripherally and in the core. Our approach yielded the first reported hit compounds of PHIP(2) with measurable IC50 values by an AlphaScreen competition assay. The follow-up libraries of four poised fragment hits improved potency into the sub-mM range while showing good ligand efficiency and detailed structural data.

Arrowsmith CH, Audia JE, Austin C, Baell J, Bennett J, Blagg J, Bountra C, Brennan PE, Brown PJ, Bunnage ME et al. 2015. The promise and peril of chemical probes (vol 11, pg 536, 2015) NATURE CHEMICAL BIOLOGY, 11 (11), pp. 887-887. | Read more

Arrowsmith CH, Audia JE, Austin C, Baell J, Bennett J, Blagg J, Bountra C, Brennan PE, Brown PJ, Bunnage ME et al. 2015. Corrigendum: The promise and peril of chemical probes. Nat Chem Biol, 11 (11), pp. 887. | Read more

Bradley AR, Wall ID, von Delft F, Green DVS, Deane CM, Marsden BD. 2015. WONKA: objective novel complex analysis for ensembles of protein-ligand structures. J Comput Aided Mol Des, 29 (10), pp. 963-973. | Show Abstract | Read more

WONKA is a tool for the systematic analysis of an ensemble of protein-ligand structures. It makes the identification of conserved and unusual features within such an ensemble straightforward. WONKA uses an intuitive workflow to process structural co-ordinates. Ligand and protein features are summarised and then presented within an interactive web application. WONKA's power in consolidating and summarising large amounts of data is described through the analysis of three bromodomain datasets. Furthermore, and in contrast to many current methods, WONKA relates analysis to individual ligands, from which we find unusual and erroneous binding modes. Finally the use of WONKA as an annotation tool to share observations about structures is demonstrated. WONKA is freely available to download and install locally or can be used online at http://wonka.sgc.ox.ac.uk.

Arrowsmith CH, Audia JE, Austin C, Baell J, Bennett J, Blagg J, Bountra C, Brennan PE, Brown PJ, Bunnage ME et al. 2015. The promise and peril of chemical probes. Nat Chem Biol, 11 (8), pp. 536-541. | Show Abstract | Read more

© 2015 Nature America, Inc. All rights reserved. Chemical probes are powerful reagents with increasing impacts on biomedical research. However, probes of poor quality or that are used incorrectly generate misleading results. To help address these shortcomings, we will create a community-driven wiki resource to improve quality and convey current best practice.

Cashman SB, Marsden BD, Dustin LB. 2014. The Humoral Immune Response to HCV: Understanding is Key to Vaccine Development. Front Immunol, 5 (NOV), pp. 550. | Show Abstract | Read more

Hepatitis C virus (HCV) remains a global problem, despite advances in treatment. The low cost and high benefit of vaccines have made them the backbone of modern public health strategies, and the fight against HCV will not be won without an effective vaccine. Achievement of this goal will benefit from a robust understanding of virus-host interactions and protective immunity in HCV infection. In this review, we summarize recent findings on HCV-specific antibody responses associated with chronic and spontaneously resolving human infection. In addition, we discuss specific epitopes within HCV's envelope glycoproteins that are targeted by neutralizing antibodies. Understanding what prompts or prevents a successful immune response leading to viral clearance or persistence is essential to designing a successful vaccine.

Bradley AR, Wall ID, Green DVS, Deane CM, Marsden BD. 2014. OOMMPPAA: a tool to aid directed synthesis by the combined analysis of activity and structural data. J Chem Inf Model, 54 (10), pp. 2636-2646. | Show Abstract | Read more

There is an ever increasing resource in terms of both structural information and activity data for many protein targets. In this paper we describe OOMMPPAA, a novel computational tool designed to inform compound design by combining such data. OOMMPPAA uses 3D matched molecular pairs to generate 3D ligand conformations. It then identifies pharmacophoric transformations between pairs of compounds and associates them with their relevant activity changes. OOMMPPAA presents this data in an interactive application providing the user with a visual summary of important interaction regions in the context of the binding site. We present validation of the tool using openly available data for CDK2 and a GlaxoSmithKline data set for a SAM-dependent methyl-transferase. We demonstrate OOMMPPAA's application in optimizing both potency and cell permeability and use OOMMPPAA to highlight nuanced and cross-series SAR. OOMMPPAA is freely available to download at http://oommppaa.sgc.ox.ac.uk/OOMMPPAA/ .

Wettstein S, Underhaug J, Perez B, Marsden BD, Yue WW, Martinez A, Blau N. 2015. Linking genotypes database with locus-specific database and genotype-phenotype correlation in phenylketonuria. Eur J Hum Genet, 23 (3), pp. 302-309. | Show Abstract | Read more

The wide range of metabolic phenotypes in phenylketonuria is due to a large number of variants causing variable impairment in phenylalanine hydroxylase function. A total of 834 phenylalanine hydroxylase gene variants from the locus-specific database PAHvdb and genotypes of 4181 phenylketonuria patients from the BIOPKU database were characterized using FoldX, SIFT Blink, Polyphen-2 and SNPs3D algorithms. Obtained data was correlated with residual enzyme activity, patients' phenotype and tetrahydrobiopterin responsiveness. A descriptive analysis of both databases was compiled and an interactive viewer in PAHvdb database was implemented for structure visualization of missense variants. We found a quantitative relationship between phenylalanine hydroxylase protein stability and enzyme activity (r(s) = 0.479), between protein stability and allelic phenotype (r(s) = -0.458), as well as between enzyme activity and allelic phenotype (r(s) = 0.799). Enzyme stability algorithms (FoldX and SNPs3D), allelic phenotype and enzyme activity were most powerful to predict patients' phenotype and tetrahydrobiopterin response. Phenotype prediction was most accurate in deleterious genotypes (≈ 100%), followed by homozygous (92.9%), hemizygous (94.8%), and compound heterozygous genotypes (77.9%), while tetrahydrobiopterin response was correctly predicted in 71.0% of all cases. To our knowledge this is the largest study using algorithms for the prediction of patients' phenotype and tetrahydrobiopterin responsiveness in phenylketonuria patients, using data from the locus-specific and genotypes database.

Fedorov O, Lingard H, Wells C, Monteiro OP, Picaud S, Keates T, Yapp C, Philpott M, Martin SJ, Felletar I et al. 2014. [1,2,4]triazolo[4,3-a]phthalazines: inhibitors of diverse bromodomains. J Med Chem, 57 (2), pp. 462-476. | Show Abstract | Read more

Bromodomains are gaining increasing interest as drug targets. Commercially sourced and de novo synthesized substituted [1,2,4]triazolo[4,3-a]phthalazines are potent inhibitors of both the BET bromodomains such as BRD4 as well as bromodomains outside the BET family such as BRD9, CECR2, and CREBBP. This new series of compounds is the first example of submicromolar inhibitors of bromodomains outside the BET subfamily. Representative compounds are active in cells exhibiting potent cellular inhibition activity in a FRAP model of CREBBP and chromatin association. The compounds described are valuable starting points for discovery of selective bromodomain inhibitors and inhibitors with mixed bromodomain pharmacology.

Canning P, Cooper CD, Krojer T, Murray JW, Pike AC, Chaikuad A, Keates T, Thangaratnarajah C, Hojzan V, Ayinampudi V et al. 2013. Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases. J Biol Chem, 288 (39), pp. 28304. | Read more

Froese DS, Forouhar F, Tran TH, Vollmar M, Kim YS, Lew S, Neely H, Seetharaman J, Shen Y, Xiao R et al. 2013. Crystal structures of malonyl-coenzyme A decarboxylase provide insights into its catalytic mechanism and disease-causing mutations. Structure, 21 (7), pp. 1182-1192. | Show Abstract | Read more

Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design.

Shintre CA, Pike ACW, Li Q, Kim J-I, Barr AJ, Goubin S, Shrestha L, Yang J, Berridge G, Ross J et al. 2013. Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states. Proc Natl Acad Sci U S A, 110 (24), pp. 9710-9715. | Show Abstract | Read more

ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters.

Canning P, Cooper CDO, Krojer T, Murray JW, Pike ACW, Chaikuad A, Keates T, Thangaratnarajah C, Hojzan V, Ayinampudi V et al. 2013. Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases. J Biol Chem, 288 (11), pp. 7803-7814. | Show Abstract | Read more

Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein "3-box" motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design.

Spencer J, Amin J, Boddiboyena R, Packham G, Cavell BE, Syed Alwi SS, Paranal RM, Heightman TD, Wang M, Marsden B et al. 2012. Back matter MedChemComm, 3 (12), pp. 1562-1562. | Read more

Fish PV, Filippakopoulos P, Bish G, Brennan PE, Bunnage ME, Cook AS, Federov O, Gerstenberger BS, Jones H, Knapp S et al. 2012. Identification of a chemical probe for bromo and extra C-terminal bromodomain inhibition through optimization of a fragment-derived hit. J Med Chem, 55 (22), pp. 9831-9837. | Show Abstract | Read more

The posttranslational modification of chromatin through acetylation at selected histone lysine residues is governed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The significance of this subset of the epigenetic code is interrogated and interpreted by an acetyllysine-specific protein-protein interaction with bromodomain reader modules. Selective inhibition of the bromo and extra C-terminal domain (BET) family of bromodomains with a small molecule is feasible, and this may represent an opportunity for disease intervention through the recently disclosed antiproliferative and anti-inflammatory properties of such inhibitors. Herein, we describe the discovery and structure-activity relationship (SAR) of a novel, small-molecule chemical probe for BET family inhibition that was identified through the application of structure-based fragment assessment and optimization techniques. This has yielded a potent, selective compound with cell-based activity (PFI-1) that may further add to the understanding of BET family function within the bromodomains.

Liu L, Zhen XT, Denton E, Marsden BD, Schapira M. 2012. ChromoHub: a data hub for navigators of chromatin-mediated signalling. Bioinformatics, 28 (16), pp. 2205-2206. | Show Abstract | Read more

UNLABELLED: The rapidly increasing research activity focused on chromatin-mediated regulation of epigenetic mechanisms is generating waves of data on writers, readers and erasers of the histone code, such as protein methyltransferases, bromodomains or histone deacetylases. To make these data easily accessible to communities of research scientists coming from diverse horizons, we have created ChromoHub, an online resource where users can map on phylogenetic trees disease associations, protein structures, chemical inhibitors, histone substrates, chromosomal aberrations and other types of data extracted from public repositories and the published literature. The interface can be used to define the structural or chemical coverage of a protein family, highlight domain architectures, interrogate disease relevance or zoom in on specific genes for more detailed information. This open-access resource should serve as a hub for cell biologists, medicinal chemists, structural biologists and other navigators that explore the biology of chromatin signalling. AVAILABILITY: http://www.thesgc.org/chromohub/.

Rose NR, Woon ECY, Tumber A, Walport LJ, Chowdhury R, Li XS, King ONF, Lejeune C, Ng SS, Krojer T et al. 2012. Plant growth regulator daminozide is a selective inhibitor of human KDM2/7 histone demethylases. J Med Chem, 55 (14), pp. 6639-6643. | Show Abstract | Read more

The JmjC oxygenases catalyze the N-demethylation of N(ε)-methyl lysine residues in histones and are current therapeutic targets. A set of human 2-oxoglutarate analogues were screened using a unified assay platform for JmjC demethylases and related oxygenases. Results led to the finding that daminozide (N-(dimethylamino)succinamic acid, 160 Da), a plant growth regulator, selectively inhibits the KDM2/7 JmjC subfamily. Kinetic and crystallographic studies reveal that daminozide chelates the active site metal via its hydrazide carbonyl and dimethylamino groups.

Woon ECY, Tumber A, Kawamura A, Hillringhaus L, Ge W, Rose NR, Ma JHY, Chan MC, Walport LJ, Che KH et al. 2012. Linking of 2-oxoglutarate and substrate binding sites enables potent and highly selective inhibition of JmjC histone demethylases. Angew Chem Int Ed Engl, 51 (7), pp. 1631-1634. | Show Abstract | Read more

Select an isoform: Linking of cosubstrate and substrate binding sites enables highly selective inhibiton of isoforms of human histone lysine demethylases. The results should provide a basis for the development of potent and selective JmjC inhibitors, possibly suitable for clinical use. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Spencer J, Amin J, Boddiboyena R, Packham G, Cavell BE, Alwi SSS, Paranal RM, Heightman TD, Wang M, Marsden B et al. 2012. Click JAHAs: conformationally restricted ferrocene-based histone deacetylase inhibitors MEDCHEMCOMM, 3 (1), pp. 61-64. | Read more

Marsden BD, Abagyan R, Lee WH. 2012. Visualisation and Efficient Communication in Structure-Based Lead Discovery pp. 176-189. | Show Abstract | Read more

A key aspect of efficient scientific research is the efficient accumulation, exchange and presentation of relevant information. Multi-disciplinary fields of research, such as structure-based lead discovery (SBLD), depend upon many different data types and require the concurrent capture and display of such heterogeneous types of data. The visualisation of such data in a more intuitive and accessible format than the underlying data capture method is often extended to provide the viewer context within a wider range of data types. In the field of SBLD, experimental protein structure determination presents a need to visualise three-dimensional data and to further annotate such visualisations with additional information. The use of high-throughput methods in both chemistry and biology has resulted in a rapid accumulation of relevant information to support and prioritise SBLD. The appropriate integration and visualisation of this data maximises the impact of the underlying information within the context of a project both not only for computational chemists and structural biologists but also for biologists and medicinal chemists. In this chapter we will discuss current approaches and outstanding issues associated with this particular challenge in the context of SBLD. © 2011 Bentham Science Publishers Ltd. All rights reserved.

Hewings DS, Wang M, Philpott M, Fedorov O, Uttarkar S, Filippakopoulos P, Picaud S, Vuppusetty C, Marsden B, Knapp S et al. 2011. 3,5-dimethylisoxazoles act as acetyl-lysine-mimetic bromodomain ligands. J Med Chem, 54 (19), pp. 6761-6770. | Show Abstract | Read more

Histone-lysine acetylation is a vital chromatin post-translational modification involved in the epigenetic regulation of gene transcription. Bromodomains bind acetylated lysines, acting as readers of the histone-acetylation code. Competitive inhibitors of this interaction have antiproliferative and anti-inflammatory properties. With 57 distinct bromodomains known, the discovery of subtype-selective inhibitors of the histone-bromodomain interaction is of great importance. We have identified the 3,5-dimethylisoxazole moiety as a novel acetyl-lysine bioisostere, which displaces acetylated histone-mimicking peptides from bromodomains. Using X-ray crystallographic analysis, we have determined the interactions responsible for the activity and selectivity of 4-substituted 3,5-dimethylisoxazoles against a selection of phylogenetically diverse bromodomains. By exploiting these interactions, we have developed compound 4d, which has IC(50) values of <5 μM for the bromodomain-containing proteins BRD2(1) and BRD4(1). These compounds are promising leads for the further development of selective probes for the bromodomain and extra C-terminal domain (BET) family and CREBBP bromodomains.

Lee WH, Yue WW, Raush E, Totrov M, Abagyan R, Oppermann U, Marsden BD. 2011. Interactive JIMD articles using the iSee concept: turning a new page on structural biology data. J Inherit Metab Dis, 34 (3), pp. 565-567. | Read more

Lee WH, Raush E, Totrov M, Abagyan R, Marsden BD, Oppermann U, Yue WW. 2011. INTERACTIVE 3D VISUALISATIONS OF PROTEIN STRUCTURES TO AID THE STUDY OF INBORN ERRORS OF METABOLISM JOURNAL OF INHERITED METABOLIC DISEASE, 34 pp. S240-S240.

Wang M, Mok MW, Harper H, Lee WH, Min J, Knapp S, Oppermann U, Marsden B, Schapira M. 2010. Structural genomics of histone tail recognition. Bioinformatics, 26 (20), pp. 2629-2630. | Show Abstract | Read more

SUMMARY: The structural genomics of histone tail recognition web server is an open access resource that presents within mini articles all publicly available experimental structures of histone tails in complex with human proteins. Each article is composed of interactive 3D slides that dissect the structural mechanism underlying the recognition of specific sequences and histone marks. A concise text html-linked to interactive graphics guides the reader through the main features of the interaction. This resource can be used to analyze and compare binding modes across multiple histone recognition modules, to evaluate the chemical tractability of binding sites involved in epigenetic signaling and design small molecule inhibitors. AVAILABILITY: http://www.thesgc.org/resources/histone_tails/ CONTACT: matthieu.schapira@utoronto.ca SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Savitsky P, Bray J, Cooper CDO, Marsden BD, Mahajan P, Burgess-Brown NA, Gileadi O. 2010. High-throughput production of human proteins for crystallization: the SGC experience. J Struct Biol, 172 (1), pp. 3-13. | Show Abstract | Read more

Producing purified human proteins with high yield and purity remains a considerable challenge. We describe the methods utilized in the Structural Genomics Consortium (SGC) in Oxford, resulting in successful purification of 48% of human proteins attempted; of those, the structures of approximately 40% were solved by X-ray crystallography. The main driver has been the parallel processing of multiple (typically 9-20) truncated constructs of each target; modest diversity in vectors and host systems; and standardized purification procedures. We provide method details as well as data on the properties of the constructs leading to crystallized proteins and the impact of methodological variants. These can be used to formulate guidelines for initial approaches to expression of new eukaryotic proteins.

Record CJ, Chaikuad A, Rellos P, Das S, Pike ACW, Fedorov O, Marsden BD, Knapp S, Lee WH. 2010. Structural comparison of human mammalian ste20-like kinases. PLoS One, 5 (8), pp. e11905. | Show Abstract | Read more

BACKGROUND: The serine/threonine mammalian Ste-20 like kinases (MSTs) are key regulators of apoptosis, cellular proliferation as well as polarization. Deregulation of MSTs has been associated with disease progression in prostate and colorectal cancer. The four human MSTs are regulated differently by C-terminal regions flanking the catalytic domains. PRINCIPAL FINDINGS: We have determined the crystal structure of kinase domain of MST4 in complex with an ATP-mimetic inhibitor. This is the first structure of an inactive conformation of a member of the MST kinase family. Comparison with active structures of MST3 and MST1 revealed a dimeric association of MST4 suggesting an activation loop exchanged mechanism of MST4 auto-activation. Together with a homology model of MST2 we provide a comparative analysis of the kinase domains for all four members of the human MST family. SIGNIFICANCE: The comparative analysis identified new structural features in the MST ATP binding pocket and has also defined the mechanism for autophosphorylation. Both structural features may be further explored for inhibitors design. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Lee WH, Lukacik P, Guo K, Ugochukwu E, Kavanagh KL, Marsden B, Oppermann U. 2009. Structure-activity relationships of human AKR-type oxidoreductases involved in bile acid synthesis: AKR1D1 and AKR1C4. Mol Cell Endocrinol, 301 (1-2), pp. 199-204. | Show Abstract | Read more

Two members of the human aldo-keto reductase (AKR) superfamily participate in the biosynthesis of bile acids by catalyzing the NADP(H) dependent reduction of 3-keto groups (AKR1C4) and Delta4 double bonds (AKR1D1) of oxysterol precursors. Structure determination of human AKR1C4 and homology modelling of AKR1D1 followed by docking experiments were used to explore active site geometries. Substrate docking resulted in ligand poses satisfying catalytic constraints, and indicates a critical role for Trp227/230 in positioning the substrate in a catalytically competent orientation. Based on the evidence gathered from our docking experiments and experimental structures, this tryptophan residue emerges as a major determinant governing substrate specificity of a subset of enzymes belonging to the AKR1 subfamily.

Bray JE, Marsden BD, Oppermann U. 2009. The human short-chain dehydrogenase/reductase (SDR) superfamily: a bioinformatics summary. Chem Biol Interact, 178 (1-3), pp. 99-109. | Show Abstract | Read more

The short-chain dehydrogenase/reductase (SDR) superfamily represents one of the largest protein superfamilies known to date. Enzymes of this family usually catalyse NAD(P)(H) dependent reactions with a substrate spectrum ranging from polyols, retinoids, steroids and fatty acid derivatives to xenobiotics. We have currently identified 73 SDR superfamily members within the human genome. A status report of the human SDR superfamily is provided in terms of 3D structure determination, co-factor preferences, subcellular localisation and functional annotation. A simple scoring system for measuring structural and functional information (SFS score) has also been introduced to monitor the status of 5 key metrics. Currently there are 17 SDR members with an SFS score of zero indicating that almost a quarter of the human SDR superfamily lacks substantial functional annotation.

Lee WH, Atienza-Herrero J, Abagyan R, Marsden BD. 2009. SGC--structural biology and human health: a new approach to publishing structural biology results. PLoS One, 4 (10), pp. e7675. | Show Abstract | Read more

The Structural Genomics Consortium (SGC) is a not-for-profit, public-private partnership established to deliver novel structural biology knowledge on proteins of medical relevance and place this information into the public domain without restriction, spearheading the concept of "Open-Source Science" to enable drug discovery. The SGC is a major provider of structural information focussed on proteins related to human health, contributing 20.5% of novel structures released by the PDB in 2008. In this article we describe the PLoS ONE Collection entitled 'Structural Biology and Human Health: Medically Relevant Proteins from the SGC'. This Collection contains a series of articles documenting many of the novel protein structures determined by the SGC and work to further characterise their function. Each article in this Collection can be read in an enhanced version where we have integrated our interactive and intuitive 3D visualisation platform, known as iSee. This publishing platform enables the communication of complex structural biology and related data to a wide audience of non-structural biologists. With the use of iSee as the first example of an interactive and intuitive 3D document publication method as part of PLoS ONE, we are pushing the boundaries of structural biology data delivery and peer-review. Our strong desire is that this step forward will encourage others to consider the need for publication of three dimensional and associated data in a similar manner. © 2009 Lee et al.

Raush E, Totrov M, Marsden BD, Abagyan R. 2009. A new method for publishing three-dimensional content. PLoS One, 4 (10), pp. e7394. | Show Abstract | Read more

A new method for electronic publishing of articles with text linked to its interactive three dimensional content is described. The method is based on a single document containing a variety of objects such as formatted text, multiple three dimensional molecular objects, textured shapes and surfaces, data tables and graphs, chemical spreadsheets, alignments, etc. The 3D article can then be published for an online web delivery using the activeICM/active X components as well as be downloaded as a single file to be browsed with all its attached objects locally with the ICM browser. Both activeICM and ICM browser are freely available for the public. This method eliminates the need for multiple methods for the web and the local off-line delivery; it offers the dramatically enhanced, customizable and interactive delivery of article's three dimensional content and data attachments in a single compact file. © 2009 Raush et al.

Marsden BD, Knapp S. 2008. Doing more than just the structure-structural genomics in kinase drug discovery. Curr Opin Chem Biol, 12 (1), pp. 40-45. | Show Abstract | Read more

Structural genomics (SG) has significantly increased the number of novel protein structures of targets with medical relevance. In the protein kinase area, SG has contributed >50% of all novel kinases structures during the past three years and determined more than 30 novel catalytic domain structures. Many of the released structures are inhibitor complexes and a number of them have identified new inhibitor binding modes and scaffolds. In addition, generated reagents, assays, and inhibitor screening data provide a diversity of chemogenomic data that can be utilized for early drug development. Here we discuss the currently available structural data for the kinase family considering novel structures as well as inhibitor complexes. Our analysis revealed that the structural coverage of many kinases families is still rather poor, and inhibitor complexes with diverse inhibitors are only available for a few kinases. However, we anticipate that with the current rate of structure determination and high throughput technologies developed by SG programs these gaps will be closed soon. In addition, the generated reagents will put SG initiatives in a unique position providing data beyond protein structure determination by identifying chemical probes, determining their binding modes and target specificity.

Fedorov O, Marsden B, Pogacic V, Rellos P, Müller S, Bullock AN, Schwaller J, Sundström M, Knapp S. 2007. A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases. Proc Natl Acad Sci U S A, 104 (51), pp. 20523-20528. | Show Abstract | Read more

Protein kinases play a pivotal role in cell signaling, and dysregulation of many kinases has been linked to disease development. A large number of kinase inhibitors are therefore currently under investigation in clinical trials, and so far seven inhibitors have been approved as anti-cancer drugs. In addition, kinase inhibitors are widely used as specific probes to study cell signaling, but systematic studies describing selectivity of these reagents across a panel of diverse kinases are largely lacking. Here we evaluated the specificity of 156 validated kinase inhibitors, including inhibitors used in clinical trials, against 60 human Ser/Thr kinases using a thermal stability shift assay. Our analysis revealed many unexpected cross-reactivities for inhibitors thought to be specific for certain targets. We also found that certain combinations of active-site residues in the ATP-binding site correlated with the detected ligand promiscuity and that some kinases are highly sensitive to inhibition using diverse chemotypes, suggesting them as preferred intervention points. Our results uncovered also inhibitor cross-reactivities that may lead to alternate clinical applications. For example, LY333'531, a PKCbeta inhibitor currently in phase III clinical trials, efficiently inhibited PIM1 kinase in our screen, a suggested target for treatment of leukemia. We determined the binding mode of this inhibitor by x-ray crystallography and in addition showed that LY333'531 induced cell death and significantly suppressed growth of leukemic cells from acute myeloid leukemia patients.

Gileadi O, Knapp S, Lee WH, Marsden BD, Müller S, Niesen FH, Kavanagh KL, Ball LJ, von Delft F, Doyle DA et al. 2007. The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins. J Struct Funct Genomics, 8 (2-3), pp. 107-119. | Show Abstract | Read more

As many of the structural genomics centers have ended their first phase of operation, it is a good point to evaluate the scientific impact of this endeavour. The Structural Genomics Consortium (SGC), operating from three centers across the Atlantic, investigates human proteins involved in disease processes and proteins from Plasmodium falciparum and related organisms. We present here some of the scientific output of the Oxford node of the SGC, where the target areas include protein kinases, phosphatases, oxidoreductases and other metabolic enzymes, as well as signal transduction proteins. The SGC has aimed to achieve extensive coverage of human gene families with a focus on protein-ligand interactions. The methods employed for effective protein expression, crystallization and structure determination by X-ray crystallography are summarized. In addition to the cumulative impact of accelerated delivery of protein structures, we demonstrate how family coverage, generic screening methodology, and the availability of abundant purified protein samples, allow a level of discovery that is difficult to achieve otherwise. The contribution of NMR to structure determination and protein characterization is discussed. To make this information available to a wide scientific audience, a new tool for disseminating annotated structural information was created that also represents an interactive platform allowing for a continuous update of the annotation by the scientific community.

Fedorov O, Sundström M, Marsden B, Knapp S. 2007. Insights for the development of specific kinase inhibitors by targeted structural genomics. Drug Discov Today, 12 (9-10), pp. 365-372. | Show Abstract | Read more

Many protein kinases are validated intervention points for drug development, however active site similarities often lead to a lack of selectivity and unwanted side effects in the clinic. To address this issue, it is desirable to increase the number of high resolution crystal structures and complexes with non-adenosine ligands available for the rational design of more selective inhibitors. Recent progress in protein crystallography and biotechnology has enabled structural genomics projects to target challenging proteins successfully, including protein kinases. As we discuss here, this effort has resulted in a considerable increase in the number of available high resolution structures and inhibitor complexes and has identified novel structural motifs that are available for drug development.

Vedadi M, Niesen FH, Allali-Hassani A, Fedorov OY, Finerty PJ, Wasney GA, Yeung R, Arrowsmith C, Ball LJ, Berglund H et al. 2006. Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc Natl Acad Sci U S A, 103 (43), pp. 15835-15840. | Show Abstract | Read more

The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins.

Eswaran J, von Kries JP, Marsden B, Longman E, Debreczeni JE, Ugochukwu E, Turnbull A, Lee WH, Knapp S, Barr AJ. 2006. Crystal structures and inhibitor identification for PTPN5, PTPRR and PTPN7: a family of human MAPK-specific protein tyrosine phosphatases. Biochem J, 395 (3), pp. 483-491. | Show Abstract | Read more

Protein tyrosine phosphatases PTPN5, PTPRR and PTPN7 comprise a family of phosphatases that specifically inactivate MAPKs (mitogen-activated protein kinases). We have determined high-resolution structures of all of the human family members, screened them against a library of 24000 compounds and identified two classes of inhibitors, cyclopenta[c]quinolinecarboxylic acids and 2,5-dimethylpyrrolyl benzoic acids. Comparative structural analysis revealed significant differences within this conserved family that could be explored for the design of selective inhibitors. PTPN5 crystallized, in two distinct crystal forms, with a sulphate ion in close proximity to the active site and the WPD (Trp-Pro-Asp) loop in a unique conformation, not seen in other PTPs, ending in a 3(10)-helix. In the PTPN7 structure, the WPD loop was in the closed conformation and part of the KIM (kinase-interaction motif) was visible, which forms an N-terminal aliphatic helix with the phosphorylation site Thr66 in an accessible position. The WPD loop of PTPRR was open; however, in contrast with the structure of its mouse homologue, PTPSL, a salt bridge between the conserved lysine and aspartate residues, which has been postulated to confer a more rigid loop structure, thereby modulating activity in PTPSL, does not form in PTPRR. One of the identified inhibitor scaffolds, cyclopenta[c]quinoline, was docked successfully into PTPRR, suggesting several possibilities for hit expansion. The determined structures together with the established SAR (structure-activity relationship) propose new avenues for the development of selective inhibitors that may have therapeutic potential for treating neurodegenerative diseases in the case of PTPRR or acute myeloblastic leukaemia targeting PTPN7.

Williams JP, Smith DC, Green BN, Marsden BD, Jennings KR, Roberts LM, Scrivens JH. 2006. Gas phase characterization of the noncovalent quaternary structure of cholera toxin and the cholera toxin B subunit pentamer. Biophys J, 90 (9), pp. 3246-3254. | Show Abstract | Read more

Cholera toxin (CTx) is an AB5 cytotonic protein that has medical relevance in cholera and as a novel mucosal adjuvant. Here, we report an analysis of the noncovalent homopentameric complex of CTx B chain (CTx B5) using electrospray ionization triple quadrupole mass spectrometry and tandem mass spectrometry and the analysis of the noncovalent hexameric holotoxin usingelectrospray ionization time-of-flight mass spectrometry over a range of pH values that correlate with those encountered by this toxin after cellular uptake. We show that noncovalent interactions within the toxin assemblies were maintained under both acidic and neutral conditions in the gas phase. However, unlike the related Escherichia coli Shiga-like toxin B5 pentamer (SLTx B), the CTx B5 pentamer was stable at low pH, indicating that additional interactions must be present within the latter. Structural comparison of the CTx B monomer interface reveals an additional alpha-helix that is absent in the SLTx B monomer. In silico energy calculations support interactions between this helix and the adjacent monomer. These data provide insight into the apparent stabilization of CTx B relative to SLTx B.

Abagyan R, Lee WH, Raush E, Budagyan L, Totrov M, Sundstrom M, Marsden BD. 2006. Disseminating structural genomics data to the public: from a data dump to an animated story. Trends Biochem Sci, 31 (2), pp. 76-78. | Read more

Marsden BD, Sundstrom M, Knapp S. 2006. High-throughput structural characterisation of therapeutic protein targets Expert Opinion on Drug Discovery, 1 (2), pp. 123-136. | Show Abstract | Read more

The overarching scientific objective for high-throughput structural biology projects targeting human proteins or relevant proteins of human pathogens is to generate a detailed structural insight of biologically relevant structural features of soluble or membrane protein families of therapeutic relevance. These data will better enable structure-guided drug discovery in pharmaceutical and biotechechnology companies. In particular, detailed structural descriptions for entire protein families will be highly valuable for the design of drugs and other bioactive compounds that are selective for individual members or groups of proteins within protein families. In addition to determined structural information, targeted high-throughput structural biology will also provide reagents, methodologies, technologies and small molecules that bind selectively to targets that are suitable as intervention points in the treatment of disease. © 2006 Informa UK Ltd.

Bullock AN, Debreczeni JE, Fedorov OY, Nelson A, Marsden BD, Knapp S. 2005. Structural basis of inhibitor specificity of the human protooncogene proviral insertion site in moloney murine leukemia virus (PIM-1) kinase. J Med Chem, 48 (24), pp. 7604-7614. | Show Abstract | Read more

The kinase PIM-1 plays a pivotal role in cytokine signaling and is implicated in the development of a number of tumors. The three-dimensional structure of PIM-1 is characterized by an unique hinge region which lacks a second hydrogen bond donor and makes it particularly important to determine how inhibitors bind to this kinase. We determined the structures of PIM-1 in complex with bisindolylmaleimide (BIM-1) and established the structure-activity relationship (SAR) for this inhibitor class. In addition, we screened a kinase targeted library and identified a number of high affinity inhibitors of PIM-1 such as imidazo[1,2-b]pyridazines, pyrazolo[1,5-a]pyrimidines, and members of the flavonoid family. In this paper we present an initial SAR of the identified scaffolds determined on the basis of a thermostability shift assay, calorimetric binding data, and biochemical assays which may find applications for the treatment of PIM-1 dependent cancer types.

Marsden B, Abagyan R. 2004. SAD--a normalized structural alignment database: improving sequence-structure alignments. Bioinformatics, 20 (15), pp. 2333-2344. | Show Abstract | Read more

MOTIVATION: We present a structural alignment database that is specifically targeted for use in derivation and optimization of sequence-structure alignment algorithms for homology modeling. We have paid attention to ensure that fold-space is properly sampled, that the structures involved in alignments are of significant resolution (better than 2.5 A) and the alignments are accurate and reliable. RESULTS: Alignments have been taken from the HOMSTRAD, BAliBASE and SCOP-based Gerstein databases along with alignments generated by a global structural alignment method described here. In order to discriminate between equivalent alignments from these different sources, we have developed a novel scoring function, Contact Alignment Quality score, which evaluates trial alignments by their statistical significance combined with their ability to reproduce conserved three-dimensional residue contacts. The resulting non-redundant, unbiased database contains 1927 alignments from across fold-space with high-resolution structures and a wide range of sequence identities. AVAILABILITY: The database can be interactively queried either over the web at http://abagyan.scripps.edu/lab/web/sad/show.cgi or by using MySQL, and is also available to download over the web.

Nentwich HA, Mustafa Z, Rugg MS, Marsden BD, Cordell MR, Mahoney DJ, Jenkins SC, Dowling B, Fries E, Milner CM et al. 2002. A novel allelic variant of the human TSG-6 gene encoding an amino acid difference in the CUB module. Chromosomal localization, frequency analysis, modeling, and expression. J Biol Chem, 277 (18), pp. 15354-15362. | Show Abstract | Read more

Tumor necrosis factor-stimulated gene-6 (TSG-6) encodes a 35-kDa protein, which is comprised of contiguous Link and CUB modules. TSG-6 protein has been detected in the articular joints of osteoarthritis (OA) patients, with little or no constitutive expression in normal adult tissues. It interacts with components of cartilage matrix (e.g. hyaluronan and aggrecan) and thus may be involved in extracellular remodeling during joint disease. In addition, TSG-6 has been found to have anti-inflammatory properties in models of acute and chronic inflammation. Here we have mapped the human TSG-6 gene to 2q23.3, a region of chromosome 2 linked with OA. A single nucleotide polymorphism was identified that involves a non-synonymous G --> A transition at nucleotide 431 of the TSG-6 coding sequence, resulting in an Arg to Gln alteration in the CUB module (at residue 144 in the preprotein). Molecular modeling of the CUB domain indicated that this amino acid change might lead to functional differences. Typing of 400 OA cases and 400 controls revealed that the A(431) variant identified here is the major TSG-6 allele in Caucasians (with over 75% being A(431) homozygotes) but that this polymorphism is not a marker for OA susceptibility in the patients we have studied. Expression of the Arg(144) and Gln(144) allotypes in Drosophila Schneider 2 cells, and functional characterization, showed that there were no significant differences in the ability of these full-length proteins to bind hyaluronan or form a stable complex with inter-alpha-inhibitor.

Damerell DR, Strain-Damerell C, Garsot S, Joyce SP, Barrett P, Marsden BD. 2019. SATurn: a modular bioinformatics framework for the design of robust maintainable web-based and standalone applications. Bioinformatics, 35 (2), pp. 349-351. | Show Abstract | Read more

Summary: SATurn is a modular, open-source, bioinformatics platform designed to specifically address the problems of maintenance and longevity commonly associated with the development of simple tools funded by academic research grants. Applications developed in SATurn can be deployed as web-based tools, standalone applications or hybrid tools which have the benefits of both. Within the Structural Genomics Consortium we have utilized SATurn to create a bioinformatics portal which routinely supports a diverse group of scientists including those interested in structural biology, cloning, glycobiology and chemical biology. Availability and implementation: https://github.com/ddamerell53/SATurn. Supplementary information: Supplementary data are available at Bioinformatics online.

Bradley AR, Wall ID, von Delft F, Green DVS, Deane CM, Marsden BD. 2015. WONKA: objective novel complex analysis for ensembles of protein-ligand structures. J Comput Aided Mol Des, 29 (10), pp. 963-973. | Show Abstract | Read more

WONKA is a tool for the systematic analysis of an ensemble of protein-ligand structures. It makes the identification of conserved and unusual features within such an ensemble straightforward. WONKA uses an intuitive workflow to process structural co-ordinates. Ligand and protein features are summarised and then presented within an interactive web application. WONKA's power in consolidating and summarising large amounts of data is described through the analysis of three bromodomain datasets. Furthermore, and in contrast to many current methods, WONKA relates analysis to individual ligands, from which we find unusual and erroneous binding modes. Finally the use of WONKA as an annotation tool to share observations about structures is demonstrated. WONKA is freely available to download and install locally or can be used online at http://wonka.sgc.ox.ac.uk.

Bradley AR, Wall ID, Green DVS, Deane CM, Marsden BD. 2014. OOMMPPAA: a tool to aid directed synthesis by the combined analysis of activity and structural data. J Chem Inf Model, 54 (10), pp. 2636-2646. | Show Abstract | Read more

There is an ever increasing resource in terms of both structural information and activity data for many protein targets. In this paper we describe OOMMPPAA, a novel computational tool designed to inform compound design by combining such data. OOMMPPAA uses 3D matched molecular pairs to generate 3D ligand conformations. It then identifies pharmacophoric transformations between pairs of compounds and associates them with their relevant activity changes. OOMMPPAA presents this data in an interactive application providing the user with a visual summary of important interaction regions in the context of the binding site. We present validation of the tool using openly available data for CDK2 and a GlaxoSmithKline data set for a SAM-dependent methyl-transferase. We demonstrate OOMMPPAA's application in optimizing both potency and cell permeability and use OOMMPPAA to highlight nuanced and cross-series SAR. OOMMPPAA is freely available to download at http://oommppaa.sgc.ox.ac.uk/OOMMPPAA/ .

Wang M, Mok MW, Harper H, Lee WH, Min J, Knapp S, Oppermann U, Marsden B, Schapira M. 2010. Structural genomics of histone tail recognition. Bioinformatics, 26 (20), pp. 2629-2630. | Show Abstract | Read more

SUMMARY: The structural genomics of histone tail recognition web server is an open access resource that presents within mini articles all publicly available experimental structures of histone tails in complex with human proteins. Each article is composed of interactive 3D slides that dissect the structural mechanism underlying the recognition of specific sequences and histone marks. A concise text html-linked to interactive graphics guides the reader through the main features of the interaction. This resource can be used to analyze and compare binding modes across multiple histone recognition modules, to evaluate the chemical tractability of binding sites involved in epigenetic signaling and design small molecule inhibitors. AVAILABILITY: http://www.thesgc.org/resources/histone_tails/ CONTACT: matthieu.schapira@utoronto.ca SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Lee WH, Atienza-Herrero J, Abagyan R, Marsden BD. 2009. SGC--structural biology and human health: a new approach to publishing structural biology results. PLoS One, 4 (10), pp. e7675. | Show Abstract | Read more

The Structural Genomics Consortium (SGC) is a not-for-profit, public-private partnership established to deliver novel structural biology knowledge on proteins of medical relevance and place this information into the public domain without restriction, spearheading the concept of "Open-Source Science" to enable drug discovery. The SGC is a major provider of structural information focussed on proteins related to human health, contributing 20.5% of novel structures released by the PDB in 2008. In this article we describe the PLoS ONE Collection entitled 'Structural Biology and Human Health: Medically Relevant Proteins from the SGC'. This Collection contains a series of articles documenting many of the novel protein structures determined by the SGC and work to further characterise their function. Each article in this Collection can be read in an enhanced version where we have integrated our interactive and intuitive 3D visualisation platform, known as iSee. This publishing platform enables the communication of complex structural biology and related data to a wide audience of non-structural biologists. With the use of iSee as the first example of an interactive and intuitive 3D document publication method as part of PLoS ONE, we are pushing the boundaries of structural biology data delivery and peer-review. Our strong desire is that this step forward will encourage others to consider the need for publication of three dimensional and associated data in a similar manner. © 2009 Lee et al.

Raush E, Totrov M, Marsden BD, Abagyan R. 2009. A new method for publishing three-dimensional content. PLoS One, 4 (10), pp. e7394. | Show Abstract | Read more

A new method for electronic publishing of articles with text linked to its interactive three dimensional content is described. The method is based on a single document containing a variety of objects such as formatted text, multiple three dimensional molecular objects, textured shapes and surfaces, data tables and graphs, chemical spreadsheets, alignments, etc. The 3D article can then be published for an online web delivery using the activeICM/active X components as well as be downloaded as a single file to be browsed with all its attached objects locally with the ICM browser. Both activeICM and ICM browser are freely available for the public. This method eliminates the need for multiple methods for the web and the local off-line delivery; it offers the dramatically enhanced, customizable and interactive delivery of article's three dimensional content and data attachments in a single compact file. © 2009 Raush et al.

Marsden BD, Knapp S. 2008. Doing more than just the structure-structural genomics in kinase drug discovery. Curr Opin Chem Biol, 12 (1), pp. 40-45. | Show Abstract | Read more

Structural genomics (SG) has significantly increased the number of novel protein structures of targets with medical relevance. In the protein kinase area, SG has contributed >50% of all novel kinases structures during the past three years and determined more than 30 novel catalytic domain structures. Many of the released structures are inhibitor complexes and a number of them have identified new inhibitor binding modes and scaffolds. In addition, generated reagents, assays, and inhibitor screening data provide a diversity of chemogenomic data that can be utilized for early drug development. Here we discuss the currently available structural data for the kinase family considering novel structures as well as inhibitor complexes. Our analysis revealed that the structural coverage of many kinases families is still rather poor, and inhibitor complexes with diverse inhibitors are only available for a few kinases. However, we anticipate that with the current rate of structure determination and high throughput technologies developed by SG programs these gaps will be closed soon. In addition, the generated reagents will put SG initiatives in a unique position providing data beyond protein structure determination by identifying chemical probes, determining their binding modes and target specificity.

Abagyan R, Lee WH, Raush E, Budagyan L, Totrov M, Sundstrom M, Marsden BD. 2006. Disseminating structural genomics data to the public: from a data dump to an animated story. Trends Biochem Sci, 31 (2), pp. 76-78. | Read more

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