I03-Macromolecular Crystallography
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Zhuoyao
Chen
,
Jinwei
Zhang
,
Adrián R.
Murillo-De-Ozores
,
María
Castañeda-Bueno
,
Francesca
D'Amico
,
Raphael
Heilig
,
Charlotte E.
Manning
,
Fiona J.
Sorrell
,
Vincenzo
D'Angiolella
,
Roman
Fischer
,
Monique P. C.
Mulder
,
Gerardo
Gamba
,
Dario R.
Alessi
,
Alex N.
Bullock
Diamond Proposal Number(s):
[10619]
Open Access
Abstract: The BTB-Kelch protein KLHL3 is a Cullin3-dependent E3 ligase that mediates the ubiquitin-dependent degradation of kinases WNK1–4 to control blood pressure and cell volume. A crystal structure of KLHL3 has defined its binding to an acidic degron motif containing a PXXP sequence that is strictly conserved in WNK1, WNK2 and WNK4. Mutations in the second proline abrograte the interaction causing the hypertension syndrome pseudohypoaldosteronism type II. WNK3 shows a diverged degron motif containing four amino acid substitutions that remove the PXXP motif raising questions as to the mechanism of its binding. To understand this atypical interaction, we determined the crystal structure of the KLHL3 Kelch domain in complex with a WNK3 peptide. The electron density enabled the complete 11-mer WNK-family degron motif to be traced for the first time revealing several conserved features not captured in previous work, including additional salt bridge and hydrogen bond interactions. Overall, the WNK3 peptide adopted a conserved binding pose except for a subtle shift to accommodate bulkier amino acid substitutions at the binding interface. At the centre, the second proline was substituted by WNK3 Thr541, providing a unique phosphorylatable residue among the WNK-family degrons. Fluorescence polarisation and structural modelling experiments revealed that its phosphorylation would abrogate the KLHL3 interaction similarly to hypertension-causing mutations. Together, these data reveal how the KLHL3 Kelch domain can accommodate the binding of multiple WNK isoforms and highlight a potential regulatory mechanism for the recruitment of WNK3.
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Mar 2022
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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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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Sandra
Röhm
,
Benedict-Tilman
Berger
,
Martin
Schröder
,
Deep
Chatterjee
,
Sebastian
Mathea
,
Andreas C.
Joerger
,
Daniel M.
Pinkas
,
Joshua C.
Bufton
,
Amelie
Tjaden
,
Lohitesh
Kovooru
,
Mark
Kudolo
,
Christian
Pohl
,
Alex N.
Bullock
,
Susanne
Müller
,
Stefan
Laufer
,
Stefan
Knapp
Abstract: Discoidin domain receptors 1 and 2 (DDR1/2) play a central role in fibrotic disorders, such as renal and pulmonary fibrosis, atherosclerosis, and various forms of cancer. Potent and selective inhibitors, so-called chemical probe compounds, have been developed to study DDR1/2 kinase signaling. However, these inhibitors showed undesired activity on other kinases such as the tyrosine protein kinase receptor TIE or tropomyosin receptor kinases, which are related to angiogenesis and neuronal toxicity. In this study, we optimized our recently published p38 mitogen-activated protein kinase inhibitor 7 toward a potent and cell-active dual DDR/p38 chemical probe and developed a structurally related negative control. The structure-guided design approach used provided insights into the P-loop folding process of p38 and how targeting of non-conserved amino acids modulates inhibitor selectivity. The developed and comprehensively characterized DDR/p38 probe, 30 (SR-302), is a valuable tool for studying the role of DDR kinase in normal physiology and in disease development.
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Sep 2021
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Aleksandra
Szykowska
,
Yu
Chen
,
Thomas B.
Smith
,
Charlotta
Preger
,
Jingjie
Yang
,
Dongming
Qian
,
Shubhashish M.
Mukhopadhyay
,
Edvard
Wigren
,
Stephen J.
Neame
,
Susanne
Gräslund
,
Helena
Persson
,
Peter J.
Atkinson
,
Elena
Di Daniel
,
Emma
Mead
,
John
Wang
,
John B.
Davis
,
Nicola A.
Burgess-Brown
,
Alex N.
Bullock
Diamond Proposal Number(s):
[15433]
Open Access
Abstract: Mutations in TREM2, a receptor expressed by microglia in the brain, are associated with an increased risk of neurodegeneration, including Alzheimer's disease. Numerous studies support a role for TREM2 in sensing damaging stimuli and triggering signaling cascades necessary for neuroprotection. Despite its significant role, ligands and regulators of TREM2 activation, and the mechanisms governing TREM2-dependent responses and its cleavage from the membrane, remain poorly characterized. Here, we present phage display generated antibody single-chain variable fragments (scFvs) to human TREM2 immunoglobulin-like domain. Co-crystal structures revealed the binding of two scFvs to an epitope on the TREM2 domain distal to the putative ligand-binding site. Enhanced functional activity was observed for oligomeric scFv species, which inhibited the production of soluble TREM2 in a HEK293 cell model. We hope that detailed characterization of their epitopes and properties will facilitate the use of these renewable binders as structural and functional biology tools for TREM2 research.
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Jul 2021
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Olivier E.
Nonga
,
Darja
Lavogina
,
Erki
Enkvist
,
Katrin
Kestav
,
Apirat
Chaikuad
,
Sarah E.
Dixon-Clarke
,
Alex N.
Bullock
,
Sergei
Kopanchuk
,
Taavi
Ivan
,
Ramesh
Ekambaram
,
Kaido
Viht
,
Stefan
Knapp
,
Asko
Uri
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)6 fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology.
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Jul 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Eleanor
Williams
,
Jana
Bagarova
,
Georgina
Kerr
,
Dong-Dong
Xia
,
Elsie S.
Place
,
Devaveena
Dey
,
Yue
Shen
,
Geoffrey A.
Bocobo
,
Agustin H.
Mohedas
,
Xiuli
Huang
,
Philip E.
Sanderson
,
Arthur
Lee
,
Wei
Zheng
,
Aris N.
Economides
,
James C.
Smith
,
Paul B.
Yu
,
Alex N.
Bullock
Diamond Proposal Number(s):
[10619]
Open Access
Abstract: Currently, no effective therapies exist for fibrodysplasia ossificans progressiva (FOP), a rare congenital syndrome in which heterotopic bone is formed in soft tissues owing to dysregulated activity of the bone morphogenetic protein (BMP) receptor kinase ALK2 (also known as ACVR1). From a screen of known biologically active compounds, we identified saracatinib as a potent ALK2 kinase inhibitor. In enzymatic and cell-based assays, saracatinib preferentially inhibited ALK2, compared with other receptors of the BMP/TGF-β signaling pathway, and induced dorsalization in zebrafish embryos consistent with BMP antagonism. We further tested the efficacy of saracatinib using an inducible ACVR1Q207D-transgenic mouse line, which provides a model of heterotopic ossification (HO), as well as an inducible ACVR1R206H-knockin mouse, which serves as a genetically and physiologically faithful FOP model. In both models, saracatinib was well tolerated and potently inhibited the development of HO, even when administered transiently following soft tissue injury. Together, these data suggest that saracatinib is an efficacious clinical candidate for repositioning in FOP treatment, offering an accelerated path to clinical proof-of-efficacy studies and potentially significant benefits to individuals with this devastating condition.
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Mar 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8421]
Open Access
Abstract: The immunophilin FKBP12 is a known inhibitor of type I BMP and TGF-β receptors that competes for binding with their substrate SMADs. FKBP12 and the close paralog FKBP12.6 additionally assemble with ryanodine receptors to control Ca2+ release. Binding of FKBP12.6 to BMP/TGF-β receptors has yet to be investigated, but appears plausible given its high sequence similarity to FKBP12. Here, we found that FKBP12.6 can assemble with BMP and TGF-β-family type I receptors, but not with type II receptors. Cellular immunoprecipitation confirmed similar binding of FKBP12 and FKBP12.6 to the BMP receptor ALK2 (ACVR1), a known target of mutations in the congenital syndrome fibrodysplasia ossificans progressiva (FOP), as well as the pediatric brain tumor diffuse intrinsic pontine glioma (DIPG). SEC-MALS analyses using purified proteins indicated a direct 1:1 interaction between FKBP12.6 and the receptor’s cytoplasmic domains. The 2.17 Å structure of this ALK2-FKBP12.6 complex bound to the inhibitor dorsomorphin showed FKBP12.6 binding to the GS domain of ALK2 in a manner equivalent to the FKBP12 complex, with ALK2 residues Phe198 and Leu199 extending into the FK506-binding pocket of FKBP12.6. These findings suggest a level of redundancy in FKBP-family regulation of BMP and TGF-β signaling.
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Jan 2021
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I03-Macromolecular Crystallography
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David
Smil
,
Jong Fu
Wong
,
Eleanor
Williams
,
Roslin J.
Adamson
,
Alison
Howarth
,
David
Mcleod
,
Ahmed
Mamai
,
Soyoung
Kim
,
Brian
Wilson
,
Taira
Kiyota
,
Ahmed
Aman
,
Julie
Owen
,
Gennady
Poda
,
Kurumi
Horiuchi
,
Ekaterina
Kuznetsova
,
Haiching
Ma
,
J. Nicole
Hamblin
,
Sue
Cramp
,
Owen
Roberts
,
Aled M.
Edwards
,
David
Uehling
,
Rima
Al-Awar
,
Alex N.
Bullock
,
Jeff
O'Meara
,
Methvin
Isaac
Open Access
Abstract: There are currently no effective chemotherapeutic drugs approved for the treatment of diffuse intrinsic pontine glioma (DIPG), an aggressive pediatric cancer resident in the pons region of the brainstem. Radiation therapy is beneficial but not curative, with the condition being uniformly fatal. Analysis of the genomic landscape surrounding DIPG has revealed that activin receptor-like kinase-2 (ALK2) constitutes a potential target for therapeutic intervention given its dysregulation in the disease. We adopted an open science approach to develop a series of potent, selective, orally bioavailable, and brain penetrant ALK2 inhibitors based on lead compound LDN-214117. Modest structural changes to the C-3, C-4, and C-5 position substituents of the core pyridine ring afforded compounds M4K2009, M4K2117, and M4K2163, each with a superior potency, selectivity, and/or blood-brain barrier (BBB) penetration profile. Robust in vivo pharmacokinetic (PK) properties and tolerability mark these inhibitors as advanced pre-clinical compounds suitable for further development and evaluation in orthotopic models of DIPG.
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Aug 2020
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[442]
Abstract: Selectivity remains a challenge for ATP-mimetic kinase inhibitors, an issue that may be overcome by targeting unique residues or binding pockets. However, to date only few strategies have been developed. Here we identify that bulky residues located N-terminal to the DFG motif (DFG-1) represent an opportunity for designing highly selective inhibitors with unexpected binding modes. We demonstrate that several diverse inhibitors exerted selective, non-canonical binding modes that exclusively target large hydrophobic DFG-1 residues present in many kinases including PIM, CK1, DAPK and CLK. Using the CLK family as a model, structural and biochemical data revealed that the DFG-1 valine controlled a non-canonical binding mode in CLK1, providing a rational for selectivity over the closely-related CLK3 which harbors a smaller DFG-1 alanine. Our data suggests that targeting the restricted back pocket in the small fraction of kinases that harbor bulky DFG-1 residues offers a versatile selectivity filter for inhibitor design.
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Aug 2020
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[15433]
Open Access
Abstract: Wnt signalling is dependent on dishevelled proteins (DVL1-3), which assemble an intracellular Wnt signalosome at the plasma membrane. The levels of DVL1-3 are regulated by multiple Cullin-RING E3 ligases that mediate their ubiquitination and degradation. The BTB-Kelch protein KLHL12 was the first E3 ubiquitin ligase to be identified for DVL1-3, but the molecular mechanisms determining its substrate interactions have remained unknown. Here, we mapped the interaction of DVL1-3 to a ‘PGXPP' motif that is conserved in other known partners and substrates of KLHL12, including PLEKHA4, PEF1, SEC31 and DRD4. To determine the binding mechanism, we solved a 2.4 Å crystal structure of the Kelch domain of KLHL12 in complex with a DVL1 peptide that bound with low micromolar affinity. The DVL1 substrate adopted a U-shaped turn conformation that enabled hydrophobic interactions with all six blades of the Kelch domain β-propeller. In cells, the mutation or deletion of this motif reduced the binding and ubiquitination of DVL1 and increased its stability confirming this sequence as a degron motif for KLHL12 recruitment. These results define the molecular mechanisms determining DVL regulation by KLHL12 and establish the KLHL12 Kelch domain as a new protein interaction module for a novel proline-rich motif.
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Jun 2020
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