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Daren
Fearon
,
Ailsa
Powell
,
Alice
Douangamath
,
Alexandre
Dias
,
Charles W. E.
Tomlinson
,
Blake H.
Balcomb
,
Jasmin C.
Aschenbrenner
,
Anthony
Aimon
,
Isabel A.
Barker
,
Jose
Brandao-Neto
,
Patrick
Collins
,
Louise E.
Dunnett
,
Michael
Fairhead
,
Richard J.
Gildea
,
Mathew
Golding
,
Tyler
Gorrie-Stone
,
Paul V.
Hathaway
,
Lizbe
Koekemoer
,
Tobias
Krojer
,
Ryan
Lithgo
,
Elizabeth M.
Maclean
,
Peter G.
Marples
,
Xiaomin
Ni
,
Rachael
Skyner
,
Romain
Talon
,
Warren
Thompson
,
Conor F.
Wild
,
Max
Winokan
,
Nathan D.
Wright
,
Graeme
Winter
,
Elizabeth J.
Shotton
,
Frank
Von Delft
Open Access
Abstract: Fragment-based drug discovery is a well-established method for the identification of chemical starting points for development into clinical candidates. Historically, crystallographic fragment screening was perceived to be low-throughput and time consuming. However, thanks to advances in synchrotron capabilities and the introduction of dedicated facilities, such as the XChem platform at Diamond Light Source, there have been substantial improvements in throughput and integration between sample preparation, data collection and hit identification. Herein we share our experiences of establishing a crystallographic fragment screening facility, our learnings from operating a user programme for ten years and our perspective on applying structural enablement to rapidly progress initial fragment hits to lead-like molecules.
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Nov 2024
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I04-1-Macromolecular Crystallography (fixed wavelength)
Krios II-Titan Krios II at Diamond
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Andre
Schutzer Godoy
,
Aline Minalli
Nakamura
,
Alice
Douangamath
,
Yun
Song
,
Gabriela
Dias Noske
,
Victor
Oliveira Gawriljuk
,
Rafaela
Sachetto Fernandes
,
Humberto
D'Muniz Pereira
,
Ketllyn irene
Zagato Oliveira
,
Daren
Fearon
,
Alexandre
Dias
,
Tobias
Krojer
,
Michael
Fairhead
,
Alisa
Powell
,
Louise
Dunnett
,
Jose
Brandao-Neto
,
Rachael
Skyner
,
Rod
Chalk
,
Dávid
Bajusz
,
Miklós
Bege
,
Anikó
Borbás
,
György Miklós
Keserű
,
Frank
Von Delft
,
Glaucius
Oliva
Diamond Proposal Number(s):
[27083, 27023]
Open Access
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.
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Apr 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Alice
Douangamath
,
Daren
Fearon
,
Paul
Gehrtz
,
Tobias
Krojer
,
Petra
Lukacik
,
C. David
Owen
,
Efrat
Resnick
,
Claire
Strain-Damerell
,
Anthony
Aimon
,
Péter
Ábrányi-Balogh
,
Jose
Brandao-Neto
,
Anna
Carbery
,
Gemma
Davison
,
Alexandre
Dias
,
Thomas D.
Downes
,
Louise
Dunnett
,
Michael
Fairhead
,
James D.
Firth
,
S. Paul
Jones
,
Aaron
Keeley
,
György M.
Keserü
,
Hanna F.
Klein
,
Mathew P.
Martin
,
Martin M.
Noble
,
Peter
O’brien
,
Ailsa
Powell
,
Rambabu N.
Reddi
,
Rachael
Skyner
,
Matthew
Snee
,
Michael J.
Waring
,
Conor
Wild
,
Nir
London
,
Frank
Von Delft
,
Martin A.
Walsh
Open Access
Abstract: COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.
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Oct 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Raysa
Khan Tareque
,
Storm
Hassell-Hart
,
Tobias
Krojer
,
Anthony
Bradley
,
Srikannathasan
Velupillai
,
Romain
Talon
,
Michael
Fairhead
,
Iain J.
Day
,
Kamlesh
Bala
,
Robert
Felix
,
Paul D.
Kemmitt
,
Paul
Brennan
,
Frank
Von Delft
,
Laura
Diaz Saez
,
Kilian
Huber
,
John
Spencer
Diamond Proposal Number(s):
[18145]
Abstract: Combined photochemical arylation, “nuisance effect” (SNAr) reaction sequences have been employed in the design of small arrays for immediate deployment in medium‐throughput X‐ray protein–ligand structure determination. Reactions were deliberately allowed to run “out of control” in terms of selectivity; for example the ortho‐arylation of 2‐phenylpyridine gave five products resulting from mono‐ and bisarylations combined with SNAr processes. As a result, a number of crystallographic hits against NUDT7, a key peroxisomal CoA ester hydrolase, have been identified.
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Aug 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Sarah L.
Kidd
,
Elaine
Fowler
,
Till
Reinhardt
,
Thomas
Compton
,
Natalia
Mateu
,
Hector
Newman
,
Dom
Bellini
,
Romain
Talon
,
Joseph
Mcloughlin
,
Tobias
Krojer
,
Anthony
Aimon
,
Anthony
Bradley
,
Michael
Fairhead
,
Paul
Brear
,
Laura
Diaz-Saez
,
Katherine
Mcauley
,
Hannah F.
Sore
,
Andrew
Madin
,
Daniel H.
O'Donovan
,
Kilian
Huber
,
Marko
Hyvonen
,
Frank
Von Delft
,
Christopher G.
Dowson
,
David R.
Spring
Diamond Proposal Number(s):
[18145, 15649, 14303, 14493]
Open Access
Abstract: Organic synthesis underpins the evolution of weak fragment hits into potent lead compounds. Deficiencies within current screening collections often result in the requirement of significant synthetic investment to enable multidirectional fragment growth, limiting the efficiency of the hit evolution process. Diversity-oriented synthesis (DOS)-derived fragment libraries are constructed in an efficient and modular fashion and thus are well-suited to address this challenge. To demonstrate the effective nature of such libraries within fragment-based drug discovery, we herein describe the screening of a 40-member DOS library against three functionally distinct biological targets using X-Ray crystallography. Firstly, we demonstrate the importance for diversity in aiding hit identification with four fragment binders resulting from these efforts. Moreover, we also exemplify the ability to readily access a library of analogues from cheap commercially available materials, which ultimately enabled the exploration of a minimum of four synthetic vectors from each molecule. In total, 10–14 analogues of each hit were rapidly accessed in three to six synthetic steps. Thus, we showcase how DOS-derived fragment libraries enable efficient hit derivatisation and can be utilised to remove the synthetic limitations encountered in early stage fragment-based drug discovery.
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May 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Efrat
Resnick
,
Anthony
Bradley
,
Jinrui
Gan
,
Alice
Douangamath
,
Tobias
Krojer
,
Ritika
Sethi
,
Paul P.
Geurink
,
Anthony
Aimon
,
Gabriel
Amitai
,
Dom
Bellini
,
James
Bennett
,
Michael
Fairhead
,
Oleg
Fedorov
,
Ronen
Gabizon
,
Jin
Gan
,
Jingxu
Guo
,
Alexander
Plotnikov
,
Nava
Reznik
,
Gian Filippo
Ruda
,
Laura
Diaz-Saez
,
Verena M.
Straub
,
Tamas
Szommer
,
Srikannathasan
Velupillai
,
Daniel
Zaidman
,
Yanling
Zhang
,
Alun R.
Coker
,
Christopher G.
Dowson
,
Haim
Barr
,
Chu
Wang
,
Kilian V. M.
Huber
,
Paul E.
Brennan
,
Huib
Ovaa
,
Frank
Von Delft
,
Nir
London
Open Access
Abstract: Covalent probes can display unmatched potency, selectivity and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered non-selective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against ten cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. By contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.
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May 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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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Abstract: The pathway of ligand dissociation and how binding sites respond to force are not well understood for any macromolecule. Force effects on biological receptors have been studied through simulation or force spectroscopy, but not by high resolution structural experiments. To investigate this challenge, we took advantage of the extreme stability of the streptavidin–biotin interaction, a paradigm for understanding non-covalent binding as well as a ubiquitous research tool. We synthesized a series of biotin-conjugates having an unchanged strong-binding biotin moiety, along with pincer-like arms designed to clash with the protein surface: ‘Love–Hate ligands’. The Love–Hate ligands contained various 2,6-di-ortho aryl groups, installed using Suzuki coupling as the last synthetic step, making the steric repulsion highly modular. We determined binding affinity, as well as solving 1.1–1.6 Å resolution crystal structures of streptavidin bound to Love–Hate ligands. Striking distortion of streptavidin’s binding contacts was found for these complexes. Hydrogen bonds to biotin’s ureido and thiophene rings were preserved for all the ligands, but biotin’s valeryl tail was distorted from the classic conformation. Streptavidin’s L3/4 loop, normally forming multiple energetically-important hydrogen bonds to biotin, was forced away by clashes with Love–Hate ligands, but Ser45 from L3/4 could adapt to hydrogen-bond to a different part of the ligand. This approach of preparing conflicted ligands represents a direct way to visualize strained biological interactions and test protein plasticity.
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Oct 2014
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I04-Macromolecular Crystallography
|
Open Access
Abstract: Streptavidin is one of the most important hubs for molecular biology, either multimerizing biomolecules, bridging one molecule to another, or anchoring to a biotinylated surface/nanoparticle. Streptavidin has the advantage of rapid ultra-stable binding to biotin. However, the ability of streptavidin to bind four biotinylated molecules in a heterogeneous manner is often limiting. Here, we present an efficient approach to isolate streptavidin tetramers with two biotin-binding sites in a precise arrangement, cis or trans. We genetically modified specific subunits with negatively charged tags, refolded a mixture of monomers, and used ion-exchange chromatography to resolve tetramers according to the number and orientation of tags. We solved the crystal structures of cis-divalent streptavidin to 1.4 Å resolution and trans-divalent streptavidin to 1.6 Å resolution, validating the isolation strategy and explaining the behavior of the Dead streptavidin variant. cis- and trans-divalent streptavidins retained tetravalent streptavidin's high thermostability and low off-rate. These defined divalent streptavidins enabled us to uncover how streptavidin binding depends on the nature of the biotin ligand. Biotinylated DNA showed strong negative cooperativity of binding to cis-divalent but not trans-divalent streptavidin. A small biotinylated protein bound readily to cis and trans binding sites. We also solved the structure of trans-divalent streptavidin bound to biotin-4-fluorescein, showing how one ligand obstructs binding to an adjacent biotin-binding site. Using a hexaglutamate tag proved a more powerful way to isolate monovalent streptavidin, for ultra-stable labeling without undesired clustering. These forms of streptavidin allow this key hub to be used with a new level of precision, for homogeneous molecular assembly.
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Oct 2013
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