I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Irene
G. Salado
,
Abhimanyu
Singh
,
Carlos
Moreno-cinos
,
Guna
Sakaine
,
Marco
Siderius
,
Pieter
Van Der Veken
,
An
Matheeussen
,
Tiffany
Van Der Meer
,
Payman
Sadek
,
Sheraz
Gul
,
Louis
Maes
,
Geert Jan
Sterk
,
Rob
Leurs
,
David
Brown
,
Koen
Augustyns
Diamond Proposal Number(s):
[16207, 15075]
Abstract: Human African trypanosomiasis is causing thousands of deaths every year in the rural areas of Africa. In this manuscript we describe the optimization of a family of phtalazinone derivatives. Phosphodiesterases have emerged as attractive molecular targets for a novel treatment for a variety of neglected parasitic diseases. Compound 1 resulted to be a potent TbrPDEB1 inhibitor with interesting activity against T. brucei in a phenotypic screen. Derivative 1 was studied in an acute in vivo mouse disease model but unfortunately showed no efficacy due to low metabolic stability. We report structural modifications to achieve compounds with an improved metabolic stability while maintaining high potency against TbrPDEB1 and T. brucei. Compound 14, presented a good microsomal stability in mouse and human microsomes and provides a good starting point for future efforts.
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Mar 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Erik
De Heuvel
,
Abhimanyu K.
Singh
,
Pierre
Boronat
,
Albert J.
Kooistra
,
Tiffany
Van Der Meer
,
Payman
Sadek
,
Antoni R.
Blaazer
,
Nathan C.
Shaner
,
Daphne S.
Bindels
,
Guy
Caljon
,
Louis
Maes
,
Geert
Jan Sterk
,
Marco
Siderius
,
Michael
Oberholzer
,
Iwan J. P.
De Esch
,
David G.
Brown
,
Rob
Leurs
Diamond Proposal Number(s):
[13689, 16207]
Open Access
Abstract: Inhibitors against Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) and B2 (TbrPDEB2) have gained interest as new treatments for human African trypanosomiasis. The recently reported alkynamide tetrahydrophthalazinones, which show submicromolar activities against TbrPDEB1 and anti-T. brucei activity, have been used as starting point for the discovery of new TbrPDEB1 inhibitors. Structure-based design indicated that the alkynamide-nitrogen atom can be readily decorated, leading to the discovery of 37, a potent TbrPDEB1 inhibitor with submicromolar activities against T. brucei parasites. Furthermore, 37 is more potent against TbrPDEB1 than hPDE4 and shows no cytotoxicity on human MRC-5 cells. The crystal structures of the catalytic domain of TbrPDEB1 co-crystalized with several different alkynamides show a bidentate interaction with key-residue Gln874, but no interaction with the parasite-specific P-pocket, despite being (uniquely) a more potent inhibitor for the parasite PDE. Incubation of blood stream form trypanosomes by 37 increases intracellular cAMP levels and results in the distortion of the cell cycle and cell death, validating phosphodiesterase inhibition as mode of action.
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Jul 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Erik
De Heuvel
,
Abhimanyu K.
Singh
,
Ewald
Edink
,
Tiffany
Van Der Meer
,
Melanie
Van Der Woude
,
Payman
Sadek
,
Mikkel P.
Krell-jørgensen
,
Toine
Van Den Bergh
,
Johan
Veerman
,
Guy
Caljon
,
Titilola D.
Kalejaiye
,
Maikel
Wijtmans
,
Louis
Maes
,
Harry P.
De Koning
,
Geert
Jan Sterk
,
Marco
Siderius
,
Iwan J. P.
De Esch
,
David
Brown
,
Rob
Leurs
Diamond Proposal Number(s):
[13689]
Open Access
Abstract: Several 3’,5’-cyclic nucleotide phosphodiesterases (PDEs) have been validated as good drug targets for a large variety of diseases. Trypanosoma brucei PDEB1 (TbrPDEB1) has been designated as a promising drug target for the treatment of human African trypanosomiasis. Recently, the first class of selective nanomolar TbrPDEB1 inhibitors was obtained by targeting the parasite specific P-pocket. However, these biphenyl-substituted tetrahydrophthalazinone-based inhibitors did not show potent cellular activity against Trypanosoma brucei (T. brucei) parasites, leaving room for further optimization. Herein, we report the discovery of a new class of potent TbrPDEB1 inhibitors that display improved activities against T. brucei parasites. Exploring different linkers between the reported tetrahydrophthalazinone core scaffold and the amide tail group resulted in the discovery of alkynamide phthalazinones as new TbrPDEB1 inhibitors, which exhibit submicromolar activities versus T. brucei parasites and no cytotoxicity to human MRC-5 cells. Elucidation of the crystal structure of alkynamide 8b (NPD-048) bound to the catalytic domain of TbrPDEB1 shows a bidentate interaction with the key-residue Gln874 and good directionality towards the P-pocket. Incubation of trypanosomes with alkynamide 8b results in an increase of intracellular cAMP, validating a PDE-mediated effect in vitro and providing a new interesting compound series for further studies towards selective TbrPDEB1 inhibitors with potent phenotypic activity.
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Jun 2019
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15075]
Abstract: Vinculin is a central component of mechanosensitive adhesive complexes that form between cells and the extracellular matrix. A myriad of infectious agents mimic vinculin binding sites (VBS), enabling them to hijack the adhesion machinery and facilitate cellular entry. Here, we report the structural and biochemical characterisation of a VBS from the chlamydial virulence factor TarP. Whilst the affinities of isolated VBS peptides from TarP and talin for vinculin are similar, their behaviour in larger fragments is markedly different. In talin, VBS are cryptic and require mechanical activation to bind vinculin, whereas the TarP VBS are located in disordered regions, and so are constitutively active. We demonstrate that the TarP VBS can uncouple talin:vinculin complexes, which may lead to adhesion destabilisation.
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Apr 2018
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Antoni R.
Blaazer
,
Abhimanyu K.
Singh
,
Erik
De Heuvel
,
Ewald
Edink
,
Kristina M.
Orrling
,
Johan J. N.
Veerman
,
Toine
Van Den Bergh
,
Chimed
Jansen
,
Erin
Balasubramaniam
,
Wouter J.
Mooij
,
Hans
Custers
,
Maarten
Sijm
,
Daniel N. A.
Tagoe
,
Titilola D.
Kalejaiye
,
Jane C.
Munday
,
Hermann
Tenor
,
An
Matheeussen
,
Maikel
Wijtmans
,
Marco
Siderius
,
Chris
De Graaf
,
Louis
Maes
,
Harry P.
De Koning
,
David S.
Bailey
,
Geert Jan
Sterk
,
Iwan J. P.
De Esch
,
David G.
Brown
,
Rob
Leurs
Abstract: Several trypanosomatid cyclic nucleotide phosphodiesterases (PDEs) possess a unique, parasite-specific cavity near the ligand-binding region that is referred to as the P-pocket. One of these enzymes, Trypanosoma brucei PDE B1 (TbrPDEB1), is considered a drug target for the treatment of African sleeping sickness. Here, we elucidate the molecular determinants of inhibitor binding and reveal that the P-pocket is amenable to directed design. By iterative cycles of design, synthesis, pharmacological evaluation, and by elucidating the structures of inhibitor-bound TbrPDEB1, hPDE4B and hPDE4D complexes, we have developed 4a,5,8,8a-tetrahydrophthalazinones as the first selective TbrPDEB1 inhibitor series. Two of these, 8 (NPD-008) and 9 (NPD-039), were potent (Ki = 100 nM) TbrPDEB1 inhibitors with antitrypanosomal effects (IC50 = 5.5 and 6.7 µM, respectively). Treatment of parasites with 8 caused an increase in intracellular cAMP levels and severe disruption of T. brucei cellular organization, chemically validating trypanosomal PDEs as therapeutic targets in trypanosomiasis.
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Apr 2018
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Andriy
Kryshtafovych
,
Reinhard
Albrecht
,
Arnaud
Basle
,
Pedro
Bule
,
Alessandro T.
Caputo
,
Ana Luisa
Carvalho
,
Kinlin L.
Chao
,
Ron
Diskin
,
Krzysztof
Fidelis
,
Carlos M. G. A.
Fontes
,
Folmer
Fredslund
,
Harry J.
Gilbert
,
Celia W.
Goulding
,
Marcus D.
Hartmann
,
Christopher S.
Hayes
,
Osnat
Herzberg
,
Johan C.
Hill
,
Andrzej
Joachimiak
,
Gert-wieland
Kohring
,
Roman I.
Koning
,
Leila
Lo Leggio
,
Marco
Mangiagalli
,
Karolina
Michalska
,
John
Moult
,
Shabir
Najmudin
,
Marco
Nardini
,
Valentina
Nardone
,
Didier
Ndeh
,
Thanh-hong
Nguyen
,
Guido
Pintacuda
,
Sandra
Postel
,
Mark J.
Van Raaij
,
Pietro
Roversi
,
Amir
Shimon
,
Abhimanyu K.
Singh
,
Eric J.
Sundberg
,
Kaspars
Tars
,
Nicole
Zitzmann
,
Torsten
Schwede
Abstract: The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment.
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Mar 2018
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I03-Macromolecular Crystallography
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Andriy
Kryshtafovych
,
John
Moult
,
Arnaud
Basle
,
Alex
Burgin
,
Timothy K.
Craig
,
Robert A.
Edwards
,
Deborah
Fass
,
Marcus
Hartmann
,
Mateusz
Korycinski
,
Rick
Lewis
,
Donald
Lorimer
,
Andrei N.
Lupas
,
Janet
Newman
,
Thomas S.
Peat
,
Kurt H.
Piepenbrink
,
Janani
Prahlad
,
Mark
Van Raaij
,
Forest
Rohwer
,
Anca M.
Segall
,
Victor
Seguritan
,
Eric J.
Sundberg
,
Abhimanyu
Singh
,
Mark A.
Wilson
,
Torsten
Schwede
Diamond Proposal Number(s):
[9948]
Open Access
Abstract: The Critical Assessment of protein Structure Prediction (CASP) experiment would not have been possible without the prediction targets provided by the experimental structural biology community. In this article, selected crystallographers providing targets for the CASP11 experiment discuss the functional and biological significance of the target proteins, highlight their most interesting structural features and assess whether these features were correctly reproduced in the predictions submitted to CASP11.
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Oct 2015
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[3808]
Open Access
Abstract: Adenoviruses are non-enveloped icosahedral viruses with trimeric fibre proteins protruding from their vertices. There are five known genera, from which only Mastadenoviruses have been widely studied. Apart from studying adenovirus as a biological model system and with a view to prevent or combat viral infection, there is a major interest in using adenovirus for vaccination, cancer therapy and gene therapy purposes. Adenoviruses from the Atadenovirus genus have been isolated from squamate reptile hosts, ruminants and birds and have a characteristic gene organization and capsid morphology. The carboxy-terminal virus-distal fibre head domains are likely responsible for primary receptor recognition. We determined the high-resolution crystal structure of the Snake Adenovirus 1 (SnAdV-1) fibre head using the multi-wavelength anomalous dispersion (MAD) method. Despite the absence of significant sequence homology, this Atadenovirus fibre head has the same beta-sandwich propeller topology as other adenovirus fibre heads. However, it is about half the size, mainly due to much shorter loops connecting the beta-strands. The detailed structure of the SnAdV-1 fibre head and other animal adenovirus fibre heads, together with the future identification of their natural receptors, may lead to the development of new strategies to target adenovirus vectors to cells of interest.
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Dec 2014
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[5860]
Abstract: The phage-proximal part of the long tail fibres of bacteriophage T4 consists of a trimer of the 1289 amino-acid gene product 34 (gp34). Different carboxy-terminal parts of gp34 have been produced and crystallized. Crystals of gp34(726-1289) diffracting X-rays to 2.9 Å resolution, crystals of gp34(781-1289) diffracting to 1.9 Å resolution and crystals of gp34(894-1289) diffracting to 3.0 and 2.0 Å resolution and belonging to different crystal forms were obtained. Native data were collected for gp34(726-1289) and gp34(894-1289), while single-wavelength anomalous diffraction data were collected for selenomethionine-containing gp34(781-1289) and gp34(894-1289). For the latter, high-quality anomalous signal was obtained.
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Jul 2014
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Abstract: Total reflection X-ray fluorescence (TXRF) spectroscopy has attracted interest in recent years. Using this technique, almost all elements ranging from sodium (Z = 11) to uranium (Z = 92) in the periodic table can be detected and analysed in a wide concentration range in a single measurement. The present state-of-the-art sensitivities of TXRF are stretched to femtogram range by employing synchrotron radiation as an excitation source. This article aims to provide a basic overview of the TXRF technique to the general readers. We describe a TXRF spectrometer developed in a laboratory source. An X-ray fluorescence-microprobe beamline for TXRF is being constructed, as an extension of this activity, on the Indian synchrotron source, Indus-2.
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Oct 2008
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