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Open Access
Abstract: Selectivity is a crucial property in small molecule development. Binding site comparisons within a protein family are a key piece of information when aiming to modulate the selectivity profile of a compound. Binding site differences can be exploited to confer selectivity for a specific target, while shared areas can provide insights into polypharmacology. As the quantity of structural data grows, automated methods are needed to process, summarize, and present these data to users. We present a computational method that provides quantitative and data-driven summaries of the available binding site information from an ensemble of structures of the same protein. The resulting ensemble maps identify the key interactions important for ligand binding in the ensemble. The comparison of ensemble maps of related proteins enables the identification of selectivity-determining regions within a protein family. We applied the method to three examples from the well-researched human bromodomain and kinase families, demonstrating that the method is able to identify selectivity-determining regions that have been used to introduce selectivity in past drug discovery campaigns. We then illustrate how the resulting maps can be used to automate comparisons across a target protein family.
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Jan 2022
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Susanne
Müller
,
Suzanne
Ackloo
,
Arij
Al Chawaf
,
Bissan
Al-Lazikani
,
Albert
Antolin
,
Jonathan B.
Baell
,
Hartmut
Beck
,
Shaunna
Beedie
,
Ulrich A. K.
Betz
,
Gustavo
Arruda Bezerra
,
Paul E.
Brennan
,
David
Brown
,
Peter J.
Brown
,
Alex N.
Bullock
,
Adrian J.
Carter
,
Apirat
Chaikuad
,
Mathilde
Chaineau
,
Alessio
Ciulli
,
Ian
Collins
,
Jan
Dreher
,
David
Drewry
,
Kristina
Edfeldt
,
Aled M.
Edwards
,
Ursula
Egner
,
Stephen V.
Frye
,
Stephen M.
Fuchs
,
Matthew D.
Hall
,
Ingo V.
Hartung
,
Alexander
Hillisch
,
Stephen H.
Hitchcock
,
Evert
Homan
,
Natarajan
Kannan
,
James R.
Kiefer
,
Stefan
Knapp
,
Milka
Kostic
,
Stefan
Kubicek
,
Andrew S.
Leach
,
Sven
Lindemann
,
Brian D.
Marsden
,
Hisanori
Matsui
,
Jordan L.
Meier
,
Daniel
Merk
,
Maurice
Michel
,
Maxwell R.
Morgan
,
Anke
Mueller-Fahrnow
,
Dafydd R.
Owen
,
Benjamin G.
Perry
,
Saul H.
Rosenberg
,
Kumar Singh
Saikatendu
,
Matthieu
Schapira
,
Cora
Scholten
,
Sujata
Sharma
,
Anton
Simeonov
,
Michael
Sundström
,
Giulio
Superti-Furga
,
Matthew H.
Todd
,
Claudia
Tredup
,
Masoud
Vedadi
,
Frank
Von Delft
,
Timothy M.
Willson
,
Georg E.
Winter
,
Paul
Workman
,
Cheryl H.
Arrowsmith
Open Access
Abstract: Twenty years after the publication of the first draft of the human genome, our knowledge of the human proteome is still fragmented. The challenge of translating the wealth of new knowledge from genomics into new medicines is that proteins, and not genes, are the primary executers of biological function. Therefore, much of how biology works in health and disease must be understood through the lens of protein function. Accordingly, a subset of human proteins has been at the heart of research interests of scientists over the centuries, and we have accumulated varying degrees of knowledge about approximately 65% of the human proteome. Nevertheless, a large proportion of proteins in the human proteome (∼35%) remains uncharacterized, and less than 5% of the human proteome has been successfully targeted for drug discovery. This highlights the profound disconnect between our abilities to obtain genetic information and subsequent development of effective medicines. Target 2035 is an international federation of biomedical scientists from the public and private sectors, which aims to address this gap by developing and applying new technologies to create by year 2035 chemogenomic libraries, chemical probes, and/or biological probes for the entire human proteome.
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Dec 2021
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I13-2-Diamond Manchester Imaging
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J.
Wade-Zhu
,
R.
Krishna
,
A. J.
Bodey
,
M.
Davies
,
N. K.
Bourne
,
C.
Rau
,
B.
Davies
,
A.
Tzelepi
,
A. N.
Jones
,
B. J.
Marsden
,
P. M.
Mummery
Diamond Proposal Number(s):
[16668]
Abstract: Herein, the first study is presented using 4D synchrotron X-ray microtomography to capture all stages of crack development in neutron irradiated and radiolytically oxidised nuclear graphite. Employing a novel loading setup, specimens of Gilsocarbon graphite, both unirradiated and irradiated at 301 °C to 19.7 × 1020 neutrons/cm2 (∼2.6 displacements/atom (dpa)), were loaded to generate a crack. All stages of the fracture process were then captured using synchrotron X-ray imaging. Reconstructed tomographic images and 3D segmented crack volumes have been used to observe and analyse the irradiation-induced evolution of the graphite microstructure as well as corresponding changes in the crack initiation, propagation, and arrest behaviour of graphite after neutron irradiation. Close examination of the applied stress-strain curves highlights the suppression of micro-crack-based damage accumulation in irradiated graphite. Moreover, as well as the crack-bridging and deflection mechanisms characteristic of unirradiated graphite, crack arrest in the irradiated graphite is shown to be significantly influenced by crack tip blunting. This change is associated with the growth of the open pore structure of graphite, specifically the enlargement and increased frequency of macro-pores, resulting from the simultaneous radiolytic oxidation of the graphite microstructure during neutron irradiation.
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Jul 2020
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I03-Macromolecular Crystallography
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Melissa
D'Ascenzio
,
Kathryn M.
Pugh
,
Rebecca
Konietzny
,
Georgina
Berridge
,
Cynthia
Tallant
,
Shaima
Hashem
,
Octovia
Monteiro
,
Jason R.
Thomas
,
Markus
Schirle
,
Stefan
Knapp
,
Brian
Marsden
,
Oleg
Fedorov
,
Chas
Bountra
,
Benedikt M.
Kessler
,
Paul E.
Brennan
Diamond Proposal Number(s):
[10619]
Open Access
Abstract: 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.
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Jan 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Anthony R.
Bradley
,
Aude
Echalier
,
Michael
Fairhead
,
Claire
Strain-Damerell
,
Paul
Brennan
,
Alex n.
Bullock
,
Nicola a.
Burgess-Brown
,
Elizabeth P.
Carpenter
,
Opher
Gileadi
,
Brian d.
Marsden
,
Wen hwa
Lee
,
Wyatt
Yue
,
Chas
Bountra
,
Frank
Von Delft
Open Access
Abstract: 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.
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Nov 2017
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[8421]
Open Access
Abstract: 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.
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May 2017
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Nicholas M.
Pearce
,
Tobias
Krojer
,
Anthony R.
Bradley
,
Patrick
Collins
,
Radoslaw P.
Nowak
,
Romain
Talon
,
Brian D.
Marsden
,
Sebastian
Kelm
,
Jiye
Shi
,
Charlotte M.
Deane
,
Frank
Von Delft
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: 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.
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Apr 2017
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Open Access
Abstract: 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[Winn, M. D. et al. (2011). Acta Cryst. D67, 235-242.]), Acta Cryst. D67, 235–242] or PHENIX [Adams et al. (2010[Adams, P. D. et al. (2010). Acta Cryst. D66, 213-221.]), 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.
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Mar 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Oakley B
Cox
,
Tobias
Krojer
,
Patrick
Collins
,
Octovia
Monteiro
,
Romain
Talon
,
Anthony
Bradley
,
Oleg
Fedorov
,
Jahangir
Amin
,
Brian D.
Marsden
,
John
Spencer
,
Frank
Von Delft
,
Paul E.
Brennan
Diamond Proposal Number(s):
[5073, 11175]
Open Access
Abstract: 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.
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Dec 2015
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: 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.
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Sep 2015
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