I03-Macromolecular Crystallography
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Takashi
Miura
,
Tika R.
Malla
,
C. David
Owen
,
Anthony
Tumber
,
Lennart
Brewitz
,
Michael A.
Mcdonough
,
Eidarus
Salah
,
Naohiro
Terasaka
,
Takayuki
Katoh
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
Halina
Mikolajek
,
Martin A.
Walsh
,
Akane
Kawamura
,
Christopher J.
Schofield
,
Hiroaki
Suga
Diamond Proposal Number(s):
[27088]
Open Access
Abstract: γ-Amino acids can play important roles in the biological activities of natural products; however, the ribosomal incorporation of γ-amino acids into peptides is challenging. Here we report how a selection campaign employing a non-canonical peptide library containing cyclic γ2,4-amino acids resulted in the discovery of very potent inhibitors of the SARS-CoV-2 main protease (Mpro). Two kinds of cyclic γ2,4-amino acids, cis-3-aminocyclobutane carboxylic acid (γ1) and (1R,3S)-3-aminocyclopentane carboxylic acid (γ2), were ribosomally introduced into a library of thioether-macrocyclic peptides. One resultant potent Mpro inhibitor (half-maximal inhibitory concentration = 50 nM), GM4, comprising 13 residues with γ1 at the fourth position, manifests a 5.2 nM dissociation constant. An Mpro:GM4 complex crystal structure reveals the intact inhibitor spans the substrate binding cleft. The γ1 interacts with the S1′ catalytic subsite and contributes to a 12-fold increase in proteolytic stability compared to its alanine-substituted variant. Knowledge of interactions between GM4 and Mpro enabled production of a variant with a 5-fold increase in potency.
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May 2023
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I03-Macromolecular Crystallography
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Radoslaw
Nowak
,
Anthony
Tumber
,
Eline
Hendrix
,
Mohammad Salik Zeya
Ansari
,
Manuela
Sabatino
,
Lorenzo
Antonini
,
Regina
Andrijes
,
Eidarus
Salah
,
Nicola
Mautone
,
Francesca Romana
Pellegrini
,
Klemensas
Simelis
,
Akane
Kawamura
,
Catrine
Johansson
,
Daniela
Passeri
,
Roberto
Pellicciari
,
Alessia
Ciogli
,
Donatella
Del Bufalo
,
Rino
Ragno
,
Mathew L.
Coleman
,
Daniela
Trisciuoglio
,
Antonello
Mai
,
Udo
Oppermann
,
Christopher J.
Schofield
,
Dante
Rotili
Diamond Proposal Number(s):
[10619]
Open Access
Abstract: MINA53 is a JmjC domain 2-oxoglutarate-dependent oxygenase that catalyzes ribosomal hydroxylation and is a target of the oncogenic transcription factor c-MYC. Despite its anticancer target potential, no small-molecule MINA53 inhibitors are reported. Using ribosomal substrate fragments, we developed mass spectrometry assays for MINA53 and the related oxygenase NO66. These assays enabled the identification of 2-(aryl)alkylthio-3,4-dihydro-4-oxoypyrimidine-5-carboxylic acids as potent MINA53 inhibitors, with selectivity over NO66 and other JmjC oxygenases. Crystallographic studies with the JmjC demethylase KDM5B revealed active site binding but without direct metal chelation; however, molecular modeling investigations indicated that the inhibitors bind to MINA53 by directly interacting with the iron cofactor. The MINA53 inhibitors manifest evidence for target engagement and selectivity for MINA53 over KDM4–6. The MINA53 inhibitors show antiproliferative activity with solid cancer lines and sensitize cancer cells to conventional chemotherapy, suggesting that further work investigating their potential in combination therapies is warranted.
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Nov 2021
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346]
Open Access
Abstract: Crystallization is the bottleneck in macromolecular crystallography; even when a protein crystallises, crystal packing often influences ligand-binding and protein–protein interaction interfaces, which are the key points of interest for functional and drug discovery studies. The human hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) readily crystallises as a homotrimer, but with a sterically blocked active site. We explored strategies aimed at altering PHD2 crystal packing by protein modification and molecules that bind at its active site and elsewhere. Following the observation that, despite weak inhibition/binding in solution, succinamic acid derivatives readily enable PHD2 crystallization, we explored methods to induce crystallization without active site binding. Cyclic peptides obtained via mRNA display bind PHD2 tightly away from the active site. They efficiently enable PHD2 crystallization in different forms, both with/without substrates, apparently by promoting oligomerization involving binding to the C-terminal region. Although our work involves a specific case study, together with those of others, the results suggest that mRNA display-derived cyclic peptides may be useful in challenging protein crystallization cases.
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Dec 2020
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I04-Macromolecular Crystallography
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Saleta
Vazquez-Rodriguez
,
Miranda
Wright
,
Catherine M.
Rogers
,
Adam P.
Cribbs
,
Srikannathasan
Velupillai
,
Martin
Philpott
,
Henry
Lee
,
James E.
Dunford
,
Kilian V. M.
Huber
,
Matthew B.
Robers
,
James D.
Vasta
,
Marie-Laetitia
Thezenas
,
Sarah
Bonham
,
Benedikt
Kessler
,
James
Bennett
,
Oleg
Fedorov
,
Florence
Raynaud
,
Adam
Donovan
,
Julian
Blagg
,
Vassilios
Bavetsias
,
Udo
Oppermann
,
Chas
Bountra
,
Akane
Kawamura
,
Paul E.
Brennan
Diamond Proposal Number(s):
[15433]
Open Access
Abstract: Histone lysine demethylases (KDMs) are involved in the dynamic regulation of gene expression and they play a critical role in several biological processes. Achieving selectivity over the different KDMs has been a major challenge for KDM inhibitor development. Here we report potent and selective KDM5 covalent inhibitors designed to target cysteine residues only present in the KDM5 sub‐family. The covalent binding to the targeted proteins was confirmed by MS and time‐dependent inhibition. Additional competition assays show that compounds were non 2‐OG competitive. Target engagement and ChIP‐seq analysis showed that the compounds inhibited the KDM5 members in cells at nano‐ to micromolar levels and induce a global increase of the H3K4me3 mark at transcriptional start sites.
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Jan 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18069]
Open Access
Abstract: N‐Methylation of lysyl residues is widely observed on histone proteins. Using isolated enzymes, we report mechanistic and structural studies on histone lysine demethylase (KDM)‐catalysed demethylation of Nε‐methylated lysine 26 on histone 1 isotype 4 (H1.4). The results reveal that methylated H1.4K26 is a substrate for all members of the KDM4 subfamily and that KDM4A‐catalysed demethylation of H1.4K26me3 peptide is similarly efficient to that of H3K9me3. Crystallographic studies of an H1.4K26me3:KDM4A complex reveal a conserved binding geometry to that of H3K9me3. In the light of the high activity of the KDM4s on this mark, our results suggest JmjC KDM‐catalysed demethylation of H1.4K26 may be as prevalent as demethylation on the H3 tail and warrants further investigation in cells.
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Oct 2018
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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
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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