I04-Macromolecular Crystallography
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Scott H.
Henderson
,
Fiona J.
Sorrell
,
James M.
Bennett
,
Oleg
Fedorov
,
Marcus T.
Hanley
,
Paulo H.
Godoi
,
Roberta
Ruela De Sousa
,
Sean
Robinson
,
Iva
Hopkins Navratilova
,
Jonathan M.
Elkins
,
Simon E.
Ward
Open Access
Abstract: elective inhibitors of DYRK1A are of interest for the treatment of cancer, Type 2 diabetes and neurological disorders. Optimization of imidazo [1,2-b]pyridazine fragment 1 through structure−activity relationship exploration and in silico drug design efforts led to the discovery of compound 17 as a potent cellular inhibitor of DYRK1A with selectivity over much of the kinome. The binding mode of compound 17 was elucidated with X-ray crystallography, facilitating the rational design of compound 29, an imidazo [1,2-b]pyridazine with improved kinase selectivity with respect to closely related CLK kinases.
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Apr 2024
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Esra
Balikci
,
Anne-Sophie M. C.
Marques
,
Ludwig G.
Bauer
,
Raina
Seupel
,
James
Bennett
,
Brigitt
Raux
,
Karly
Buchan
,
Klemensas
Simelis
,
Usha
Singh
,
Catherine
Rogers
,
Jennifer
Ward
,
Carol
Cheng
,
Tamas
Szommer
,
Kira
Schützenhofer
,
Jonathan M.
Elkins
,
David L.
Sloman
,
Ivan
Ahel
,
Oleg
Fedorov
,
Paul E.
Brennan
,
Kilian V. M.
Huber
Diamond Proposal Number(s):
[19301, 28172]
Open Access
Abstract: Cofactor mimicry represents an attractive strategy for the development of enzyme inhibitors but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome. Here, we uncover the ADP-ribose (ADPr) hydrolase NUDT5 as an unexpected, noncovalent, off-target of clinical BTK inhibitors. Using a combination of biochemical, biophysical, and intact cell NanoBRET assays as well as X-ray crystallography, we confirm catalytic inhibition and cellular target engagement of NUDT5 and reveal an unusual binding mode that is independent of the reactive acrylamide warhead. Further investigation of the prototypical BTK inhibitor ibrutinib also revealed potent inhibition of the largely unstudied NUDIX hydrolase family member NUDT14. By exploring structure–activity relationships (SARs) around the core scaffold, we identify a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor. Cocrystallization experiments yielded new insights into the NUDT14 hydrolase active site architecture and inhibitor binding, thus providing a basis for future chemical probe design.
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Apr 2024
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Brigitt
Raux
,
Karly A.
Buchan
,
James
Bennett
,
Thomas
Christott
,
Matthew S.
Dowling
,
Gillian
Farnie
,
Oleg
Fedorov
,
Vicki
Gamble
,
Carina
Gileadi
,
Charline
Giroud
,
Kilian V. M.
Huber
,
Magdalena
Korczynska
,
Chris
Limberakis
,
Arjun
Narayanan
,
Dafydd R.
Owen
,
Laura
Diaz Saez
,
Ingrid A.
Stock
,
Allyn T.
Londregan
Abstract: Epigenetic proteins containing YEATS domains (YD) are an emerging target class in drug discovery. Described herein are the discovery and characterization efforts associated with PFI-6, a new chemical probe for the YD of MLLT1 (ENL/YEATS1) and MLLT3 (AF9/YEATS3). For hit identification, fragment-like mimetics of endogenous YD ligands (crotonylated histone-containing proteins), were synthesized via parallel medicinal chemistry (PMC) and screened for MLLT1 binding. Subsequent SAR studies led to iterative MLLT1/3 binding and selectivity improvements, culminating in the discovery of PFI-6. PFI-6 demonstrates good affinity and selectivity for MLLT1/3 vs. other human YD proteins (YEATS2/4) and engages MLLT3 in cells. Small-molecule X-ray co-crystal structures of two molecules, including PFI-6, bound to the YD of MLLT1/3 are also described. PFI-6 may be a useful tool molecule to better understand the biological effects associated with modulation of MLLT1/3.
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Nov 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459]
Open Access
Abstract: The worldwide public health and socioeconomic consequences caused by the COVID-19 pandemic highlight the importance of increasing preparedness for viral disease outbreaks by providing rapid disease prevention and treatment strategies. The NSP3 macrodomain of coronaviruses including SARS-CoV-2 is among the viral protein repertoire that was identified as a potential target for the development of antiviral agents, due to its critical role in viral replication and consequent pathogenicity in the host. By combining virtual and biophysical screening efforts, we discovered several experimental small molecules and FDA-approved drugs as inhibitors of the NSP3 macrodomain. Analogue characterisation of the hit matter and crystallographic studies confirming binding modes, including that of the antibiotic compound aztreonam, to the active site of the macrodomain provide valuable structure–activity relationship information that support current approaches and open up new avenues for NSP3 macrodomain inhibitor development.
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Feb 2023
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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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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15433]
Open Access
Abstract: Kinases represent one of the most intensively pursued groups of targets in modern-day drug discovery. Often it is desirable to achieve selective inhibition of the kinase of interest over the remaining ∼500 kinases in the human kinome. This is especially true when inhibitors are intended to be used to study the biology of the target of interest. We present a pipeline of open-source software that analyzes public domain data to repurpose compounds that have been used in previous kinase inhibitor development projects. We define the dual-specificity tyrosine-regulated kinase 1A (DYRK1A) as the kinase of interest, and by addition of a single methyl group to the chosen starting point we remove glycogen synthase kinase β (GSK3β) and cyclin-dependent kinase (CDK) inhibition. Thus, in an efficient manner we repurpose a GSK3β/CDK chemotype to deliver 8b, a highly selective DYRK1A inhibitor.
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Aug 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Vincent
Fagan
,
Catrine
Johansson
,
Carina
Gileadi
,
Octovia
Monteiro
,
James Edward
Dunford
,
Reshma
Nibhani
,
Martin
Philpott
,
Jessica
Malzahn
,
Graham
Wells
,
Ruth
Farham
,
Adam
Cribbs
,
Nadia
Halidi
,
Fengling
Li
,
Irene
Chau
,
Holger
Greschik
,
Srikannathasan
Velupillai
,
Abdellah
Allali-Hassani
,
James M.
Bennett
,
Thomas
Christott
,
Charline
Giroud
,
Andrew M.
Lewis
,
Kilian V. M.
Huber
,
Nick
Athanasou
,
Chas
Bountra
,
Manfred
Jung
,
Roland
Schüle
,
Masoud
Vedadi
,
Cheryl H.
Arrowsmith
,
Yan
Xiong
,
Jian
Jin
,
Oleg
Fedorov
,
Gillian
Farnie
,
Paul E.
Brennan
,
Udo C. T.
Oppermann
Diamond Proposal Number(s):
[10619, 15433]
Abstract: Modifications of histone tails, including lysine/arginine methylation, provide the basis of a 'chromatin or histone code'. Proteins that con-tain 'reader' domains can bind to these modifications and form specific effector complexes, which ultimately mediate chromatin function. The spindlin1 (SPIN1) protein contains three Tudor methyl-lysine/arginine reader domains and was identified as a putative onco-gene and transcriptional co-activator. Here we report a SPIN1 chemi-cal probe inhibitor with low nanomolar in vitro activity, exquisite selectivity on a panel of methyl reader and writer proteins, and with submicromolar cellular activity. X-ray crystallography showed that this Tudor domain chemical probe simultaneously engages Tudor domains 1 and 2 via a bidentate binding mode. Small molecule inhibition and siRNA knockdown of SPIN1, as well as chemoproteomic studies, iden-tified genes which are transcriptionally regulated by SPIN1 in squa-mous cell carcinoma and suggest that SPIN1 may have a roll in cancer related inflammation and/or cancer metastasis.
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Sep 2019
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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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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Yann-Vai
Le Bihan
,
Rachel M.
Lanigan
,
Butrus
Atrash
,
Mark G.
Mclaughlin
,
Srikannathasan
Velupillai
,
Andrew G.
Malcolm
,
Katherine S.
England
,
Gian Filippo
Ruda
,
N. Yi
Mok
,
Anthony
Tumber
,
Kathy
Tomlin
,
Harry
Saville
,
Erald
Shehu
,
Craig
Mcandrew
,
Leanne
Carmichael
,
James M.
Bennett
,
Fiona
Jeganathan
,
Paul
Eve
,
Adam
Donovan
,
Angela
Hayes
,
Francesca
Wood
,
Florence I.
Raynaud
,
Oleg
Fedorov
,
Paul
Brennan
,
Rosemary
Burke
,
Rob
Van Montfort
,
Olivia W.
Rossanese
,
Julian
Blagg
,
Vassilios
Bavetsias
Diamond Proposal Number(s):
[20145]
Open Access
Abstract: Residues in the histone substrate binding sites that differ between the KDM4 and KDM5 subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one series was designed to rationally exploit these residue differences between the histone substrate binding sites in order to improve affinity for the KDM4-subfamily over KDM5-subfamily enzymes. In particular, residues E169 and V313 (KDM4A numbering) were targeted. Additionally, the conformational restriction of the flexible pyridopyrimidinone C8-substituent was investigated. These approaches yielded potent and cell-penetrant dual KDM4/5-subfamily inhibitors including 19a (KDM4A and KDM5B Ki = 0.004 and 0.007 μM, respectively). Compound cellular profiling in two orthogonal target engagement assays revealed a significant reduction from biochemical to cell-based activity across multiple analogues; this decrease was shown to be consistent with 2OG competition, and suggest that sub-nanomolar biochemical potency will be required with C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one compounds to achieve sub-micromolar target inhibition in cells.
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May 2019
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I02-Macromolecular Crystallography
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Megan J.
Agajanian
,
Matthew P.
Walker
,
Alison D.
Axtman
,
Roberta R.
Ruela-De-Sousa
,
D. Stephen
Serafin
,
Alex D.
Rabinowitz
,
David M.
Graham
,
Meagan B.
Ryan
,
Tigist
Tamir
,
Yuko
Nakamichi
,
Melissa V.
Gammons
,
James M.
Bennett
,
Rafael M.
Counago
,
David H.
Drewry
,
Jonathan M.
Elkins
,
Carina
Gileadi
,
Opher
Gileadi
,
Paulo H.
Godoi
,
Nirav
Kapadia
,
Susanne
Müller
,
André S.
Santiago
,
Fiona J.
Sorrell
,
Carrow I.
Wells
,
Oleg
Fedorov
,
Timothy M.
Willson
,
William J.
Zuercher
,
Michael B.
Major
Open Access
Abstract: β-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity.
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Jan 2019
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