I04-1-Macromolecular Crystallography (fixed wavelength)
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Muhamamd
Faheem
,
Napoleao
Fonseca Valadares
,
Jose
Brandao-Neto
,
Domenico
Bellini
,
Patrick
Collins
,
Nicholas M.
Pearce
,
Louise
Bird
,
Juliana
Torini De Souza
,
Raymond
Owens
,
Humberto
Pereira
,
Frank
Von Delft
,
João Alexandre Ribeiro Gonçalves
Barbosa
Diamond Proposal Number(s):
[11175]
Open Access
Abstract: Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness – which is limited to the parasite’s adult form – and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. 14 of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.
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Sep 2021
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Nathan David
Wright
,
Patrick
Collins
,
Lizbe
Koekemoer
,
Tobias
Krojer
,
Romain
Talon
,
Elliot
Nelson
,
Mingda
Ye
,
Radoslaw
Nowak
,
Joseph
Newman
,
Jia Tsing
Ng
,
Nick
Mitrovic
,
Helton
Wiggers
,
Frank
Von Delft
Open Access
Abstract: Despite the tremendous success of X-ray cryo-crystallography in recent decades, the transfer of crystals from the drops in which they are grown to diffractometer sample mounts remains a manual process in almost all laboratories. Here, the Shifter, a motorized, interactive microscope stage that transforms the entire crystal-mounting workflow from a rate-limiting manual activity to a controllable, high-throughput semi-automated process, is described. By combining the visual acuity and fine motor skills of humans with targeted hardware and software automation, it was possible to transform the speed and robustness of crystal mounting. Control software, triggered by the operator, manoeuvres crystallization plates beneath a clear protective cover, allowing the complete removal of film seals and thereby eliminating the tedium of repetitive seal cutting. The software, either upon request or working from an imported list, controls motors to position crystal drops under a hole in the cover for human mounting at a microscope. The software automatically captures experimental annotations for uploading to the user's data repository, removing the need for manual documentation. The Shifter facilitates mounting rates of 100–240 crystals per hour in a more controlled process than manual mounting, which greatly extends the lifetime of the drops and thus allows a dramatic increase in the number of crystals retrievable from any given drop without loss of X-ray diffraction quality. In 2015, the first in a series of three Shifter devices was deployed as part of the XChem fragment-screening facility at Diamond Light Source, where they have since facilitated the mounting of over 120 000 crystals. The Shifter was engineered to have a simple design, providing a device that could be readily commercialized and widely adopted owing to its low cost. The versatile hardware design allows use beyond fragment screening and protein crystallography.
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Jan 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Fragment based methods are now widely used to identify starting points in drug discovery and generation of tools for chemical biology. A significant challenge is optimization of these weak binding fragments to hit and lead compounds. We have developed an approach where individual reaction mixtures of analogues of hits can be evaluated without purification of the product. Here, we describe experiments to optimise the processes and then assess such mixtures in the high throughput crystal structure determination facility, XChem. Diffraction data for crystals of the proteins Hsp90 and PDHK2 soaked individually with 83 crude reaction mixtures are analysed manually or with the automated XChem procedures. The results of structural analysis are compared with binding measurements from other biophysical techniques. This approach can transform early hit to lead optimisation and the lessons learnt from this study provide a protocol that can be used by the community.
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Sep 2020
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Abstract: The XChem facility at Diamond Light Source offers fragment screening by X-ray crystallography as a general access user program. The main advantage of X-ray crystallography as a primary fragment screen is that it yields directly the location and pose of the fragment hits, whether within pockets of interest or merely on surface sites: this is the key information for structure-based design and for enabling synthesis of follow-up molecules. Extensive streamlining of the screening experiment at XChem has engendered a very active user program that is generating large amounts of data: in 2017, 36 academic and industry groups generated 35,000 datasets of uniquely soaked crystals. It has also generated a large number of learnings concerning the main remaining bottleneck, namely, obtaining a suitable crystal system that will support a successful fragment screen. Here we discuss the practicalities of generating screen-ready crystals that have useful electron density maps, and how to ensure they will be successfully reproduced and usable at a facility outside the home lab.
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Oct 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: The productive exploration of chemical space is an enduring challenge in chemical biology and medicinal chemistry. Natural products are biologically relevant, and their frameworks have facilitated chemical tool and drug discovery. A “top-down” synthetic approach is described that enabled a range of complex bridged intermediates to be converted with high step efficiency into 26 diverse sp3-rich scaffolds. The scaffolds have local natural product-like features, but are only distantly related to specific natural product frameworks. To assess biological relevance, a set of 52 fragments was prepared, and screened by high-throughput crystallography against three targets from two protein families (ATAD2, BRD1 and JMJD2D). In each case, 3D fragment hits were identified that would serve as distinctive starting points for ligand discovery. This demonstrates that frameworks that are distantly related to natural products can facilitate discovery of new biologically relevant regions within chemical space.
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Oct 2017
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I04-Macromolecular Crystallography
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Rachel J.
Harding
,
Renato
Ferreira De Freitas
,
Patrick
Collins
,
Ivan
Franzoni
,
Mani
Ravichandran
,
Hui
Ouyang
,
Kevin A.
Juarez-Ornelas
,
Mark
Lautens
,
Matthieu
Schapira
,
Frank
Von Delft
,
Vijayaratnam
Santhakumar
,
Cheryl H.
Arrowsmith
Abstract: Inhibitors of HDAC6 have attractive potential in numerous cancers. HDAC6 inhibitors to date target the catalytic domains, but targeting the unique zinc-finger ubiquitin-binding domain (Zf-UBD) of HDAC6 may be an attractive alternative strategy. We developed X-ray crystallography and biophysical assays to identify and characterize small molecules capable of binding to the Zf-UBD and competing with ubiquitin binding. Our results revealed two adjacent ligand-able pockets of HDAC6 Zf-UBD and the first functional ligands for this domain.
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Oct 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Patrick J.
Mcintyre
,
Patrick
Collins
,
Lukáš
Vrzal
,
Kristian
Birchall
,
Laurence H.
Arnold
,
Chido
Mpamhanga
,
Peter J.
Coombs
,
Selena G.
Burgess
,
Mark W.
Richards
,
Anja
Winter
,
Václav
Veverka
,
Frank
Von Delft
,
Andy
Merritt
,
Richard
Bayliss
Diamond Proposal Number(s):
[14331]
Abstract: The mitotic kinase Aurora-A and its partner protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2) are overexpressed in cancers, and it has been proposed that they work together as an oncogenic holoenzyme. TPX2 is responsible for activating Aurora-A during mitosis, ensuring proper cell division. Disruption of the interface with TPX2 is therefore a potential target for novel anticancer drugs that exploit the increased sensitivity of cancer cells to mitotic stress. Here, we investigate the interface using coprecipitation assays and isothermal titration calorimetry to quantify the energetic contribution of individual residues of TPX2. Residues Tyr8, Tyr10, Phe16, and Trp34 of TPX2 are shown to be crucial for robust complex formation, suggesting that the interaction could be abrogated through blocking any of the three pockets on Aurora-A that complement these residues. Phosphorylation of Aurora-A on Thr288 is also necessary for high-affinity binding, and here we identify arginine residues that communicate the phosphorylation of Thr288 to the TPX2 binding site. With these findings in mind, we conducted a high-throughput X-ray crystallography-based screen of 1255 fragments against Aurora-A and identified 59 hits. Over three-quarters of these hits bound to the pockets described above, both validating our identification of hotspots and demonstrating the druggability of this protein–protein interaction. Our study exemplifies the potential of high-throughput crystallography facilities such as XChem to aid drug discovery. These results will accelerate the development of chemical inhibitors of the Aurora-A/TPX2 interaction.
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Oct 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)
I04-Macromolecular Crystallography
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Xiaoli
Xiong
,
Stephen
Martin
,
Lesley F.
Haire
,
Stephen A.
Wharton
,
Rodney S.
Daniels
,
Michael S.
Bennett
,
John W.
Mccauley
,
Patrick J.
Collins
,
Philip A.
Walker
,
John
Skehel
,
Steven
Gamblin
Diamond Proposal Number(s):
[7707]
Abstract: Of the 132 people known to have been infected with H7N9 influenza viruses in China, 37 died, and many were severely ill1. Infection seems to have involved contact with infected poultry2, 3. We have examined the receptor-binding properties of this H7N9 virus and compared them with those of an avian H7N3 virus. We find that the human H7 virus has significantly higher affinity for ?-2,6-linked sialic acid analogues (‘human receptor’) than avian H7 while retaining the strong binding to ?-2,3-linked sialic acid analogues (‘avian receptor’) characteristic of avian viruses. The human H7 virus does not, therefore, have the preference for human versus avian receptors characteristic of pandemic viruses. X-ray crystallography of the receptor-binding protein, haemagglutinin (HA), in complex with receptor analogues indicates that both human and avian receptors adopt different conformations when bound to human H7 HA than they do when bound to avian H7 HA. Human receptor bound to human H7 HA exits the binding site in a different direction to that seen in complexes formed by HAs from pandemic viruses4, 5 and from an aerosol-transmissible H5 mutant6. The human-receptor-binding properties of human H7 probably arise from the introduction of two bulky hydrophobic residues by the substitutions Gln226Leu and Gly186Val. The former is shared with the 1957 H2 and 1968 H3 pandemic viruses and with the aerosol-transmissible H5 mutant. We conclude that the human H7 virus has acquired some of the receptor-binding characteristics that are typical of pandemic viruses, but its retained preference for avian receptor may restrict its further evolution towards a virus that could transmit efficiently between humans, perhaps by binding to avian-receptor-rich mucins in the human respiratory tract7 rather than to cellular receptors.
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Jun 2013
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