I04-Macromolecular Crystallography
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Mujtaba
Hassan
,
Sjors
Van Klaveren
,
Maria
Håkansson
,
Carl
Diehl
,
Rebeka
Kovačič
,
Floriane
Baussière
,
Anders
Sundin
,
Jaka
Dernovšek
,
Björn
Walse
,
Fredrik
Zetterberg
,
Hakon
Leffler
,
Marko
Anderluh
,
Tihomir
Tomašič
,
Žiga
Jakopin
,
Ulf J.
Nilsson
Diamond Proposal Number(s):
[23282]
Abstract: We have obtained the X-ray crystal structure of the galectin-8 N-terminal domain (galectin-8N) with a previously reported quinoline–galactoside ligand at a resolution of 1.6 Å. Based on this X-ray structure, a collection of galactosides derivatised at O3 with triazole, benzimidazole, benzothiazole, and benzoxazole moieties were designed and synthesised. This led to the discovery of a 3-O-(N-methylbenzimidazolylmethyl)–galactoside with a Kd of 1.8 μM for galectin-8N, the most potent selective synthetic galectin-8N ligand to date. Molecular dynamics simulations showed that benzimidazole–galactoside derivatives bind the non-conserved amino acid Gln47, accounting for the higher selectivity for galectin-8N. Galectin-8 is a carbohydrate-binding protein that plays a key role in pathological lymphangiogenesis, modulation of the immune system, and autophagy. Thus, the benzimidazole-derivatised galactosides represent promising compounds for studies of the pathological implications of galectin-8, as well as a starting point for the development of anti-tumour and anti-inflammatory therapeutics targeting galectin-8.
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Jun 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Anka
Lucic
,
Philip
Hinchliffe
,
Tika R.
Malla
,
Catherine L.
Tooke
,
Jurgen
Brem
,
Karina
Calvopina
,
Christopher T.
Lohans
,
Patrick
Rabe
,
Michael A.
Mcdonough
,
Timothy
Armistead
,
Allen M.
Orville
,
James
Spencer
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[17212, 23269, 18069]
Abstract: Penems have demonstrated potential as antibacterials and β-lactamase inhibitors; however, their clinical use has been limited, especially in comparison with the structurally related carbapenems. Faropenem is an orally active antibiotic with a C2 tetrahydrofuran (THF) ring, which is resistant to hydrolysis by some β-lactamases. We report studies on the reactions of faropenem with carbapenem-hydrolysing β-lactamases, focusing on the class A serine β-lactamase KPC-2 and the metallo β-lactamases (MBLs) VIM-2 (a subclass B1 MBL) and L1 (a B3 MBL). Kinetic studies show that faropenem is a substrate for all three β-lactamases, though it is less efficiently hydrolysed by KPC-2. Crystallographic analyses on faropenem-derived complexes reveal the opening of the β-lactam ring with formation of an imine with KPC-2, VIM-2, and L1. In the cases of the KPC-2 and VIM-2 structures, the THF ring is opened to give an alkene, but with L1 the THF ring remains intact. Solution state studies, employing NMR, were performed on L1, KPC-2, VIM-2, VIM-1, NDM-1, OXA-23, OXA-10, and OXA-48. The solution results reveal, in all cases, formation of imine products in which the THF ring is opened; formation of a THF ring-closed imine product was only observed with VIM-1 and VIM-2. An enamine product with a closed THF ring was also observed in all cases, at varying levels. Combined with previous reports, the results exemplify the potential for different outcomes in the reactions of penems with MBLs and SBLs and imply further structure-activity relationship studies are worthwhile to optimise the interactions of penems with β-lactamases. They also exemplify how crystal structures of β-lactamase substrate/inhibitor complexes do not always reflect reaction outcomes in solution.
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Feb 2021
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I03-Macromolecular Crystallography
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Sandra
Röhm
,
Martin
Schroeder
,
Jessica E.
Dwyer
,
Caroline S.
Widdowson
,
Apirat
Chaikuad
,
Benedict-Tilman
Berger
,
Andreas C.
Joerger
,
Andreas
Krämer
,
Jule
Harbig
,
Daniel
Dauch
,
Mark
Kudolo
,
Stefan
Laufer
,
Mark C.
Bagley
,
Stefan
Knapp
Diamond Proposal Number(s):
[10619]
Abstract: The p38 MAPK cascade is a key signaling pathway linked to a multitude of physiological functions and of central importance in inflammatory and autoimmune diseases. Although studied extensively, little is known about how conformation-specific inhibitors alter signaling outcomes. Here, we have explored the highly dynamic back pocket of p38 MAPK with allosteric urea fragments. However, screening against known off-targets showed that these fragments maintained the selectivity issues of their parent compound BIRB-796, while combination with the hinge-binding motif of VPC-00628 greatly enhanced inhibitor selectivity. Further efforts focused therefore on the exploration of the αC-out pocket of p38 MAPK, yielding compound 137 as a highly selective type-II inhibitor. Even though 137 is structurally related to a recent p38 type-II chemical probe, SR-318, the data presented here provide valuable insights into back-pocket interactions that are not addressed in SR-318 and it provides an alternative chemical tool with good cellular activity targeting also the p38 back pocket.
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Dec 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Lucía
Serran Aguilera
,
Elena
Mariotto
,
Gianluca
Rubbini
,
Francisco Fermín
Castro Navas
,
Carmen
Marco
,
María Paz
Carrasco-Jiménez
,
Marco
Ballarotto
,
Antonio
Macchiarulo
,
Ramon
Hurtado-Guerrero
,
Giampietro
Viola
,
Luisa Carlota
Lopez-Cara
Diamond Proposal Number(s):
[8035]
Abstract: Seeking for new anticancer drugs with strong antiproliferative activity and simple molecular structure, we designed a novel series of compounds based on our previous reported pharmacophore model composed of five moieties. Antiproliferative assays on four tumoral cell lines and evaluation of Human Choline Kinase CKα1 enzymatic activity was performed for these compounds. Among tested molecules, those ones with biphenyl spacer showed betters enzymatic and antiproliferative activities (n-v). Docking and crystallization studies validate the hypothesis and confirm the results. The most active compound (t) induces a significant arrest of the cell cycle in G0/G1 phase that ultimately lead to apoptosis, following the mitochondrial pathway, as demonstrated for other choline kinase inhibitors. However additional assays reveal that the inhibition of choline uptake could also be involved in the antiproliferative outcome of this class of compounds.
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Dec 2020
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I04-Macromolecular Crystallography
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Sonia
Martínez-González
,
Ana
Belén García
,
M. Isabel
Albarrán
,
Antonio
Cebriá
,
Adrián
Amezquita-Alves
,
Francisco Javier
García-Campos
,
Jaime
Martínez-Gago
,
Jorge
Martínez-Torrecuadrada
,
Ines
Munoz
,
Carmen
Blanco-Aparicio
,
Joaquín
Pastor
Diamond Proposal Number(s):
[16252]
Abstract: CDK8 is a cyclin-dependent kinase that forms part of the mediator complex, and modulates the transcriptional output from distinct transcription factors involved in oncogenic control. Overexpression of CDK8 has been observed in various cancers, representing a potential target for developing novel CDK8 inhibitors in cancer therapeutics. In the course of our investigations to discover new CDK8 inhibitors, we designed and synthesized tricyclic pyrido[2,3-b][1,5]benzoxazepin-5(6H)-one derivatives, by introduction of chemical complexity in the multi-kinase inhibitor Sorafenib taking into account the flexibility of the P-loop motif of CDK8 protein observed after analysis of structural information of co-crystallized CDK8 inhibitors. In vitro evaluation of the inhibitory activity of the prepared compounds against CDK8 led us to identify compound 2 as the most potent inhibitor of the series (IC50 = 8.25 nM). Co-crystal studies and the remarkable selectivity profile of compound 2 are presented. Compound 2 showed moderate reduction of phosphorylation of CDK8 substrate STAT1 in cells, in line with other reported Type II CDK8 inhibitors. We propose herein an alternative to find a potential therapeutic use for this chemical series.
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Sep 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Open Access
Abstract: Rising numbers of cases of multidrug- and extensively drug-resistant Pseudomonas aeruginosa over recent years have created an urgent need for novel therapeutic approaches to cure potentially fatal infections. One such approach is virulence attenuation where anti-virulence compounds, designed to reduce pathogenicity without affording bactericidal effects, are employed to treat infections. P. aeruginosa uses the pqs quorum sensing (QS) system, to coordinate the expression of a large number of virulence determinants as well as bacterial-host interactions and hence represents an excellent anti-virulence target.
We report the synthesis and identification of a new series of thiazole-containing quinazolinones capable of inhibiting PqsR, the transcriptional regulator of the pqs QS system. The compounds demonstrated high potency (IC50 < 300 nM) in a whole-cell assay, using a mCTX::PpqsA-lux-based bioreporter for the P. aeruginosa PAO1-L and PA14 strains. Structural evaluation defined the binding modes of four analogues in the ligand-binding domain of PqsR through X-ray crystallography. Further work showed the ability of 6-chloro-3((2-pentylthiazol-4-yl)methyl)quinazolin-4(3H)-one (18) and 6-chloro-3((2-hexylthiazol-4-yl)methyl)quinazolin-4(3H)-one (19) to attenuate production of the PqsR-regulated virulence factor pyocyanin. Compounds 18 and 19 showed a low cytotoxic profile in the A549 human epithelial lung cell line making them suitable candidates for further pre-clinical evaluation.
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Aug 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Chalada
Suebsuwong
,
Bing
Dai
,
Daniel M.
Pinkas
,
Anantha Lakshmi
Duddupudi
,
Li
Li
,
Joshua C.
Bufton
,
Lisa
Schlicher
,
Mads
Gyrd-Hansen
,
Ming
Hu
,
Alex N.
Bullock
,
Alexei
Degterev
,
Gregory D.
Cuny
Diamond Proposal Number(s):
[15433]
Abstract: Receptor-interacting protein kinase 2 (RIPK2) is a key mediator of nucleotide-binding oligomerization domain (NOD) cell signaling that has been implicated in various chronic inflammatory conditions. A new class of RIPK2 kinase/NOD signaling inhibitors based on a 3,5-diphenyl-2-aminopyridine scaffold was developed. Several co-crystal structures of RIPK2•inhibitor complexes were analyzed to provide insights into inhibitor selectivity versus the structurally related activin receptor-like kinase 2 (ALK2) demonstrating that the inhibitor sits deeper in the hydrophobic binding pocket of RIPK2 perturbing the orientation of the DFG motif. In addition, the structure-activity relationship study revealed that in addition to anchoring to the hinge and DFG via the 2-aminopyridine and 3-phenylsulfonamide, respectively, appropriate occupancy of the region between the gatekeeper and the αC-helix provided by substituents in the 4- and 5-positions of the 3-phenylsulfonamide were necessary to achieve potent NOD cell signaling inhibition. For example, compound 18t (e.g. CSLP37) displayed potent biochemical RIPK2 kinase inhibition (IC50 = 16 ± 5 nM), >20-fold selectivity versus ALK2 and potent NOD cell signaling inhibition (IC50 = 26 ± 4 nM) in the HEKBlue assay. Finally, in vitro ADME and pharmacokinetic characterization of 18t further supports the prospects of the 3,5-diphenyl-2-aminopyridine scaffold for the generation of in vivo pharmacology probes of RIPK2 kinase and NOD cell signaling functions.
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May 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Yann-vai
Le Bihan
,
Rachel M.
Lanigan
,
Butrus
Atrash
,
Mark G.
Mclaughlin
,
Srikannathasan
Velupillai
,
Andrew G.
Malcolm
,
Katherine S.
England
,
Gian Filippo
Ruda
,
N. Yi
Mok
,
Anthony
Tumber
,
Kathy
Tomlin
,
Harry
Saville
,
Erald
Shehu
,
Craig
Mcandrew
,
Leanne
Carmichael
,
James M.
Bennett
,
Fiona
Jeganathan
,
Paul
Eve
,
Adam
Donovan
,
Angela
Hayes
,
Francesca
Wood
,
Florence I.
Raynaud
,
Oleg
Fedorov
,
Paul
Brennan
,
Rosemary
Burke
,
Rob
Van Montfort
,
Olivia W.
Rossanese
,
Julian
Blagg
,
Vassilios
Bavetsias
Diamond Proposal Number(s):
[20145]
Open Access
Abstract: Residues in the histone substrate binding sites that differ between the KDM4 and KDM5 subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one series was designed to rationally exploit these residue differences between the histone substrate binding sites in order to improve affinity for the KDM4-subfamily over KDM5-subfamily enzymes. In particular, residues E169 and V313 (KDM4A numbering) were targeted. Additionally, the conformational restriction of the flexible pyridopyrimidinone C8-substituent was investigated. These approaches yielded potent and cell-penetrant dual KDM4/5-subfamily inhibitors including 19a (KDM4A and KDM5B Ki = 0.004 and 0.007 μM, respectively). Compound cellular profiling in two orthogonal target engagement assays revealed a significant reduction from biochemical to cell-based activity across multiple analogues; this decrease was shown to be consistent with 2OG competition, and suggest that sub-nanomolar biochemical potency will be required with C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one compounds to achieve sub-micromolar target inhibition in cells.
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May 2019
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Stephanie
Myers
,
Duncan C.
Miller
,
Lauren
Molyneux
,
Mercedes
Arasta
,
Ruth H.
Bawn
,
Timothy
Blackburn
,
Simon J.
Cook
,
Noel
Edwards
,
Jane A.
Endicott
,
Bernard T.
Golding
,
Roger J.
Griffin
,
Tim
Hammonds
,
Ian R.
Hardcastle
,
Suzannah J.
Harnor
,
Amy
Heptinstall
,
Pamela
Lochhead
,
Mathew P.
Martin
,
Nick C.
Martin
,
David R.
Newell
,
Paul J.
Owen
,
Leon C.
Pang
,
Tristan
Reuillon
,
Laurent J. M.
Rigoreau
,
Huw
Thomas
,
Julie A.
Tucker
,
Lan-zhen
Wang
,
Ai-ching
Wong
,
Martin E. M.
Noble
,
Stephen R.
Wedge
,
Celine
Cano
Diamond Proposal Number(s):
[9948, 13587]
Abstract: Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC50 values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (∼3-fold) against ERK5, but importantly attenuated inhibition of p38α. Systematic variation of the substituents on the aroyl group led to the selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC50 0.82 μM for ERK5; IC50 > 120 μM for p38α). The crystal structure (PDB 5O7I) of this compound in complex with ERK5 has been solved. This compound was orally bioavailable and inhibited bFGF-driven Matrigel plug angiogenesis and tumour xenograft growth. The selective ERK5 inhibitor described herein provides a lead for further development into a tool compound for more extensive studies seeking to examine the role of ERK5 signalling in cancer and other diseases.
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May 2019
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I24-Microfocus Macromolecular Crystallography
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Vinayak
Singh
,
Angela
Pacitto
,
Stefano
Donini
,
Davide M.
Ferraris
,
Sándor
Boros
,
Eszter
Illyés
,
Bálint
Szokol
,
Menico
Rizzi
,
Tom L.
Blundell
,
David B.
Ascher
,
Janos
Pato
,
Valerie
Mizrahi
Abstract: Tuberculosis (TB) is a major infectious disease associated increasingly with drug resistance. Thus, new anti-tubercular agents with novel mechanisms of action are urgently required for the treatment of drug-resistant TB. In prior work, we identified compound 1 (cyclohexyl(4-(isoquinolin-5-ylsulfonyl)piperazin-1-yl)methanone) and showed that its anti-tubercular activity is attributable to inhibition of inosine-5′-monophosphate dehydrogenase (IMPDH) in Mycobacterium tuberculosis. In the present study, we explored the structure–activity relationship around compound 1 by synthesizing and evaluating the inhibitory activity of analogues against M. tuberculosis IMPDH in biochemical and whole-cell assays. X-ray crystallography was performed to elucidate the mode of binding of selected analogues to IMPDH. We establish the importance of the cyclohexyl, piperazine and isoquinoline rings for activity, and report the identification of an analogue with IMPDH-selective activity against a mutant of M. tuberculosis that is highly resistant to compound 1. We also show that the nitrogen in urea analogues is required for anti-tubercular activity and identify benzylurea derivatives as promising inhibitors that warrant further investigation.
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Apr 2019
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