I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Joseph A
Newman
,
Angeline E.
Gavard
,
Nergis
Imprachim
,
Hazel
Aitkenhead
,
Hadley E.
Sheppard
,
Robert
Te Poele
,
Paul A.
Clarke
,
Mohammad Anwar
Hossain
,
Louisa
Temme
,
Hans J.
Oh
,
Carrow I.
Wells
,
Zachary W.
Davis-Gilbert
,
Paul
Workman
,
Opher
Gileadi
,
David H.
Drewry
Diamond Proposal Number(s):
[18145, 19301, 28172]
Open Access
Abstract: Brachyury is a transcription factor that plays an essential role in tumour growth of the rare bone cancer chordoma and is implicated in other solid tumours. Brachyury is minimally expressed in healthy tissues, making it a potential therapeutic target. Unfortunately, as a ligandless transcription factor, brachyury has historically been considered undruggable. To investigate direct targeting of brachyury by small molecules, we determine the structure of human brachyury both alone and in complex with DNA. The structures provide insights into DNA binding and the context of the chordoma associated G177D variant. We use crystallographic fragment screening to identify hotspots on numerous pockets on the brachyury surface. Finally, we perform follow-up chemistry on fragment hits and describe the progression of a thiazole chemical series into binders with low µM potency. Thus we show that brachyury is ligandable and provide an example of how crystallographic fragment screening may be used to target protein classes that are difficult to address using other approaches.
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Feb 2025
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I04-Macromolecular Crystallography
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Frances M.
Bashore
,
Sophia M.
Min
,
Xiangrong
Chen
,
Stefanie
Howell
,
Caroline H.
Rinderle
,
Gabriel
Morel
,
Josie A.
Silvaroli
,
Carrow I.
Wells
,
Bruce A.
Bunnell
,
David H.
Drewry
,
Navjot S.
Pabla
,
Sila K.
Ultanir
,
Alex N.
Bullock
,
Alison D.
Axtman
Open Access
Abstract: Acylaminoindazole-based inhibitors of CDKL2 were identified via analyses of cell-free binding and selectivity data. Compound 9 was selected as a CDKL2 chemical probe based on its potent inhibition of CDKL2 enzymatic activity, engagement of CDKL2 in cells, and excellent kinome-wide selectivity, especially when used in cells. Compound 16 was designed as a negative control to be used alongside compound 9 in experiments to interrogate CDKL2-mediated biology. A solved cocrystal structure of compound 9 bound to CDKL2 highlighted key interactions it makes within its ATP-binding site. Inhibition of downstream phosphorylation of EB2, a CDKL2 substrate, in rat primary neurons provided evidence that engagement of CDKL2 by compound 9 in cells resulted in inhibition of its activity. When used at relevant concentrations, compound 9 does not impact the viability of rat primary neurons or certain breast cancer cells nor elicit consistent changes in the expression of proteins involved in epithelial–mesenchymal transition.
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Aug 2024
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I04-Macromolecular Crystallography
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Han Wee
Ong
,
Yi
Liang
,
William
Richardson
,
Emily R.
Lowry
,
Carrow I.
Wells
,
Xiangrong
Chen
,
Margaux
Silvestre
,
Kelvin
Dempster
,
Josie A.
Silvaroli
,
Jeffery L.
Smith
,
Hynek
Wichterle
,
Navjot S.
Pabla
,
Sila K.
Ultanir
,
Alex N.
Bullock
,
David H.
Drewry
,
Alison D.
Axtman
Diamond Proposal Number(s):
[28172]
Open Access
Abstract: Despite mediating several essential processes in the brain, including during development, cyclin-dependent kinase-like 5 (CDKL5) remains a poorly characterized human protein kinase. Accordingly, its substrates, functions, and regulatory mechanisms have not been fully described. We realized that availability of a potent and selective small molecule probe targeting CDKL5 could enable illumination of its roles in normal development as well as in diseases where it has become aberrant due to mutation. We prepared analogs of AT-7519, a compound that has advanced to phase II clinical trials and is a known inhibitor of several cyclin-dependent kinases (CDKs) and cyclin-dependent kinase-like kinases (CDKLs). We identified analog 2 as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog 2 demonstrates excellent selectivity and only retains GSK3α/β affinity. We next demonstrated the inhibition of downstream CDKL5 and GSK3α/β signaling and solved a co-crystal structure of analog 2 bound to human CDKL5. A structurally similar analog (4) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3α/β, making it a suitable negative control. Finally, we used our chemical probe pair (2 and 4) to demonstrate that inhibition of CDKL5 and/or GSK3α/β promotes the survival of human motor neurons exposed to endoplasmic reticulum stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.
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Apr 2023
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Susanne
Müller
,
Suzanne
Ackloo
,
Arij
Al Chawaf
,
Bissan
Al-Lazikani
,
Albert
Antolin
,
Jonathan B.
Baell
,
Hartmut
Beck
,
Shaunna
Beedie
,
Ulrich A. K.
Betz
,
Gustavo
Arruda Bezerra
,
Paul E.
Brennan
,
David
Brown
,
Peter J.
Brown
,
Alex N.
Bullock
,
Adrian J.
Carter
,
Apirat
Chaikuad
,
Mathilde
Chaineau
,
Alessio
Ciulli
,
Ian
Collins
,
Jan
Dreher
,
David
Drewry
,
Kristina
Edfeldt
,
Aled M.
Edwards
,
Ursula
Egner
,
Stephen V.
Frye
,
Stephen M.
Fuchs
,
Matthew D.
Hall
,
Ingo V.
Hartung
,
Alexander
Hillisch
,
Stephen H.
Hitchcock
,
Evert
Homan
,
Natarajan
Kannan
,
James R.
Kiefer
,
Stefan
Knapp
,
Milka
Kostic
,
Stefan
Kubicek
,
Andrew S.
Leach
,
Sven
Lindemann
,
Brian D.
Marsden
,
Hisanori
Matsui
,
Jordan L.
Meier
,
Daniel
Merk
,
Maurice
Michel
,
Maxwell R.
Morgan
,
Anke
Mueller-Fahrnow
,
Dafydd R.
Owen
,
Benjamin G.
Perry
,
Saul H.
Rosenberg
,
Kumar Singh
Saikatendu
,
Matthieu
Schapira
,
Cora
Scholten
,
Sujata
Sharma
,
Anton
Simeonov
,
Michael
Sundström
,
Giulio
Superti-Furga
,
Matthew H.
Todd
,
Claudia
Tredup
,
Masoud
Vedadi
,
Frank
Von Delft
,
Timothy M.
Willson
,
Georg E.
Winter
,
Paul
Workman
,
Cheryl H.
Arrowsmith
Open Access
Abstract: Twenty years after the publication of the first draft of the human genome, our knowledge of the human proteome is still fragmented. The challenge of translating the wealth of new knowledge from genomics into new medicines is that proteins, and not genes, are the primary executers of biological function. Therefore, much of how biology works in health and disease must be understood through the lens of protein function. Accordingly, a subset of human proteins has been at the heart of research interests of scientists over the centuries, and we have accumulated varying degrees of knowledge about approximately 65% of the human proteome. Nevertheless, a large proportion of proteins in the human proteome (∼35%) remains uncharacterized, and less than 5% of the human proteome has been successfully targeted for drug discovery. This highlights the profound disconnect between our abilities to obtain genetic information and subsequent development of effective medicines. Target 2035 is an international federation of biomedical scientists from the public and private sectors, which aims to address this gap by developing and applying new technologies to create by year 2035 chemogenomic libraries, chemical probes, and/or biological probes for the entire human proteome.
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Dec 2021
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I03-Macromolecular Crystallography
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Benjamin J.
Eduful
,
Sean N.
O'Byrne
,
Louisa
Temme
,
Christopher R. M.
Asquith
,
Yi
Liang
,
Alfredo
Picado
,
Joseph R.
Pilotte
,
Mohammad Anwar
Hossain
,
Carrow I.
Wells
,
William J.
Zuercher
,
Carolina M. C.
Catta-Preta
,
Priscila
Zonzini Ramos
,
André De S.
Santiago
,
Rafael M.
Counago
,
Christopher G.
Langendorf
,
Kévin
Nay
,
Jonathan S.
Oakhill
,
Thomas L.
Pulliam
,
Chenchu
Lin
,
Dominik
Awad
,
Timothy M.
Willson
,
Daniel E.
Frigo
,
John W.
Scott
,
David H.
Drewry
Diamond Proposal Number(s):
[10619]
Open Access
Abstract: CAMKK2 is a serine/threonine kinase and an activator of AMPK whose dysregulation is linked with multiple diseases. Unfortunately, STO-609, the tool inhibitor commonly used to probe CAMKK2 signaling, has limitations. To identify promising scaffolds as starting points for the development of high-quality CAMKK2 chemical probes, we utilized a hinge-binding scaffold hopping strategy to design new CAMKK2 inhibitors. Starting from the potent but promiscuous disubstituted 7-azaindole GSK650934, a total of 32 compounds, composed of single-ring, 5,6-, and 6,6-fused heteroaromatic cores, were synthesized. The compound set was specifically designed to probe interactions with the kinase hinge-binding residues. Compared to GSK650394 and STO-609, 13 compounds displayed similar or better CAMKK2 inhibitory potency in vitro, while compounds 13g and 45 had improved selectivity for CAMKK2 across the kinome. Our systematic survey of hinge-binding chemotypes identified several potent and selective inhibitors of CAMKK2 to serve as starting points for medicinal chemistry programs.
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Jul 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[105433]
Open Access
Abstract: Extracellular signal-regulated kinase 3 (ERK3), known also as mitogen-activated protein kinase 6 (MAPK6), is an atypical member of MAPK kinase family, which has been poorly studied. Little is known regarding its function in biological processes, yet this atypical kinase has been suggested to play important roles in the migration and invasiveness of certain cancers. The lack of tools, such as a selective inhibitor, hampers the study of ERK3 biology. Here, we report the crystal structure of the kinase domain of this atypical MAPK kinase, providing molecular insights into its distinct ATP binding pocket compared to the classical MAPK ERK2, explaining differences in their inhibitor binding properties. Medium-scale small molecule screening identified a number of inhibitors, several of which unexpectedly exhibited remarkably high inhibitory potencies. The crystal structure of CLK1 in complex with CAF052, one of the most potent inhibitors identified for ERK3, revealed typical type-I binding mode of the inhibitor, which by structural comparison could likely be maintained in ERK3. Together with the presented structural insights, these diverse chemical scaffolds displaying both reversible and irreversible modes of action, will serve as a starting point for the development of selective inhibitors for ERK3, which will be beneficial for elucidating the important functions of this understudied kinase.
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Nov 2020
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I02-Macromolecular Crystallography
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Alfredo
Picado
,
Apirat
Chaikuad
,
Carrow I.
Wells
,
Safal
Shrestha
,
William J.
Zuercher
,
Julie E.
Pickett
,
Frank E.
Kwarcinski
,
Parvathi
Sinha
,
Chandi S.
De Silva
,
Reena
Zutshi
,
Shubin
Liu
,
Natarajan
Kannan
,
Stefan
Knapp
,
David H.
Drewry
,
Timothy M.
Willson
Diamond Proposal Number(s):
[442]
Abstract: STK17B is a member of the death-associated protein kinase family and has been genetically linked to the development of diverse diseases. However, the role of STK17B in normal and disease pathology is poorly defined. Here, we present the discovery of thieno[3,2-d] pyrimidine SGC-STK17B-1 (11s), a high-quality chemical probe for this understudied “dark” kinase. 11s is an ATP-competitive inhibitor that showed remarkable selectivity over other kinases including the closely related STK17A. X-ray crystallography of 11s and related thieno[3,2-d]pyrimidines bound to STK17B revealed a unique P-loop conformation characterized by a salt bridge between R41 and the carboxylic acid of the inhibitor. Molecular dynamic simulations of STK17B revealed the flexibility of the P-loop and a wide range of R41 conformations available to the apo-protein. The isomeric thieno[2,3-d]pyrimidine SGC-STK17B-1N (19g) was identified as a negative control compound. The >100-fold lower activity of 19g on STK17B was attributed to the reduced basicity of its pyrimidine N1.
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Nov 2020
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I24-Microfocus Macromolecular Crystallography
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Gerson S.
Profeta
,
Caio V.
Dos Reis
,
André Da S.
Santiago
,
Paulo H. C.
Godoi
,
Angela M.
Fala
,
Carrow I.
Wells
,
Roger
Sartori
,
Anita P. T.
Salmazo
,
Priscila Z.
Ramos
,
Katlin B.
Massirer
,
Jonathan M.
Elkins
,
David H.
Drewry
,
Opher
Gileadi
,
Rafael M.
Counago
Diamond Proposal Number(s):
[16171]
Open Access
Abstract: Calcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no selective small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homolog, CAMKK1, are thought to have overlapping biological roles. Here we present six new co-structures of potent ligands bound to CAMKK2 identified from a library of commercially-available kinase inhibitors. Enzyme assays confirmed that most of these compounds are equipotent inhibitors of both human CAMKKs and isothermal titration calorimetry (ITC) revealed that binding to some of these molecules to CAMKK2 is enthalpy driven. We expect our results to advance current efforts to discover small molecule kinase inhibitors selective to each human CAMKK.
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Nov 2019
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I03-Macromolecular Crystallography
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Carrow
Wells
,
Rafael M.
Counago
,
Juanita C.
Limas
,
Tuanny L.
Almeida
,
Jeanette Gowen
Cook
,
David H
Drewry
,
Jonathan M.
Elkins
,
Opher
Gileadi
,
Nirav R.
Kapadia
,
Alvaro
Lorente-Macias
,
Julie E.
Pickett
,
Alexander
Riemen
,
Roberta R.
Ruela-De-Sousa
,
Timothy M.
Willson
,
Cunyu
Zhang
,
William J
Zuercher
,
Reena
Zutshi
,
Alison D.
Axtman
Diamond Proposal Number(s):
[14664]
Abstract: Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2K inhibitors. Optimization furnished a small molecule chemical probe (SGC-AAK1-1, 25) that is potent and selective for AAK1/BMP2K over other NAK family members, demonstrates narrow activity in a kinome-wide screen, and is functionally active in cells. This inhibitor represents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.
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Oct 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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