I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Shifali
Shishodia
,
Marina
Demetriades
,
Dong
Zhang
,
Nok Yin
Tam
,
Pratheesh
Maheswaran
,
Caitlin
Clunie-O'Connor
,
Anthony
Tumber
,
Ivanhoe K. H.
Leung
,
Yi Min
Ng
,
Thomas M.
Leissing
,
Afaf H.
El-Sagheer
,
Eidarus
Salah
,
Tom
Brown
,
Wei Shen
Aik
,
Michael A.
Mcdonough
,
Christopher J.
Schofield
Open Access
Abstract: FTO catalyzes the Fe(II) and 2-oxoglutarate (2OG)-dependent modification of nucleic acids, including the demethylation of N6-methyladenosine (m6A) in mRNA. FTO is a proposed target for anti-cancer therapy. Using information from crystal structures of FTO in complex with 2OG and substrate mimics, we designed and synthesized two series of FTO inhibitors, which were characterized by turnover and binding assays, and by X-ray crystallography with FTO and the related bacterial enzyme AlkB. A potent inhibitor employing binding interactions spanning the FTO 2OG and substrate binding sites was identified. Selectivity over other clinically targeted 2OG oxygenases was demonstrated, including with respect to the hypoxia-inducible factor prolyl and asparaginyl hydroxylases (PHD2 and FIH) and selected JmjC histone demethylases (KDMs). The results illustrate how structure-based design can enable the identification of potent and selective 2OG oxygenase inhibitors and will be useful for the development of FTO inhibitors for use in vivo.
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Nov 2021
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Tzu-Lan
Yeh
,
Thomas m.
Leissing
,
Martine I.
Abboud
,
Cyrille C.
Thinnes
,
Onur
Atasoylu
,
James P.
Holt-Martyn
,
Dong
Zhang
,
Anthony
Tumber
,
Kerstin
Lippl
,
Christopher T.
Lohans
,
Ivanhoe K. H.
Leung
,
Helen
Morcrette
,
Ian J.
Clifton
,
Timothy D. W.
Claridge
,
Akane
Kawamura
,
Emily
Flashman
,
Xin
Lu
,
Peter J.
Ratcliffe
,
Rasheduzzaman
Chowdhury
,
Christopher W.
Pugh
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[12346, 9306]
Open Access
Abstract: Inhibition of the human 2-oxoglutarate (2OG) dependent hypoxia inducible factor (HIF) prolyl hydroxylases (human PHD1–3) causes upregulation of HIF, thus promoting erythropoiesis and is therefore of therapeutic interest. We describe cellular, biophysical, and biochemical studies comparing four PHD inhibitors currently in clinical trials for anaemia treatment, that describe their mechanisms of action, potency against isolated enzymes and in cells, and selectivities versus representatives of other human 2OG oxygenase subfamilies. The ‘clinical’ PHD inhibitors are potent inhibitors of PHD catalyzed hydroxylation of the HIF-α oxygen dependent degradation domains (ODDs), and selective against most, but not all, representatives of other human 2OG dependent dioxygenase subfamilies. Crystallographic and NMR studies provide insights into the different active site binding modes of the inhibitors. Cell-based results reveal the inhibitors have similar effects on the upregulation of HIF target genes, but differ in the kinetics of their effects and in extent of inhibition of hydroxylation of the N- and C-terminal ODDs; the latter differences correlate with the biophysical observations.
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Sep 2017
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Rasheduzzaman
Chowdhury
,
Ivanhoe K. H.
Leung
,
Ya-Min
Tian
,
Martine I.
Abboud
,
Wei
Ge
,
Carmen
Domene
,
François-Xavier
Cantrelle
,
Isabelle
Landrieu
,
Adam P.
Hardy
,
Christopher W.
Pugh
,
Peter J.
Ratcliffe
,
Timothy D. W.
Claridge
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[12346]
Open Access
Abstract: The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1–3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel–Lindau protein (VHL)–elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.
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Aug 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Mun Chiang
Chan
,
Onur
Atasoylu
,
Emma
Hodson
,
Anthony
Tumber
,
Ivanhoe K. H.
Leung
,
Rasheduzzaman
Chowdhury
,
Verónica
Gómez-Pérez
,
Marina
Demetriades
,
Anna M.
Rydzik
,
James
Holt-Martyn
,
Ya-Min
Tian
,
Tammie
Bishop
,
Timothy D. W.
Claridge
,
Akane
Kawamura
,
Christopher W.
Pugh
,
Peter J.
Ratcliffe
,
Christopher J.
Schofield
,
Sonia
Rocha
Open Access
Abstract: As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs). Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs). Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4) induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.
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Sep 2015
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I02-Macromolecular Crystallography
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Abstract: The prolyl hydroxylase domain proteins (PHDs) catalyse the post-translational hydroxylation of the hypoxia-inducible factor (HIF), a modification that regulates the hypoxic response in humans. The PHDs are Fe(II)/2-oxoglutarate (2OG) oxygenases; their catalysis is proposed to provide a link between cellular HIF levels and changes in O2 availability. Transient kinetic studies have shown that purified PHD2 reacts slowly with O2 compared with some other studied 2OG oxygenases, a property which may be related to its hypoxia-sensing role. PHD2 forms a stable complex with Fe(II) and 2OG; crystallographic and kinetic analyses indicate that an Fe(II)-co-ordinated water molecule, which must be displaced before O2 binding, is relatively stable in the active site of PHD2. We used active site substitutions to investigate whether these properties are related to the slow reaction of PHD2 with O2. While disruption of 2OG binding in a R383K variant did not accelerate O2 activation, we found that substitution of the Fe(II)-binding aspartate for a glutamate residue (D315E) manifested significantly reduced Fe(II) binding, yet maintained catalytic activity with a 5-fold faster reaction with O2. The results inform on how the precise active site environment of oxygenases can affect rates of O2 activation and provide insights into limiting steps in PHD catalysis
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Nov 2014
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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John S.
Scotti
,
Ivanhoe K. H.
Leung
,
Wei
Ge
,
Michael A.
Bentley
,
Jordi
Paps
,
Holger B.
Kramer
,
Joongoo
Lee
,
Weishen
Aik
,
Hwanho
Choi
,
Steinar M.
Paulsen
,
Lesley A. H.
Bowman
,
Nikita D.
Loik
,
Shoichiro
Horita
,
Chia-Hua
Ho
,
Nadia J.
Kershaw
,
Christoph M.
Tang
,
Timothy D. W.
Claridge
,
Gail M.
Preston
,
Michael
Mcdonough
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[7495]
Abstract: The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins.
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Sep 2014
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I19-Small Molecule Single Crystal Diffraction
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Diamond Proposal Number(s):
[7768]
Abstract: In the search for synthetic mimics of protein secondary structures relevant to the mediation of protein-protein interactions, we have synthesized a series of tetrasubstituted diphenylacetylenes that display -sheet structures in two directions. Extensive X-ray crystallographic and NMR solution phase studies are consistent with these proteomimetics adopting sheet structures, displaying both hydrophobic and hydrophilic amino acid side chains.
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Apr 2014
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Sander S.
Van Berkel
,
Joanne
Nettleship
,
Ivanhoe K. H.
Leung
,
Jurgen
Brem
,
Hwanho
Choi
,
David I.
Stuart
,
Timothy D. W.
Claridge
,
Michael A.
Mcdonough
,
Raymond J.
Owens
,
Jingshan
Ren
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[8423]
Abstract: β-Lactam antibiotics react with penicillin binding proteins (PBPs) to form relatively stable acyl-enzyme complexes. We describe structures derived from the reaction of piperacillin with PBP3 (Pseudomonas aeruginosa) including not only the anticipated acyl-enzyme complex but also an unprecedented complex with (5S)-penicilloic acid, which was formed by C-5 epimerization of the nascent (5R)-penicilloic acid product. Formation of the complex was confirmed by solution studies, including NMR. Together, these results will be useful in the design of new PBP inhibitors and raise the possibility that noncovalent PBP inhibition by penicilloic acids may be of clinical relevance.
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Aug 2013
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Nathan
Rose
,
Esther C. Y.
Woon
,
Anthony
Tumber
,
Louise J.
Walport
,
Rasheduzzaman
Chowdhury
,
Xuan Shirley
Li
,
Oliver N. F.
King
,
Clarisse
Lejeune
,
Stanley
Ng
,
Tobias
Krojer
,
Mun Chiang
Chan
,
Anna M.
Rydzik
,
Richard J.
Hopkinson
,
Ka Hing
Che
,
Michelle
Daniel
,
Claire
Strain-Damerell
,
Carina
Gileadi
,
Grazyna
Kochan
,
Ivanhoe K. H.
Leung
,
James
Dunford
,
Kar Kheng
Yeo
,
Peter J.
Ratcliffe
,
Nicola
Burgess-Brown
,
Frank
Von Delft
,
Susanne
Muller
,
Brian
Marsden
,
Paul. E.
Brennan
,
Michael A.
Mcdonough
,
Udo
Oppermann
,
Robert J.
Klose
,
Christopher J.
Schofield
,
Akane
Kawamura
Diamond Proposal Number(s):
[7495]
Abstract: The JmjC oxygenases catalyze the N-demethylation of Nε-methyl lysine residues in histones and are current therapeutic targets. A
SET of human 2-oxoglutarate analogues were screened using a unified assay platform for JmjC demethylases and related oxygenases. Results led to the finding that daminozide (N-(dimethylamino)succinamic acid, 160 Da), a plant growth regulator, selectively inhibits the KDM2/7 JmjC subfamily. Kinetic and crystallographic studies reveal that daminozide chelates the active site metal via its hydrazide carbonyl and dimethylamino groups.
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Jul 2012
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[442]
Abstract: The final step in carnitine biosynthesis is catalyzed by gamma-butyrobetaine (gamma BB) hydroxylase (BBOX), an iron/2-oxoglutarate (2OG) dependent oxygenase. BBOX is inhibited by trimethylhydrazine-propionate (THP), a clinically used compound. We report structural and mechanistic studies on BBOX and its reaction with THP. Crystallographic and sequence analyses reveal that BBOX and trimethyllysine hydroxylase form a subfamily of 2OG oxygenases that dimerize using an N-terminal domain. The crystal structure reveals the active site is enclosed and how THP competes with gamma BB. THP is a substrate giving formaldehyde (supporting structural links with histone demethylases), dimethylamine, malonic acid semi-aldehyde, and an unexpected product with an additional carbon-carbon bond resulting from N-demethylation coupled to oxidative rearrangement, likely via an unusual radical mechanism. The results provide a basis for development of improved BBOX inhibitors and may inspire the discovery of additional rearrangement reactions.
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Dec 2010
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