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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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Tamar
Skaist Mehlmam
,
Justin T.
Biel
,
Syeda Maryam
Azeem
,
Elliot R.
Nelson
,
Sakib
Hossain
,
Louise
Dunnett
,
Neil G.
Paterson
,
Alice
Douangamath
,
Romain
Talon
,
Danny
Axford
,
Helen
Orins
,
Frank
Von Delft
,
Daniel A.
Keedy
Diamond Proposal Number(s):
[15751, 18340, 23570]
Open Access
Abstract: Much of our current understanding of how small-molecule ligands interact with proteins stems from X-ray crystal structures determined at cryogenic (cryo) temperature. For proteins alone, room-temperature (RT) crystallography can reveal previously hidden, biologically relevant alternate conformations. However, less is understood about how RT crystallography may impact the conformational landscapes of protein-ligand complexes. Previously, we showed that small-molecule fragments cluster in putative allosteric sites using a cryo crystallographic screen of the therapeutic target PTP1B (Keedy et al., 2018). Here, we have performed two RT crystallographic screens of PTP1B using many of the same fragments, representing the largest RT crystallographic screens of a diverse library of ligands to date, and enabling a direct interrogation of the effect of data collection temperature on protein-ligand interactions. We show that at RT, fewer ligands bind, and often more weakly – but with a variety of temperature-dependent differences, including unique binding poses, changes in solvation, new binding sites, and distinct protein allosteric conformational responses. Overall, this work suggests that the vast body of existing cryo-temperature protein-ligand structures may provide an incomplete picture, and highlights the potential of RT crystallography to help complete this picture by revealing distinct conformational modes of protein-ligand systems. Our results may inspire future use of RT crystallography to interrogate the roles of protein-ligand conformational ensembles in biological function.
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Mar 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[16816, 18582, 19190, 25586, 30588]
Open Access
Abstract: Polyomaviruses are a family of ubiquitous double-stranded DNA viruses many of which are human pathogens. These include BK polyomavirus which causes severe urinary tract infection in immunocompromised patients and Merkel cell polyomavirus associated with aggressive cancers. The small genome of polyomaviruses lacks conventional drug targets, and no specific drugs are available at present. Here we focus on the main structural protein VP1 of BK polyomavirus which is responsible for icosahedral capsid formation. To provide a foundation towards rational drug design, we crystallized truncated VP1 pentamers and subjected them to a high-throughput screening for binding drug-like fragments through a direct X-ray analysis. To enable a highly performant screening, rigorous optimization of the crystallographic pipeline and processing with the latest generation PanDDA2 software were necessary. As a result, a total of 144 binding hits were established. Importantly, the hits are well clustered in six surface pockets. Three pockets are located on the outside of the pentamer and map on the regions where the ‘invading’ C-terminal arm of another pentamer is attached upon capsid assembly. Another set of three pockets is situated within the wide pore along the five-fold axis of the VP1 pentamer. These pockets are situated at the interaction interface with the minor capsid protein VP2 which is indispensable for normal functioning of the virus. Here we systematically analyse the three outside pockets which are highly conserved across various polyomaviruses, while point mutations in these pockets are detrimental for viral replication. We show that one of the pockets can accommodate antipsychotic drug trifluoperazine. For each pocket, we derive pharmacophore features which enable the design of small molecules preventing the interaction between VP1 pentamers and therefore inhibiting capsid assembly. Our data lay a foundation towards a rational development of first-in-class drugs targeting polyomavirus capsid.
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May 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Joseph A.
Newman
,
Alice
Douangamath
,
Setayesh
Yadzani
,
Yuliana
Yosaatmadja
,
Anthony
Aimon
,
Jose
Brandao-Neto
,
Louise
Dunnett
,
Tyler
Gorrie-Stone
,
Rachael
Skyner
,
Daren
Fearon
,
Matthieu
Schapira
,
Frank
Von Delft
,
Opher
Gileadi
Diamond Proposal Number(s):
[26998]
Open Access
Abstract: There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two “druggable” pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.
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Aug 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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G. D.
Noske
,
A. M.
Nakamura
,
V. O.
Gawriljuk
,
R. S.
Fernandes
,
G.
M. A. Lima
,
H. V. D.
Rosa
,
H. D.
Pereira
,
A. C. M.
Zeri
,
A. A. F. Z.
Nascimento
,
M. C. L. C.
Freire
,
D.
Fearon
,
A.
Douangamath
,
F.
Von Delft
,
G.
Oliva
,
A. S.
Godoy
Diamond Proposal Number(s):
[27963]
Abstract: SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral Mpro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. The lack of structural information for intermediary forms of Mpro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 Mpro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the Mpro bound to its endogenous N and C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.
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Jun 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Daniel
Zaidman
,
Paul
Gehrtz
,
Mihajlo
Filep
,
Daren
Fearon
,
Ronen
Gabizon
,
Alice
Douangamath
,
Jaime
Prilusky
,
Shirly
Duberstein
,
Galit
Cohen
,
C. David
Owen
,
Efrat
Resnick
,
Claire
Strain-Damerell
,
Petra
Lukacik
,
Haim
Barr
,
Martin A.
Walsh
,
Frank
Von Delft
,
Nir
London
Diamond Proposal Number(s):
[18145, 27963]
Abstract: Designing covalent inhibitors is increasingly important, although it remains challenging. Here, we present covalentizer, a computational pipeline for identifying irreversible inhibitors based on structures of targets with non-covalent binders. Through covalent docking of tailored focused libraries, we identify candidates that can bind covalently to a nearby cysteine while preserving the interactions of the original molecule. We found ∼11,000 cysteines proximal to a ligand across 8,386 complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In a prospective evaluation, five out of nine predicted covalent kinase inhibitors showed half-maximal inhibitory concentration (IC50) values between 155 nM and 4.5 μM. Application against an existing SARS-CoV Mpro reversible inhibitor led to an acrylamide inhibitor series with low micromolar IC50 values against SARS-CoV-2 Mpro. The docking was validated by 12 co-crystal structures. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.
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Jun 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Martin A.
Redhead
,
C. David
Owen
,
Lennart
Brewitz
,
Amelia H.
Collette
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
Sean W.
Robinson
,
Patrick M.
Collins
,
Philipp
Schäfer
,
Mark
Swindells
,
Chris J.
Radoux
,
Iva Navratilova
Hopkins
,
Daren
Fearon
,
Alice
Douangamath
,
Frank
Von Delft
,
Tika R.
Malla
,
Laura
Vangeel
,
Thomas
Vercruysse
,
Jan
Thibaut
,
Pieter
Leyssen
,
Tu-Trinh
Nguyen
,
Mitchell
Hull
,
Anthony
Tumber
,
David J.
Hallett
,
Christopher J.
Schofield
,
David I.
Stuart
,
Andrew L.
Hopkins
,
Martin A.
Walsh
Open Access
Abstract: Effective agents to treat coronavirus infection are urgently required, not only to treat COVID-19, but to prepare for future outbreaks. Repurposed anti-virals such as remdesivir and human anti-inflammatories such as barcitinib have received emergency approval but their overall benefits remain unclear. Vaccines are the most promising prospect for COVID-19, but will need to be redeveloped for any future coronavirus outbreak. Protecting against future outbreaks requires the identification of targets that are conserved between coronavirus strains and amenable to drug discovery. Two such targets are the main protease (Mpro) and the papain-like protease (PLpro) which are essential for the coronavirus replication cycle. We describe the discovery of two non-antiviral therapeutic agents, the caspase-1 inhibitor SDZ 224015 and Tarloxotinib that target Mpro and PLpro, respectively. These were identified through extensive experimental screens of the drug repurposing ReFRAME library of 12,000 therapeutic agents. The caspase-1 inhibitor SDZ 224015, was found to be a potent irreversible inhibitor of Mpro (IC50 30 nM) while Tarloxotinib, a clinical stage epidermal growth factor receptor inhibitor, is a sub micromolar inhibitor of PLpro (IC50 300 nM, Ki 200 nM) and is the first reported PLpro inhibitor with drug-like properties. SDZ 224015 and Tarloxotinib have both undergone safety evaluation in humans and hence are candidates for COVID-19 clinical evaluation.
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Jun 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Dávid
Bajusz
,
Warren S.
Wade
,
Grzegorz
Satała
,
Andrzej J.
Bojarski
,
Janez
Ilaš
,
Jessica
Ebner
,
Florian
Grebien
,
Henrietta
Papp
,
Ferenc
Jakab
,
Alice
Douangamath
,
Daren
Fearon
,
Frank
Von Delft
,
Marion
Schuller
,
Ivan
Ahel
,
Amanda
Wakefield
,
Sándor
Vajda
,
János
Gerencsér
,
Péter
Pallai
,
György M.
Keserű
Diamond Proposal Number(s):
[27001, 18145, 27963]
Open Access
Abstract: Fragment-based drug design has introduced a bottom-up process for drug development, with improved sampling of chemical space and increased effectiveness in early drug discovery. Here, we combine the use of pharmacophores, the most general concept of representing drug-target interactions with the theory of protein hotspots, to develop a design protocol for fragment libraries. The SpotXplorer approach compiles small fragment libraries that maximize the coverage of experimentally confirmed binding pharmacophores at the most preferred hotspots. The efficiency of this approach is demonstrated with a pilot library of 96 fragment-sized compounds (SpotXplorer0) that is validated on popular target classes and emerging drug targets. Biochemical screening against a set of GPCRs and proteases retrieves compounds containing an average of 70% of known pharmacophores for these targets. More importantly, SpotXplorer0 screening identifies confirmed hits against recently established challenging targets such as the histone methyltransferase SETD2, the main protease (3CLPro) and the NSP3 macrodomain of SARS-CoV-2.
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May 2021
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NONE-No attached Diamond beamline
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Alice
Douangamath
,
Alisa
Powell
,
Daren
Fearon
,
Patrick M.
Collins
,
Romain
Talon
,
Tobias
Krojer
,
Rachael
Skyner
,
Jose
Brandao-Neto
,
Louise
Dunnett
,
Alexandre
Dias
,
Anthony
Aimon
,
Nicholas M.
Pearce
,
Conor
Wild
,
Tyler J.
Gorrie-Stone
,
Frank
Von Delft
Open Access
Abstract: In fragment-based drug discovery, hundreds or often thousands of compounds smaller than ~300 Da are tested against the protein of interest to identify chemical entities that can be developed into potent drug candidates. Since the compounds are small, interactions are weak, and the screening method must therefore be highly sensitive; moreover, structural information tends to be crucial for elaborating these hits into lead-like compounds. Therefore, protein crystallography has always been a gold-standard technique, yet historically too challenging to find widespread use as a primary screen.
Initial XChem experiments were demonstrated in 2014 and then trialed with academic and industrial collaborators to validate the process. Since then, a large research effort and significant beamtime have streamlined sample preparation, developed a fragment library with rapid follow-up possibilities, automated and improved the capability of I04-1 beamline for unattended data collection, and implemented new tools for data management, analysis and hit identification.
XChem is now a facility for large-scale crystallographic fragment screening, supporting the entire crystals-to-deposition process, and accessible to academic and industrial users worldwide. The peer-reviewed academic user program has been actively developed since 2016, to accommodate projects from as broad a scientific scope as possible, including well-validated as well as exploratory projects. Academic access is allocated through biannual calls for peer-reviewed proposals, and proprietary work is arranged by Diamond's Industrial Liaison group. This workflow has already been routinely applied to over a hundred targets from diverse therapeutic areas, and effectively identifies weak binders (1%-30% hit rate), which both serve as high-quality starting points for compound design and provide extensive structural information on binding sites. The resilience of the process was demonstrated by continued screening of SARS-CoV-2 targets during the COVID-19 pandemic, including a 3-week turn-around for the main protease.
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May 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Marion
Schuller
,
Galen J.
Correy
,
Stefan
Gahbauer
,
Daren
Fearon
,
Taiasean
Wu
,
Roberto Efraín
Díaz
,
Iris D.
Young
,
Luan
Carvalho Martins
,
Dominique H.
Smith
,
Ursula
Schulze-Gahmen
,
Tristan W.
Owens
,
Ishan
Deshpande
,
Gregory E.
Merz
,
Aye C.
Thwin
,
Justin T.
Biel
,
Jessica K.
Peters
,
Michelle
Moritz
,
Nadia
Herrera
,
Huong T.
Kratochvil
,
Anthony
Aimon
,
James
Bennett
,
Jose
Brandao Neto
,
Aina E.
Cohen
,
Alexandre
Dias
,
Alice
Douangamath
,
Louise
Dunnett
,
Oleg
Fedorov
,
Matteo P.
Ferla
,
Martin R.
Fuchs
,
Tyler J.
Gorrie-Stone
,
James M.
Holton
,
Michael G.
Johnson
,
Tobias
Krojer
,
George
Meigs
,
Alisa J.
Powell
,
Johannes Gregor Matthias
Rack
,
Victor
Rangel
,
Silvia
Russi
,
Rachael E.
Skyner
,
Clyde A.
Smith
,
Alexei S.
Soares
,
Jennifer L.
Wierman
,
Kang
Zhu
,
Peter
O’brien
,
Natalia
Jura
,
Alan
Ashworth
,
John J.
Irwin
,
Michael C.
Thompson
,
Jason E.
Gestwicki
,
Frank
Von Delft
,
Brian K.
Shoichet
,
James S.
Fraser
,
Ivan
Ahel
Diamond Proposal Number(s):
[27001]
Open Access
Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate–ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.
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Apr 2021
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