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
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Hanna
Tarhonskaya
,
Radoslaw P.
Nowak
,
Catrine
Johansson
,
Aleksandra
Szykowska
,
Anthony
Tumber
,
Rebecca L.
Hancock
,
Pauline
Lang
,
Emily
Flashman
,
Udo
Oppermann
,
Christopher J.
Schofield
,
Akane
Kawamura
Diamond Proposal Number(s):
[11175, 10619]
Open Access
Abstract: Methylation of lysine-4 of histone H3 (H3K4men) is an important regulatory factor in eukaryotic transcription. Removal of the transcriptionally activating H3K4 methylation is catalysed by histone demethylases, including the JmjC KDM5 subfamily. The JmjC KDMs are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases, some of which are associated with cancer. Altered levels of TCA cycle intermediates, and the associated metabolites D- and L-2-hydroxyglutarate (2HG), can cause changes in chromatin methylation status. We report comprehensive biochemical, structural and cellular studies on the interaction of TCA cycle intermediates with KDM5B which is a current medicinal chemistry target for cancer. The tested TCA intermediates were poor or moderate KDM5B inhibitors, except for oxaloacetate and succinate, which were shown to compete for binding with 2OG. D- and L-2HG were moderately potent inhibitors at levels which might be relevant in cancer cells bearing isocitrate dehydrogenase mutations. Crystallographic analyses with succinate, fumarate, L-malate, oxaloacetate, pyruvate, D- and L-2HG support the kinetic studies showing competition with 2OG. An unexpected binding mode for oxaloacetate was observed in which it coordinates the active site metal via its C-4 carboxylate rather than the C-1 carboxylate/C-2 keto groups. Studies employing immunofluorescence antibody-based assays reveal no changes in H3K4me3 levels in cells ectopically overexpressing KDM5B in response to dosing with TCA cycle metabolite pro-drug esters, suggesting that the high levels of cellular 2OG may preclude inhibition. The combined results reveal the potential for KDM5B inhibition by TCA cycle intermediates, but suggest that in cells such inhibition will normally be effectively competed by 2OG.
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Aug 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Akane
Kawamura
,
Martin
Münzel
,
Tatsuya
Kojima
,
Clarence
Yapp
,
Bhaskar
Bhushan
,
Yuki
Goto
,
Anthony
Tumber
,
Takayuki
Katoh
,
Oliver N. F.
King
,
Toby
Passioura
,
Louise J.
Walport
,
Stephanie B.
Hatch
,
Sarah
Madden
,
Susanne
Müller
,
Paul E.
Brennan
,
Rasheduzzaman
Chowdhury
,
Richard J.
Hopkinson
,
Hiroaki
Suga
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[1230, 9306]
Open Access
Abstract: The JmjC histone demethylases (KDMs) are linked to tumour cell proliferation and are current cancer targets; however, very few highly selective inhibitors for these are available. Here we report cyclic peptide inhibitors of the KDM4A-C with selectivity over other KDMs/2OG oxygenases, including closely related KDM4D/E isoforms. Crystal structures and biochemical analyses of one of the inhibitors (CP2) with KDM4A reveals that CP2 binds differently to, but competes with, histone substrates in the active site. Substitution of the active site binding arginine of CP2 to N-ɛ-trimethyl-lysine or methylated arginine results in cyclic peptide substrates, indicating that KDM4s may act on non-histone substrates. Targeted modifications to CP2 based on crystallographic and mass spectrometry analyses results in variants with greater proteolytic robustness. Peptide dosing in cells manifests KDM4A target stabilization. Although further development is required to optimize cellular activity, the results reveal the feasibility of highly selective non-metal chelating, substrate-competitive inhibitors of the JmjC KDMs.
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Apr 2017
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I02-Macromolecular Crystallography
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Anthony
Tumber
,
Andrea
Nuzzi
,
Edward S.
Hookway
,
Stephanie B.
Hatch
,
Srikannathasan
Velupillai
,
Catrine
Johansson
,
Akane
Kawamura
,
Pavel
Savitsky
,
Clarence
Yapp
,
Aleksandra
Szykowska
,
Na
Wu
,
Chas
Bountra
,
Claire
Strain-Damerell
,
Nicola A.
Burgess-Brown
,
Gian Filippo
Ruda
,
Oleg
Fedorov
,
Shonagh
Munro
,
Katherine S.
England
,
Radoslaw P.
Nowak
,
Christopher J.
Schofield
,
Nicholas B.
La Thangue
,
Charlotte
Pawlyn
,
Faith
Davies
,
Gareth
Morgan
,
Nick
Athanasou
,
Susanne
Müller
,
Udo
Oppermann
,
Paul E.
Brennan
Open Access
Abstract: Methylation of lysine residues on histone tail is a dynamic epigenetic modification that plays a key role in chromatin structure and gene regulation. Members of the KDM5 (also known as JARID1) sub-family are 2-oxoglutarate (2-OG) and Fe2+-dependent oxygenases acting as histone 3 lysine 4 trimethyl (H3K4me3) demethylases, regulating proliferation, stem cell self-renewal, and differentiation. Here we present the characterization of KDOAM-25, an inhibitor of KDM5 enzymes. KDOAM-25 shows biochemical half maximal inhibitory concentration values of <100 nM for KDM5A-D in vitro, high selectivity toward other 2-OG oxygenases sub-families, and no off-target activity on a panel of 55 receptors and enzymes. In human cell assay systems, KDOAM-25 has a half maximal effective concentration of ∼50 μM and good selectivity toward other demethylases. KDM5B is overexpressed in multiple myeloma and negatively correlated with the overall survival. Multiple myeloma MM1S cells treated with KDOAM-25 show increased global H3K4 methylation at transcriptional start sites and impaired proliferation.
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Mar 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12346]
Open Access
Abstract: While the oxygen-dependent reversal of lysine Nɛ-methylation is well established, the existence of bona fide Nω-methylarginine demethylases (RDMs) is controversial. Lysine demethylation, as catalysed by two families of lysine demethylases (the flavin-dependent KDM1 enzymes and the 2-oxoglutarate- and oxygen-dependent JmjC KDMs, respectively), proceeds via oxidation of the N-methyl group, resulting in the release of formaldehyde. Here we report detailed biochemical studies clearly demonstrating that, in purified form, a subset of JmjC KDMs can also act as RDMs, both on histone and non-histone fragments, resulting in formaldehyde release. RDM catalysis is studied using peptides of wild-type sequences known to be arginine-methylated and sequences in which the KDM’s methylated target lysine is substituted for a methylated arginine. Notably, the preferred sequence requirements for KDM and RDM activity vary even with the same JmjC enzymes. The demonstration of RDM activity by isolated JmjC enzymes will stimulate efforts to detect biologically relevant RDM activity.
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Jun 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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I03-Macromolecular Crystallography
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Open Access
Abstract: The JmjC-domain-containing 2-oxoglutarate-dependent oxygenases catalyze protein hydroxylation and Nemethyllysine
demethylation via hydroxylation. A subgroup of this family, the JmjC lysine demethylases (JmjC KDMs) are
involved in histone modifications at multiple sites. There are conflicting reports as to the substrate selectivity of some
JmjC oxygenases with respect to KDM activities. In this study, a panel of modified histone H3 peptides was tested for
demethylation against 15 human JmjC-domain-containing proteins. The results largely confirmed known Nemethyllysine
substrates. However, the purified KDM4 catalytic domains showed greater substrate promiscuity than
previously reported (i.e., KDM4A was observed to catalyze demethylation at H3K27 as well as H3K9/K36).
Crystallographic analyses revealed that the Ne-methyllysine of an H3K27me3 peptide binds similarly to Ne-methyllysines of
H3K9me3/H3K36me3 with KDM4A. A subgroup of JmjC proteins known to catalyze hydroxylation did not display
demethylation activity. Overall, the results reveal that the catalytic domains of the KDM4 enzymes may be less selective than
previously identified. They also draw a distinction between the Ne-methyllysine demethylation and hydroxylation activities
within the JmjC subfamily. These resultswill be of use to thoseworkingon functional studies of the JmjCenzymes.
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Jan 2015
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I04-Macromolecular Crystallography
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Jurgen
Brem
,
Sander S.
Van Berkel
,
Wei Shen
Aik
,
Anna M.
Rydzik
,
Matthew B.
Avison
,
Ilaria
Pettinati
,
Klaus-Daniel
Umland
,
Akane
Kawamura
,
Jim
Spencer
,
Timothy D. W.
Claridge
,
Michael
Mcdonough
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[8922]
Abstract: The use of beta-lactam antibiotics is compromised by resistance, which is provided by beta-lactamases belonging to both metallo (MBL)- and serine (SBL)-beta-lactamase subfamilies. The rhodanines are one of very few compound classes that inhibit penicillin-binding proteins (PBPs), SBLs and, as recently reported, MBLs. Here, we describe crystallographic analyses of the mechanism of inhibition of the clinically relevant VIM-2 MBL by a rhodanine, which reveal that the rhodanine ring undergoes hydrolysis to give a thioenolate. The thioenolate is found to bind via di-zinc chelation, mimicking the binding of intermediates in beta-lactam hydrolysis. Crystallization of VIM-2 in the presence of the intact rhodanine led to observation of a ternary complex of MBL, a thioenolate fragment and rhodanine. The crystallographic observations are supported by kinetic and biophysical studies, including 19F NMR analyses, which reveal the rhodanine-derived thioenolate to be a potent broad-spectrum MBL inhibitor and a lead structure for the development of new types of clinically useful MBL inhibitors.
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Nov 2014
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Katherine S.
England
,
Anthony
Tumber
,
Tobias
Krojer
,
Guiseppe
Scozzafava
,
Stanley
Ng
,
Michelle
Daniel
,
Aleksandra
Szykowska
,
Kahing
Che
,
Frank
Von Delft
,
Nicola A.
Burgess-Brown
,
Akane
Kawamura
,
Christopher J.
Schofield
,
Paul E.
Brennan
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: A potent inhibitor of the JmjC histone lysine demethylase KDM2A (compound 35, pIC50 7.2) with excellent selectivity over representatives from other KDM subfamilies has been developed; the discovery that a triazolopyridine compound binds to the active site of JmjC KDMs was followed by optimisation of the triazole substituent for KDM2A inhibition and selectivity.
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Sep 2014
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I02-Macromolecular Crystallography
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Abstract: Carnitine is essential for fatty acid metabolism, but is associated with both health benefits and risks, especially heart disease. We report the identification of potent, selective and cell active inhibitors of γ-butyrobetaine hydroxylase (BBOX), which catalyses the final step of carnitine biosynthesis in animals. A crystal structure of BBOX in complex with a lead inhibitor reveals that it binds in two modes, one of which adopts an unusual ‘U-shape’ conformation stabilised by inter- and intra-molecular π-stacking interactions. Conformational changes observed on binding of the inhibitor to BBOX likely reflect those occurring in catalysis; they also rationalise the inhibition of BBOX by high levels of its substrate γ-butyrobetaine (GBB), as observed both with isolated BBOX protein and in cellular studies.
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Feb 2014
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