Publications by authors named "Matthias T Ehebauer"

12 Publications

  • Page 1 of 1

A novel high-content imaging-based technique for measuring binding of Dickkopf-1 to low-density lipoprotein receptor-related protein 6.

J Pharmacol Toxicol Methods 2019 Jan - Feb;95:47-55. Epub 2018 Nov 23.

Alzheimer's Research UK Oxford Drug Discovery Institute, University of Oxford, NDM Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7FZ, UK. Electronic address:

Introduction: Dickkopf-related protein 1 (Dkk1) is a secreted protein ligand of low-density lipoprotein receptor-related protein 6 (LRP6), which antagonises canonical Wnt signalling. Elevated Dkk1 levels have been linked to Alzheimer's disease (AD), with protein blockade protective in pre-clinical AD models, suggesting inhibitors of Dkk1-LRP6 binding may have therapeutic utility against AD. Cell-based Dkk1-LRP6 assays reported in the literature use either modified Dkk1 protein and/or do not possess suitable throughput for drug screening. Here we report a novel immunocytochemical-based assay utilising high-content imaging (HCI) and automated data analysis suitable for the screening of protein and small-molecule inhibitors of Dkk1-LRP6 binding.

Methods: We developed an immunocytochemical (ICC) protocol to detect specific binding of exogenous human Dkk1 protein to human LRP6 transiently expressed in HEK293 cells. Images were generated using the PerkinElmer Operetta HCI System, after which quantitative data was generated using the PerkinElmer Columbus™ System.

Results: Our ICC technique and analysis pipeline allowed measurement of cell membrane-localised, LRP6-specific Dkk1 binding, normalised at individual cellular events. Saturation binding demonstrated concentration-dependent Dkk1 binding to LRP6, with a K in keeping with reported values. Association kinetic experiments demonstrated the utility of the technique to investigate Dkk1 binding kinetics. Human Dkk members Dkk2 and Dkk4 fully displaced Dkk1 binding in a competition assay, while Dkk3 and Soggy-1/DkkL1 exhibited non-complete displacement of Dkk1. Finally gallocyanine, a previously reported inhibitor of Dkk1-LRP6 binding, fully displaced Dkk1 near the expected IC.

Discussion: In conclusion, we provide a validated cell-based assay, suitable for the screening of inhibitors of Dkk1-LRP6 binding, and provide the basis for additional assay development, investigating Dkk1-LRP6 pharmacology.
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http://dx.doi.org/10.1016/j.vascn.2018.11.003DOI Listing
March 2019

Small molecule inhibitors of RAS-effector protein interactions derived using an intracellular antibody fragment.

Nat Commun 2018 08 9;9(1):3169. Epub 2018 Aug 9.

Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, UK.

Targeting specific protein-protein interactions (PPIs) is an attractive concept for drug development, but hard to implement since intracellular antibodies do not penetrate cells and most small-molecule drugs are considered unsuitable for PPI inhibition. A potential solution to these problems is to select intracellular antibody fragments to block PPIs, use these antibody fragments for target validation in disease models and finally derive small molecules overlapping the antibody-binding site. Here, we explore this strategy using an anti-mutant RAS antibody fragment as a competitor in a small-molecule library screen for identifying RAS-binding compounds. The initial hits are optimized by structure-based design, resulting in potent RAS-binding compounds that interact with RAS inside the cells, prevent RAS-effector interactions and inhibit endogenous RAS-dependent signalling. Our results may aid RAS-dependent cancer drug development and demonstrate a general concept for developing small compounds to replace intracellular antibody fragments, enabling rational drug development to target validated PPIs.
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http://dx.doi.org/10.1038/s41467-018-05707-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085350PMC
August 2018

Discovery of a PCAF Bromodomain Chemical Probe.

Angew Chem Int Ed Engl 2017 01 14;56(3):827-831. Epub 2016 Dec 14.

Structural Genomics Consortium & Target Discovery Institute, University of Oxford, NDM Research Building, Roosevelt Drive, Oxford, OX3 7DQ and OX3 7FZ, UK.

The p300/CBP-associated factor (PCAF) and related GCN5 bromodomain-containing lysine acetyl transferases are members of subfamily I of the bromodomain phylogenetic tree. Iterative cycles of rational inhibitor design and biophysical characterization led to the discovery of the triazolopthalazine-based L-45 (dubbed L-Moses) as the first potent, selective, and cell-active PCAF bromodomain (Brd) inhibitor. Synthesis from readily available (1R,2S)-(-)-norephedrine furnished L-45 in enantiopure form. L-45 was shown to disrupt PCAF-Brd histone H3.3 interaction in cells using a nanoBRET assay, and a co-crystal structure of L-45 with the homologous Brd PfGCN5 from Plasmodium falciparum rationalizes the high selectivity for PCAF and GCN5 bromodomains. Compound L-45 shows no observable cytotoxicity in peripheral blood mononuclear cells (PBMC), good cell-permeability, and metabolic stability in human and mouse liver microsomes, supporting its potential for in vivo use.
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http://dx.doi.org/10.1002/anie.201610816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412877PMC
January 2017

ATP half-sites in RadA and RAD51 recombinases bind nucleotides.

FEBS Open Bio 2016 05 6;6(5):372-85. Epub 2016 Apr 6.

Department of Biochemistry University of Cambridge UK.

Homologous recombination is essential for repair of DNA double-strand breaks. Central to this process is a family of recombinases, including archeal RadA and human RAD51, which form nucleoprotein filaments on damaged single-stranded DNA ends and facilitate their ATP-dependent repair. ATP binding and hydrolysis are dependent on the formation of a nucleoprotein filament comprising RadA/RAD51 and single-stranded DNA, with ATP bound between adjacent protomers. We demonstrate that truncated, monomeric Pyrococcus furiosus RadA and monomerised human RAD51 retain the ability to bind ATP and other nucleotides with high affinity. We present crystal structures of both apo and nucleotide-bound forms of monomeric RadA. These structures reveal that while phosphate groups are tightly bound, RadA presents a shallow, poorly defined binding surface for the nitrogenous bases of nucleotides. We suggest that RadA monomers would be constitutively bound to nucleotides in the cell and that the bound nucleotide might play a structural role in filament assembly.
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http://dx.doi.org/10.1002/2211-5463.12052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856416PMC
May 2016

Characterization of the mycobacterial acyl-CoA carboxylase holo complexes reveals their functional expansion into amino acid catabolism.

PLoS Pathog 2015 Feb 19;11(2):e1004623. Epub 2015 Feb 19.

European Molecular Biology Laboratory, Hamburg Unit, Hamburg, Germany; Center for Structural Systems Biology, Hamburg, Germany.

Biotin-mediated carboxylation of short-chain fatty acid coenzyme A esters is a key step in lipid biosynthesis that is carried out by multienzyme complexes to extend fatty acids by one methylene group. Pathogenic mycobacteria have an unusually high redundancy of carboxyltransferase genes and biotin carboxylase genes, creating multiple combinations of protein/protein complexes of unknown overall composition and functional readout. By combining pull-down assays with mass spectrometry, we identified nine binary protein/protein interactions and four validated holo acyl-coenzyme A carboxylase complexes. We investigated one of these--the AccD1-AccA1 complex from Mycobacterium tuberculosis with hitherto unknown physiological function. Using genetics, metabolomics and biochemistry we found that this complex is involved in branched amino-acid catabolism with methylcrotonyl coenzyme A as the substrate. We then determined its overall architecture by electron microscopy and found it to be a four-layered dodecameric arrangement that matches the overall dimensions of a distantly related methylcrotonyl coenzyme A holo complex. Our data argue in favor of distinct structural requirements for biotin-mediated γ-carboxylation of α-β unsaturated acid esters and will advance the categorization of acyl-coenzyme A carboxylase complexes. Knowledge about the underlying structural/functional relationships will be crucial to make the target category amenable for future biomedical applications.
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http://dx.doi.org/10.1371/journal.ppat.1004623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4347857PMC
February 2015

Active site conformational changes upon reaction intermediate biotinyl-5'-AMP binding in biotin protein ligase from Mycobacterium tuberculosis.

Protein Sci 2014 Jul 22;23(7):932-9. Epub 2014 Apr 22.

European Molecular Biology Laboratory, EMBL-Hamburg, c/o DESY, Building 25A, Notkestrasse 85, 22603, Hamburg, Germany.

Protein biotinylation, a rare form of post-translational modification, is found in enzymes required for lipid biosynthesis. In mycobacteria, this process is essential for the formation of their complex and distinct cell wall and has become a focal point of drug discovery approaches. The enzyme responsible for this process, biotin protein ligase, substantially varies in different species in terms of overall structural organization, regulation of function and substrate specificity. To advance the understanding of the molecular mechanism of biotinylation in Mycobacterium tuberculosis we have biochemically and structurally characterized the corresponding enzyme. We report the high-resolution crystal structures of the apo-form and reaction intermediate biotinyl-5'-AMP-bound form of M. tuberculosis biotin protein ligase. Binding of the reaction intermediate leads to clear disorder-to-order transitions. We show that a conserved lysine, Lys138, in the active site is essential for biotinylation.
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http://dx.doi.org/10.1002/pro.2475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4088977PMC
July 2014

Using a fragment-based approach to target protein-protein interactions.

Chembiochem 2013 Feb 23;14(3):332-42. Epub 2013 Jan 23.

Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.

The ability to identify inhibitors of protein-protein interactions represents a major challenge in modern drug discovery and in the development of tools for chemical biology. In recent years, fragment-based approaches have emerged as a new methodology in drug discovery; however, few examples of small molecules that are active against chemotherapeutic targets have been published. Herein, we describe the fragment-based approach of targeting the interaction between the tumour suppressor BRCA2 and the recombination enzyme RAD51; it makes use of a screening pipeline of biophysical techniques that we expect to be more generally applicable to similar targets. Disruption of this interaction in vivo is hypothesised to give rise to cellular hypersensitivity to radiation and genotoxic drugs. We have used protein engineering to create a monomeric form of RAD51 by humanising a thermostable archaeal orthologue, RadA, and used this protein for fragment screening. The initial fragment hits were thoroughly validated biophysically by isothermal titration calorimetry (ITC) and NMR techniques and observed by X-ray crystallography to bind in a shallow surface pocket that is occupied in the native complex by the side chain of a phenylalanine from the conserved FxxA interaction motif found in BRCA2. This represents the first report of fragments or any small molecule binding at this protein-protein interaction site.
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http://dx.doi.org/10.1002/cbic.201200521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3594973PMC
February 2013

The PE/PPE multigene family codes for virulence factors and is a possible source of mycobacterial antigenic variation: perhaps more?

Biochimie 2012 Jan 12;94(1):110-6. Epub 2011 Oct 12.

European Molecular Biology Laboratory - Hamburg, c/o DESY, Notkestrasse 85, Building 25A, D-22603 Hamburg, Germany.

The PE/PPE multigene family codes for approximately 10% of the Mycobacterium tuberculosis proteome and is encoded by 176 open reading frames. These proteins possess, and have been named after, the conserved proline-glutamate (PE) or proline-proline-glutamate (PPE) motifs at their N-terminus. Their genes have a conserved structure and repeat motifs that could be a potential source of antigenic variation in M. tuberculosis. PE/PPE genes are scattered throughout the genome and PE/PPE pairs are usually encoded in bicistronic operons although this is not universally so. This gene family has evolved by specific gene duplication events. PE/PPE proteins are either secreted or localized to the cell surface. Several are thought to be virulence factors, which participate in evasion of the host immune response. This review summarizes the current knowledge about the gene family in order to better understand its biological function.
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http://dx.doi.org/10.1016/j.biochi.2011.09.026DOI Listing
January 2012

The progress made in determining the Mycobacterium tuberculosis structural proteome.

Proteomics 2011 Aug 14;11(15):3128-33. Epub 2011 Jun 14.

European Molecular Biology Laboratory - Hamburg, c/o DESY, Hamburg, Germany.

Mycobacterium tuberculosis is a highly infectious pathogen that is still responsible for millions of deaths annually. Effectively treating this disease typically requires a course of antibiotics, most of which were developed decades ago. These drugs are, however, not effective against persistent tubercle bacilli and the emergence of drug-resistant stains threatens to make many of them obsolete. The identification of new drug targets, allowing the development of new potential drugs, is therefore imperative. Both proteomics and structural biology have important roles to play in this process, the former as a means of identifying promising drug targets and the latter allowing understanding of protein function and protein-drug interactions at atomic resolution. The determination of M. tuberculosis protein structures has been a goal of the scientific community for the last decade, who have aimed to supply a large amount of structural data that can be used in structure-based approaches for drug discovery and design. Only since the genome sequence of M. tuberculosis has been available has the determination of large numbers of tuberculosis protein structures been possible. Currently, the molecular structures of 8.5% of all the pathogen's protein-encoding ORFs have been determined. In this review, we look at the progress made in determining the M. tuberculosis structural proteome and the impact this has had on the development of potential new drugs, as well as the discovery of the function of crucial mycobaterial proteins.
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http://dx.doi.org/10.1002/pmic.201000787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3345573PMC
August 2011

Improved mycobacterial protein production using a Mycobacterium smegmatis groEL1ΔC expression strain.

BMC Biotechnol 2011 Mar 25;11:27. Epub 2011 Mar 25.

European Molecular Biology Laboratory (EMBL), Hamburg Outstation, c/o DESY, Building 25a, Notkestrasse 85, 22603 Hamburg, Germany.

Background: The non-pathogenic bacterium Mycobacterium smegmatis is widely used as a near-native expression host for the purification of Mycobacterium tuberculosis proteins. Unfortunately, the Hsp60 chaperone GroEL1, which is relatively highly expressed, is often co-purified with polyhistidine-tagged recombinant proteins as a major contaminant when using this expression system. This is likely due to a histidine-rich C-terminus in GroEL1.

Results: In order to improve purification efficiency and yield of polyhistidine-tagged mycobacterial target proteins, we created a mutant version of GroEL1 by removing the coding sequence for the histidine-rich C-terminus, termed GroEL1ΔC. GroEL1ΔC, which is a functional protein, is no longer able to bind nickel affinity beads. Using a selection of challenging test proteins, we show that GroEL1ΔC is no longer present in protein samples purified from the groEL1ΔC expression strain and demonstrate the feasibility and advantages of purifying and characterising proteins produced using this strain.

Conclusions: This novel Mycobacterium smegmatis expression strain allows efficient expression and purification of mycobacterial proteins while concomitantly removing the troublesome contaminant GroEL1 and consequently increasing the speed and efficiency of protein purification.
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http://dx.doi.org/10.1186/1472-6750-11-27DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3076238PMC
March 2011

The structural and functional determinants of the Axin and Dishevelled DIX domains.

BMC Struct Biol 2009 Nov 12;9:70. Epub 2009 Nov 12.

European Molecular Biology Laboratory, Notkestrasse 85, 22603 Hamburg, Germany.

Background: The dishevelled and axin genes encode multi-domain proteins that play key roles in WNT signalling. Dishevelled prevents beta-catenin degradation by interfering with the interaction of beta-catenin with the degradation-mediating Axin-APC-GSK3beta complex. This interference leads to an accumulation of cytoplasmic beta-catenin, which enters the nucleus and interacts with transcription factors that induce expression of Wnt-target genes. Axin, as a component of the degradation-mediating complex, is a potent negative regulator of Wnt signalling, whereas Dishevelled is a potent activator. Both Dishevelled and Axin possess a DIX (Dishevelled/Axin) domain, which mediates protein-protein interactions, specifically homodimerization.

Results: An evolutionary trace analysis of DIX domains identified conserved residues which, when mapped onto the crystal structure of the Axin DIX domain and a comparative model of the Dishevelled DIX domain, allow their categorization as residues of either structural or functional importance. We identify residues that are structural and functional determinants of the DIX domain fold, as well as those that are specific to homodimerization of Axin and Dishevelled.

Conclusion: This report provides the first explanation of the mutant phenotypes caused by non-synonymous substitutions in the Dishevelled and Axin DIX domain by correlating their presumed functional significance with molecular structure.
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http://dx.doi.org/10.1186/1472-6807-9-70DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2780430PMC
November 2009

High-resolution crystal structure of the human Notch 1 ankyrin domain.

Biochem J 2005 Nov;392(Pt 1):13-20

Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.

The Notch receptor is part of a highly conserved signalling system of central importance to animal development. Its ANK (ankyrin) domain is required for Notch-mediated signal transduction. The crystal structure of the human Notch 1 ANK domain was solved by molecular replacement at 1.9 A (1 A=0.1 nm) resolution, and it shows that the features identified in the Drosophila homologue are conserved. The domain has six of the seven ANK repeats predicted from sequence. The putative first repeat, which has only part of the consensus and a long insertion, is disordered in both molecules in the asymmetric unit, possibly due to the absence of the RAM (RBPJkappa-associated molecule) region N-terminal to it. The exposed hydrophobic core is involved in intermolecular interactions in the crystal. Evolutionary trace analysis identified several residues that map to the hairpins of the structure and may be of functional importance. Based on the Notch 1 ANK structure and analysis of homologous Notch ANK sequences, we predict two possible binding sites on the domain: one on the concave surface of repeat 2 and the other below the hairpins of repeats 6-7.
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http://dx.doi.org/10.1042/BJ20050515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1317659PMC
November 2005