I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Luís A. R.
Carvalho
,
Vanessa T.
Almeida
,
Jose A.
Brito
,
Kenneth M.
Lum
,
Tânia F.
Oliveira
,
Rita C.
Guedes
,
Lídia M.
Gonçalves
,
Susana D.
Lucas
,
Benjamin F.
Cravatt
,
Margarida
Archer
,
Rui
Moreira
Diamond Proposal Number(s):
[20161]
Abstract: 3-Oxo-β-sultams are four-membered ring ambident electrophiles that can react with nucleophiles either at the carbonyl carbon or at the sulfonyl sulfur atoms, and that have been reported to inhibit serine hydrolases via acylation of the active-site serine residue. We have developed a panel of 3-oxo-β-sultam inhibitors and show, through crystallographic data, that they are regioselective sulfonylating electrophiles, covalently binding to the catalytic serine of human and porcine elastases through the sulfur atom. Application of 3-oxo-β-sultam-derived activity-based probes in a human proteome revealed their potential to label disease-related serine hydrolases and proteasome subunits. Activity-based protein profiling applications of 3-oxo-β-sultams should open up new opportunities to investigate these classes of enzymes in complex proteomes and expand the toolbox of available sulfur-based covalent protein modifiers in chemical biology.
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Mar 2020
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Abstract: Although a previously developed bump-hole approach has proven powerful in generating specific inhibitors for mapping functions of protein kinases, its application is limited by the intolerance of the large-to-small mutation by certain kinases and the inability to control two kinases separately in the same cells. Herein, we describe the development of an alternative chemical-genetic approach to overcome these limitations. Our approach features the use of an engineered cysteine residue at a particular position as a reactive feature to sensitize a kinase of interest to selective covalent blockade by electrophilic inhibitors and is thus termed Ele-Cys approach. We successfully applied the Ele-Cys approach to identify selective covalent inhibitors of a receptor tyrosine kinase EphB1, and solved cocrystal structures to determine the mode of covalent binding. Importantly, the Ele-Cys and bump-hole approaches afforded orthogonal inhibition of two distinct kinases in the cell, opening the door to their combined use in the study of multi-kinase signaling pathways.
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May 2017
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I24-Microfocus Macromolecular Crystallography
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Michael D.
Urbaniak
,
Iain T.
Collie
,
Wenxia
Fang
,
Tonia
Aristotelous
,
Susanne
Eskilsson
,
Olawale G.
Raimi
,
Justin
Harrison
,
Iva Hopkins
Navratilova
,
Julie A.
Frearson
,
Daan M. F.
Van Aalten
,
Michael A. J.
Ferguson
Abstract: Bacteriocin; P aeruginosa.; pyocin S2; pyocin AP41; immunity protein Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final reaction in the biosynthesis of UDP-GlcNAc, an essential metabolite in many organisms including Trypanosoma brucei, the etiological agent of Human African Trypanosomiasis. High-throughput screening of recombinant T. brucei UAP identified a UTP-competitive inhibitor with selectivity over the human counterpart despite the high level of conservation of active site residues. Biophysical characterization of the UAP enzyme kinetics revealed that the human and trypanosome enzymes both display a strictly ordered bi−bi mechanism, but with the order of substrate binding reversed. Structural characterization of the T. brucei UAP−inhibitor complex revealed that the inhibitor binds at an allosteric site absent in the human homologue that prevents the conformational rearrangement required to bind UTP.
The identification of a selective inhibitory allosteric binding site in the parasite enzyme has therapeutic potential.
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Sep 2013
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I04-Macromolecular Crystallography
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Anders E. G.
Lindgren
,
Tobias
Karlberg
,
Ann-gerd
Thorsell
,
Mareike
Hesse
,
Sara
Spjut
,
Torun
Ekblad
,
C. David
Andersson
,
Ana Filipa
Pinto
,
Johan
Weigelt
,
Michael O.
Hottiger
,
Anna
Linusson
,
Mikael
Elofsson
,
Herwig
Schüler
Diamond Proposal Number(s):
[6603]
Abstract: Inhibiting ADP-ribosyl transferases with PARP-inhibitors is considered a promising strategy for the treatment of many cancers and ischemia, but most of the cellular targets are poorly characterized. Here, we describe an inhibitor of ADP-ribosyltransferase-3/poly(ADP-ribose) polymerase-3 (ARTD3), a regulator of DNA repair and mitotic progression. In vitro profiling against 12 members of the enzyme family suggests selectivity for ARTD3, and crystal structures illustrate the molecular basis for inhibitor selectivity. The compound is active in cells, where it elicits ARTD3-specific effects at submicromolar concentration. Our results show that by targeting the nicotinamide binding site, selective inhibition can be achieved among the closest relatives of the validated clinical target, ADP-ribosyltransferase-1/poly(ADP-ribose) polymerase-1.
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Jun 2013
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10369]
Open Access
Abstract: Inhibition of protein kinases using ATP-competitive compounds is an important strategy in drug discovery. In contrast, the allosteric regulation of kinases through the disruption of protein–protein interactions has not been widely adopted, despite the potential for selective targeting. Aurora-A kinase regulates mitotic entry and mitotic spindle assembly and is a promising target for anticancer therapy. The microtubule-associated protein TPX2 activates Aurora-A through binding to two sites. Aurora-A recognition is mediated by two motifs within the first 43 residues of TPX2, connected by a flexible linker. To characterize the contributions of these three structural elements, we prepared a series of TPX2 proteomimetics and investigated their binding affinity for Aurora-A using isothermal titration calorimetry. A novel stapled TPX2 peptide was developed that has improved binding affinity for Aurora-A and mimics the function of TPX2 in activating Aurora-A’s autophosphorylation. We conclude that the helical region of TPX2 folds upon binding Aurora-A, and that stabilization of this helix does not compromise Aurora-A activation. This study demonstrates that the preparation of these proteomimetics using modern synthesis methods is feasible and their biochemical evaluation demonstrates the power of proteomimetics as tool compounds for investigating PPIs involving intrinsically disordered regions of proteins.
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Dec 2016
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I24-Microfocus Macromolecular Crystallography
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Abstract: Enterobactin (ENT) is a siderophore (iron-chelating compound) produced by Escherichia coli in order to gain access to iron, an essential nutriment for bacterial growth. ENT is used as an exosiderophore by the opportunistic human pathogen Pseudomonas aeruginosa with transport of ferri-ENT across the bacterial outer membrane by the transporter PfeA. Next to pfeA gene on the chromosome is localized a gene encoding for an esterase, PfeE, whose transcription is regulated, as for pfeA, by the presence of ENT in bacterial environment. Purified PfeE hydrolyzed ferri-ENT into three molecules of 2,3 DHBS (2,3 dihydroxybenzoylserine) still complexed with ferric iron, and complete dissociation of iron from ENT chelating groups was only possible in the presence of both PfeE and an iron reducer, such as DTT. The crystal structure of PfeE and an inactive PfeE mutant complexed with ferri-ENT or a non-hydrolysable ferri-catechol complex allowed identification of the enzyme binding site and the catalytic triad. Finally, cell fractionation and fluorescence microscopy showed periplasmic localization of PfeE in P. aeruginosa cells. Thus, the molecular mechanism of iron release from ENT in P. aeruginosa differs from that previously described in E. coli. In P. aeruginosa, siderophore hydrolysis occurs in the periplasm, with ENT never reaching the bacterial cytoplasm. In E. coli, ferri-ENT crosses the inner membrane via the ABC transporter FepBCD and ferri-ENT is hydrolyzed by the esterase Fes only once it is in the cytoplasm.
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Aug 2018
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Mathieu
Métifiot
,
Kasthuraiah
Maddali
,
Barry C.
Johnson
,
Stephen
Hare
,
Steven J.
Smith
,
Xue Zhi
Zhao
,
Christophe
Marchand
,
Terrence R.
Burke
,
Stephen H.
Hughes
,
Peter
Cherepanov
,
Yves
Pommier
Abstract: On the basis of a series of lactam and phthalimide derivatives that inhibit HIV-1 integrase, we developed a new molecule, XZ-259, with biochemical and antiviral activities comparable to raltegravir. We determined the crystal structures of XZ-259 and four other derivatives in complex with the prototype foamy virus intasome. The compounds bind at the integrase-Mg2+-DNA interface of the integrase active site. In biochemical and antiviral assays, XZ-259 inhibits raltegravir-resistant HIV-1 integrases harboring the Y143R mutation. Molecular modeling is also presented suggesting that XZ-259 can bind in the HIV-1 intasome with its dimethyl sulfonamide group adopting two opposite orientations. Molecular dynamics analyses of the HIV-1 intasome highlight the importance of the viral DNA in drug potency
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Dec 2012
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Magnus
Alphey
,
Lisa
Pirrie
,
Leah S.
Torrie
,
Wassila Abdelli
Boulkeroua
,
Mary
Gardiner
,
Aurijit
Sarkar
,
Marko
Maringer
,
Wulf
Oehlmann
,
Ruth
Brenk
,
Michael S.
Scherman
,
Michael
Mcneil
,
Martin
Rejzek
,
Robert A.
Field
,
Mahavir
Singh
,
David
Gray
,
Nicholas J.
Westwood
,
James H.
Naismith
Open Access
Abstract: Glucose-1-phosphate thymidylyltransferase (RmlA) catalyzes the condensation of glucose-1-phosphate (G1P) with deoxy-thymidine triphosphate (dTTP) to yield dTDP-d-glucose and pyrophosphate. This is the first step in the l-rhamnose biosynthetic pathway. l-Rhamnose is an important component of the cell wall of many microorganisms, including Mycobacterium tuberculosis and Pseudomonas aeruginosa. Here we describe the first nanomolar inhibitors of P. aeruginosa RmlA. These thymine analogues were identified by high-throughput screening and subsequently optimized by a combination of protein crystallography, in silico screening, and synthetic chemistry. Some of the inhibitors show inhibitory activity against M. tuberculosis. The inhibitors do not bind at the active site of RmlA but bind at a second site remote from the active site. Despite this, the compounds act as competitive inhibitors of G1P but with high cooperativity. This novel behavior was probed by structural analysis, which suggests that the inhibitors work by preventing RmlA from undergoing the conformational change key to its ordered bi-bi mechanism.
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Nov 2012
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Mahboob
Salim
,
Timothy J.
Knowles
,
Alfie T.
Baker
,
Martin S.
Davey
,
Mark
Jeeves
,
Pooja
Sridhar
,
John
Wilkie
,
Carrie R.
Willcox
,
Hachemi
Kadri
,
Taher E.
Taher
,
Pierre
Vantourout
,
Adrian
Hayday
,
Youcef
Mehellou
,
Fiyaz
Mohammed
,
Benjamin E.
Willcox
Diamond Proposal Number(s):
[14692]
Abstract: Human Vγ9/Vδ2 T-cells detect tumour cells and microbial infections by recognising small phosphorylated prenyl metabolites termed phosphoantigens (P-Ag). The type-1 transmembrane protein Butyrophilin 3A1 (BTN3A1) is critical to the P-Ag-mediated activation of Vγ9/Vδ2 T-cells, however, the molecular mechanisms involved in BTN3A1-mediated metabolite sensing are unclear, including how P-Ag are discriminated from non-antigenic small molecules. Here, we utilised NMR and X-ray crystallography to probe P-Ag sensing by BTN3A1. Whereas the BTN3A1 Immunoglobulin Variable domain failed to bind P-Ag, the intracellular B30.2 domain bound a range of negatively-charged small molecules, including P-Ag, in a positively-charged surface pocket. However, NMR chemical shift perturbations indicated BTN3A1 discriminated P-Ag from non-antigenic small molecules by their ability to induce a specific conformational change in the B30.2 domain that propagated from the P-Ag binding site to distal parts of the domain. These results suggest BTN3A1 selectively detects P-Ag intracellularly via a conformational antigenic sensor in its B30.2 domain, and have implications for rational design of antigens for Vγ9/Vδ2 -based T-cell immunotherapies.
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Sep 2017
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I24-Microfocus Macromolecular Crystallography
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Ratana
Charoenwattanasatien
,
Salila
Pengthaisong
,
Imogen
Breen
,
Risa
Mutoh
,
Sompong
Sansenya
,
Yanling
Hua
,
Anupong
Tankrathok
,
Liang
Wu
,
Chomphunuch
Songsiriritthigul
,
Hideaki
Tanaka
,
Spencer J.
Williams
,
Gideon
Davies
,
Genji
Kurisu
,
James R. Ketudat
Cairns
Abstract: Human glucosylcerebrosidase 2 (GBA2) of the CAZy family GH116 is responsible for the breakdown of
glycosphingolipids on the cytoplasmic face of the endoplasmic reticulum and Golgi apparatus. Genetic defects in GBA2 result in
spastic paraplegia and cerebellar ataxia, while cross-talk between GBA2 and GBA1 glucosylceramidases may affect Gaucher
disease. Here, we report the first three-dimensional structure for any GH116 enzyme, Thermoanaerobacterium xylanolyticum
TxGH116 β-glucosidase, alone and in complex with diverse ligands. These structures allow identification of the glucoside binding
and active site residues, which are shown to be conserved with GBA2. Mutagenic analysis of TxGH116 and structural modeling
of GBA2 provide a detailed structural and functional rationale for pathogenic missense mutations of GBA2.
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May 2016
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