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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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diego A.
Leonardo
,
Italo A.
Cavini
,
Fernanda A.
Sala
,
Deborah C.
Mendonça
,
Higor
V. D. Rosa
,
Patricia S.
Kumagai
,
Edson
Crusca Jr
,
Napoleao F.
Valadares
,
Ivo A.
Marques
,
Jose
Brandao-Neto
,
Claudia E.
Munte
,
Hans R.
Kalbitzer
,
Nicolas
Soler
,
Isabel
Uson
,
Ingemar
André
,
Ana
P. U. Araujo
,
Humberto
D'Muniz Pereira
,
Richard C.
Garratt
Diamond Proposal Number(s):
[6397, 23570, 25296]
Abstract: Septins are an example of subtle molecular recognition whereby different paralogues must correctly assemble into functional filaments important for essential cellular events such as cytokinesis. Most possess C-terminal domains capable of forming coiled coils which are believed to be involved in filament formation and bundling. Here, we report an integrated structural approach which aims to unravel their architectural diversity and in so doing provide direct structural information for the coiled-coil regions of five human septins. Unexpectedly, we encounter dimeric structures presenting both parallel and antiparallel arrangements which are in consonance with molecular modelling suggesting that both are energetically accessible. These sequences therefore code for two metastable states of different orientations which employ different but overlapping interfaces. The antiparallel structures present a mixed coiled-coil interface, one side of which is dominated by a continuous chain of core hydrophilic residues. This unusual type of coiled coil could be used to expand the toolkit currently available to the protein engineer for the design of previously unforeseen coiled-coil based assemblies. Within a physiological context, our data provide the first atomic details related to the assumption that the parallel orientation is likely formed between septin monomers from the same filament whilst antiparallelism may participate in the widely described interfilament cross-bridges necessary for higher order structures and thereby septin function.
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Feb 2021
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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-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Abstract: The assembly of a septin filament requires that homologous monomers must distinguish between one another in establishing appropriate interfaces with their neighbors. To understand this phenomenon at the molecular level, we present the first four crystal structures of heterodimeric septin complexes. We describe in detail the two distinct types of G-interface present within the octameric particles which must polymerize to form filaments. These are formed between SEPT2 and SEPT6 and between SEPT7 and SEPT3, and their description permits an understanding of the structural basis for the selectivity necessary for correct filament assembly. By replacing SEPT6 by SEPT8 or SEPT11, it is possible to rationalize Kinoshita's postulate which predicts the exchangeability of septins from within a subgroup. Switches I and II, which in classical small GTPases provide a mechanism for nucleotide-dependent conformational change, have been repurposed in septins to play a fundamental role in molecular recognition. Specifically, it is switch I which holds the key to discriminating between the two different G-interfaces. Moreover, residues which are characteristic for a given subgroup play subtle, but pivotal, roles in guaranteeing that the correct interfaces are formed.
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Sep 2020
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14493, 5810]
Open Access
Abstract: Human septins 3, 9 and 12 are the only members of a specific subgroup of septins that display several unusual features, including the absence of a C-terminal coiled coil. This particular subgroup (the SEPT3 septins) are present in rod-like octameric protofilaments but are lacking in similar hexameric assemblies, which only contain representatives of the three remaining subgroups. Both hexamers and octamers can self-assemble into mixed filaments by end-to-end association, implying that the SEPT3 septins may facilitate polymerization but not necessarily function. These filaments frequently associate into higher order complexes which associate with biological membranes, triggering a wide range of cellular events. In the present work, a complete compendium of crystal structures for the GTP-binding domains of all of the SEPT3 subgroup members when bound to either GDP or to a GTP analogue is provided. The structures reveal a unique degree of plasticity at one of the filamentous interfaces (dubbed NC). Specifically, structures of the GDP and GTPγS complexes of SEPT9 reveal a squeezing mechanism at the NC interface which would expel a polybasic region from its binding site and render it free to interact with negatively charged membranes. On the other hand, a polyacidic region associated with helix α5′, the orientation of which is particular to this subgroup, provides a safe haven for the polybasic region when retracted within the interface. Together, these results suggest a mechanism which couples GTP binding and hydrolysis to membrane association and implies a unique role for the SEPT3 subgroup in this process. These observations can be accounted for by constellations of specific amino-acid residues that are found only in this subgroup and by the absence of the C-terminal coiled coil. Such conclusions can only be reached owing to the completeness of the structural studies presented here.
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May 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Juliana Roverta
Torini
,
Adriano
De Freitas Fernandes
,
Vitor Hugo
Balasco Serrao
,
Larissa
Romanello
,
Louise
Bird
,
Joanne E.
Nettleship
,
Raymond J.
Owens
,
Jose
Brandao-neto
,
Ana Eliza
Zeraik
,
Ricardo
Demarco
,
Humberto
D'muniz Pereira
Diamond Proposal Number(s):
[5073, 14493]
Abstract: Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease.
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Jul 2019
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14496]
Abstract: Septins are GTP-binding proteins that will often spontaneously assemble into filaments. In some species, particularly budding yeast, it is well known that these are capable of associating with membranes in order to fulfill their cellular role as a component of the cytoskeleton. Different from other human septins, SEPT7 appears to be unique in that it is an essential component of all hetero-oligomeric complexes described to date. As a step towards understanding the molecular basis of filament assembly, here we present two high-resolution structures of the SEPT7 GTPase domain complexed with GDP. One of these reveals a previously unreported coordination for the magnesium ion involving four water molecules and only a tenuous connection to the protein. The higher resolution structures provide unambiguous insight into the interactions at the G-interface where a structural motif based on an antiparallel β-bridge allows for the rationalization of why some septins show nucleotide-dependent β-strand slippage and others do not.
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Apr 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Larissa
Romanello
,
Ana Eliza
Zeraik
,
Adriano
De Freitas Fernandes
,
Juliana Roberta
Torini
,
Louise E.
Bird
,
Joanne E.
Nettleship
,
Heather
Rada
,
Yamini
Reddivari
,
Ray J.
Owens
,
Vitor Hugo Balasco
Serrão
,
Ricardo
Demarco
,
Jose
Brandao-neto
,
Humberto
D'muniz Pereira
Diamond Proposal Number(s):
[5073]
Abstract: Schistosoma mansoni, the parasite responsible for schistosomiasis, lacks the “de novo” purine biosynthetic pathway and depends entirely on the purine salvage pathway for the supply of purines. Numerous reports of praziquantel resistance have been described, as well as stimulated efforts to develop new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine salvage pathway. Here, we describe a crystallographic structure of the S. mansoni HPGRT-1 (SmHGPRT), complexed with IMP at a resolution of 2.8Ǻ. Four substitutions were identified in the region of the active site between SmHGPRT-1 and human HGPRT. We also present data from RNA-Seq and WISH, suggesting that some isoforms of HGPRT might be involved in the process related to sexual maturation and reproduction in worms; furthermore, its enzymatic assays show that the isoform SmHGPRT-3 does not present the same catalytic efficiency as other isoforms. Finally, although other studies have previously suggested this enzyme as a potential antischistosomal chemotherapy target, the kinetics parameters reveal the impossibility to use SmHGPRT as an efficient chemotherapeutic target.
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Feb 2019
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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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