I03-Macromolecular Crystallography
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Gijs
Ruijgrok
,
Wendy A.
Offen
,
Isabelle B.
Pickles
,
Deepa
Raju
,
Thanasis
Patsos
,
Casper
De Boer
,
Tim
Ofman
,
Joep
Rompa
,
Daan
Van Oord
,
Eleanor J.
Dodson
,
Alexander
Beekers
,
Thijs
Voskuilen
,
Michela
Ferrari
,
Liang
Wu
,
Antonius P. A.
Janssen
,
Jeroen D. C.
Codée
,
P. Lynne
Howell
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[32736]
Open Access
Abstract: During infection, the human opportunistic pathogen Pseudomonas aeruginosa forms protective biofilms, whose matrix consists of proteins, nucleic acids, and polysaccharides such as alginate, Psl, and Pel. Psl, a polymeric pentasaccharide composed of mannose, rhamnose, and glucose, is produced during the early stages of biofilm formation, serving as a protective barrier against antibiotics and the immune system. The Psl biosynthesis gene cluster, besides encoding various glycosyltransferases, also includes an endoglycosidase, PslG. Here, we show, by activity-based protein profiling, structural studies on enzyme–inhibitor complexes, and defined substrate processing, that PslG is not, as previously suggested, an endo-β-mannosidase but instead a retaining endo-β-glucosidase. This insight allows the design of both competitive and covalent PslG inhibitors, as we show for repeating pentasaccharide mimetics featuring either a reducing end deoxynojirimycin or cyclophellitol moiety. This work provides valuable tools to deepen the understanding of Psl biosynthesis, its function in biofilm formation, and its contribution to antibiotic resistance. We demonstrate the enzyme’s actual endo−β–glucosidase activity, a means to monitor PslG activity in P. aeruginosa biofilms, and a blueprint for inhibitor design.
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Feb 2025
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I04-Macromolecular Crystallography
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Qin
Su
,
Max
Louwerse
,
Rob F.
Lammers
,
Elmer
Maurits
,
Max
Janssen
,
Rolf G.
Boot
,
Valentina
Borlandelli
,
Wendy A.
Offen
,
Daniël
Linzel
,
Sybrin P.
Schröder
,
Gideon J.
Davies
,
Herman S.
Overkleeft
,
Marta
Artola
,
Johannes M. F. G.
Aerts
Open Access
Abstract: GBA2, the non-lysosomal β-glucosylceramidase, is an enzyme involved in glucosylceramide metabolism. Pharmacological inhibition of GBA2 by N-alkyl iminosugars is well tolerated and benefits patients suffering from Sandhoff and Niemann–Pick type C diseases, and GBA2 inhibitors have been proposed as candidate-clinical drugs for the treatment of parkinsonism. With the ultimate goal to unravel the role of GBA2 in (patho)physiology, we sought to develop a GBA2-specific activity-based probe (ABP). A library of probes was tested for activity against GBA2 and the two other cellular retaining β-glucosidases, lysosomal GBA1 and cytosolic GBA3. We show that β-D-arabinofuranosyl cyclitol aziridine (β-D-Araf aziridine) reacts with the GBA2 active site nucleophile to form a covalent and irreversible bond. Fluorescent β-D-Araf aziridine probes potently and selectively label GBA2 both in vitro and in cellulo, allowing for visualization of the localization of overexpressed GBA2 using fluorescence microscopy. Co-staining with an antibody selective for the lysosomal β-glucosylceramidase GBA1, shows distinct subcellular localization of the two enzymes. We proffer our ABP technology for further delineating the role and functioning of GBA2 in disease and propose the β-D-Araf aziridine scaffold as a good starting point for the development of GBA2-specific inhibitors for clinical development.
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Sep 2024
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I03-Macromolecular Crystallography
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Valentina
Borlandelli
,
Wendy
Offen
,
Olga
Moroz
,
Alba
Nin-Hill
,
Nicholas
Mcgregor
,
Lars
Binkhorst
,
Akihiro
Ishiwata
,
Zachary
Armstrong
,
Marta
Artola
,
Carme
Rovira
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: GH127 and GH146 microorganismal retaining β-l-arabinofuranosidases, expressed by human gut microbiomes, feature an atypical catalytic domain and an unusual mechanism of action. We recently reported that both Bacteroides thetaiotaomicron BtGH146 and Bifidobacterium longum HypBA1 are inhibited by β-l-arabinofuranosyl cyclophellitol epoxide, supporting the action of a zinc-coordinated cysteine as a catalytic nucleophile, where in most retaining GH families, an aspartate or glutamate is employed. This work presents a panel of β-l-arabinofuranosyl cyclophellitol epoxides and aziridines as mechanism-based BtGH146/HypBA1 inhibitors and activity-based probes. The β-l-arabinofuranosyl cyclophellitol aziridines both inhibit and label β-l-arabinofuranosidase efficiently (however with different activities), whereas the epoxide-derived probes favor BtGH146 over HypBA1. These findings are accompanied by X-ray structural analysis of the unmodified β-l-arabinofuranosyl cyclophellitol aziridine in complex with both isozymes, which were shown to react by nucleophilic opening of the aziridine, at the pseudoanomeric carbon, by the active site cysteine nucleophile to form a stable thioether bond. Altogether, our activity-based probes may serve as chemical tools for the detection and identification of low-abundance β-l-arabinofuranosidases in complex biological samples.
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Dec 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[24948, 18598]
Open Access
Abstract: Bacteria and yeasts grow on biomass polysaccharides by expressing and excreting a complex array of glycoside hydrolase (GH) enzymes. Identification and annotation of such GH pools, which are valuable commodities for sustainable energy and chemistries, by conventional means (genomics, proteomics) are complicated, as primary sequence or secondary structure alignment with known active enzymes is not always predictive for new ones. Here we report a “low-tech”, easy-to-use, and sensitive multiplexing activity-based protein-profiling platform to characterize the xyloglucan-degrading GH system excreted by the soil saprophyte, Cellvibrio japonicus, when grown on xyloglucan. A suite of activity-based probes bearing orthogonal fluorophores allows for the visualization of accessory exo-acting glycosidases, which are then identified using biotin-bearing probes. Substrate specificity of xyloglucanases is directly revealed by imbuing xyloglucan structural elements into bespoke activity-based probes. Our ABPP platform provides a highly useful tool to dissect xyloglucan-degrading systems from various sources and to rapidly select potentially useful ones. The observed specificity of the probes moreover bodes well for the study of other biomass polysaccharide-degrading systems, by modeling probe structures to those of desired substrates.
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Nov 2023
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I03-Macromolecular Crystallography
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Chi-Lin
Kuo
,
Qin
Su
,
Adrianus M. C. H.
Van Den Nieuwendijk
,
Thomas J. M.
Beenakker
,
Wendy A.
Offen
,
Lianne I.
Willems
,
Rolf. G.
Boot
,
Alexi J.
Sarris
,
André R. A.
Marques
,
Jeroen D. C.
Codée
,
Gijsbert A.
Van Der Marel
,
Bogdan I.
Florea
,
Gideon J.
Davies
,
Herman S.
Overkleeft
,
Johannes M. F. G.
Aerts
Abstract: Acid β-galactosidase (GLB1) and galactocerebrosidase (GALC) are retaining exo-β-galactosidases involved in lysosomal glycoconjugate metabolism. Deficiency of GLB1 may result in the lysosomal storage disorders GM1 gangliosidosis, Morquio B syndrome, and galactosialidosis, and deficiency of GALC may result in Krabbe disease. Activity-based protein profiling (ABPP) is a powerful technique to assess the activity of retaining glycosidases in relation to health and disease. This work describes the use of fluorescent and biotin-carrying activity-based probes (ABPs) to assess the activity of both GLB1 and GALC in cell lysates, culture media, and tissue extracts. The reported ABPs, which complement the growing list of retaining glycosidase ABPs based on configurational isomers of cyclophellitol, should assist in fundamental and clinical research on various β-galactosidases, whose inherited deficiencies cause debilitating lysosomal storage disorders.
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Oct 2023
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I03-Macromolecular Crystallography
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Nicholas G. S.
Mcgregor
,
Chi-Lin
Kuo
,
Thomas
Beenakker
,
Chun-Sing
Wong
,
Wendy A.
Offen
,
Zachary
Armstrong
,
Bobby I.
Florea
,
Jeroen D.
Codee
,
Herman S.
Overkleeft
,
Hans
Aerts
,
Gideon
Davies
Diamond Proposal Number(s):
[24948, 18598]
Open Access
Abstract: Exo-β-mannosidases are a broad class of stereochemically retaining hydrolases that are essential for the breakdown of complex carbohydrate substrates found in all kingdoms of life. Yet the detection of exo-β-mannosidases in complex biological samples remains challenging, necessitating the development of new methodologies. Cyclophellitol and its analogues selectively label the catalytic nucleophiles of retaining glycoside hydrolases, making them valuable tool compounds. Furthermore, cyclophellitol can be readily redesigned to enable the incorporation of a detection tag, generating activity-based probes (ABPs) that can be used to detect and identify specific glycosidases in complex biological samples. Towards the development of ABPs for exo-β-mannosidases, we present a concise synthesis of β-manno-configured cyclophellitol, cyclophellitol aziridine, and N-alkyl cyclophellitol aziridines. We show that these probes covalently label exo-β-mannosidases from GH families 2, 5, and 164. Structural studies of the resulting complexes support a canonical mechanism-based mode of action in which the active site nucleophile attacks the pseudo-anomeric centre to form a stable ester linkage, mimicking the glycosyl enzyme intermediate. Furthermore, we demonstrate activity- based protein profiling using an N-alkyl aziridine derivative by specifically labelling MANBA in mouse kidney tissue. Together, these results show that synthetic manno-configured cyclophellitol analogues hold promise for detecting exo-β-mannosidases in biological and biomedical research.
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Dec 2021
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Gideon J.
Davies
,
Rhianna J.
Rowland
,
Yurong
Chen
,
Imogen
Breen
,
Liang
Wu
,
Wendy A.
Offen
,
Thomas
Beenakker
,
Qin
Su
,
Adrianus M. C. H.
Van Den Nieuwendijk
,
Johannes M. F. G.
Aerts
,
Marta
Artola
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587, 18598]
Open Access
Abstract: Gaucher disease (GD) is a lysosomal storage disorder caused by inherited deficiencies in β-glucocerebrosidase (GBA). Current treatments require rapid disease diagnosis and a means of monitoring therapeutic efficacy, both of which may be supported by the use of GBA-targeting activity-based probes (ABPs). Here, we report the synthesis and structural analysis of a range of cyclophellitol epoxide and aziridine inhibitors and ABPs for GBA. We demonstrate their covalent mechanism-based mode of action and uncover binding of the new N- functionalised aziridines to the ligand binding cleft. These inhibitors became scaffolds for the development of ABPs; the O6-fluorescent tags of which bind in an allosteric site at the dimer interface. Considering GBA’s preference for O6- and N -functionalised reagents, we synthesised a bi-functional aziridine ABP which we hoped would offer a more powerful imaging agent. Whilst this ABP binds to two unique active site clefts of GBA, no further benefit in potency was achieved over our first generation ABPs. Nevertheless, such ABPs should serve useful in the study of GBA in relation to GD and inform the design of future probes.
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Sep 2021
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I04-Macromolecular Crystallography
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Hermen S.
Overkleeft
,
Sybrin
Schröder
,
Wendy
Offen
,
Alexandra
Males
,
Yi
Jin
,
Casper
De Boer
,
Jacopo
Enotarpi
,
Gijs
Van Der Marel
,
Bogdan
Florea
,
Jeroen
Codée
,
Gideon
Davies
Diamond Proposal Number(s):
[13587, 18598]
Open Access
Abstract: There is a vast genomic resource for enzymes active on carbohydrates. Lagging far behind, however, are functional chemical tools for the rapid characterization of carbohydrate‐active enzymes. Activity‐based probes (ABPs) offer one chemical solution to these issues with ABPs based upon cyclophellitol epoxide and aziridine covalent and irreversible inhibitors representing a potent and widespread approach. Such inhibitors for enzymes active on polysaccharides are potentially limited by the requirement for several glycosidic bonds, themselves substrates for the enzyme targets. Here we show that non‐hydrolysable trisaccharide can be synthesized and applied even to enzymes with challenging subsite requirements. We find that incorporation of carbasugar moieties, which we accomplished by cuprate‐assisted regioselective trans‐diaxial epoxide opening of carba‐mannal we synthesised for this purpose, yields inactivators that act as powerful activity‐based inhibitors for a‐1,6 endo‐mannanases. 3‐D structures at 1.35 – 1.47 Å resolutions confirm the design rationale and binding to the enzymatic nucleophile. Carbasugar oligosaccharide cyclophellitols offer a powerful new approach for the design of robust endoglycosidase inhibitors, while the synthesis procedures presented here should allow adaptation towards activity‐based endoglycosidase probes as well as configurational isosteres targeting other endoglycosidase families.
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Apr 2021
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Patrick
Weber
,
Martin
Thonhofer
,
Summer
Averill
,
Gideon J.
Davies
,
Andres Gonzalez
Santana
,
Philipp
Müller
,
Seyed A.
Nasseri
,
Wendy A.
Offen
,
Bettina M.
Pabst
,
Eduard
Paschke
,
Michael
Schalli
,
Ana
Torvisco
,
Marion
Tschernutter
,
Christina
Tysoe
,
Werner
Windischhofer
,
Stephen G.
Withers
,
Andreas
Wolfsgruber
,
Tanja M.
Wrodnigg
,
Arnold E.
Stütz
Open Access
Abstract: Glycosidase inhibitors have shown great potential as pharmacological chaperones for lysosomal storage diseases. In light of this, a series of new cyclopentanoid β-galactosidase inhibitors were prepared and their inhibitory and pharmacological chaperoning activities determined and compared with those of lipophilic analogs of the potent β-d-galactosidase inhibitor 4-epi-isofagomine. Structure-activity relationships were investigated by X-ray crystallography as well as by alterations in the cyclopentane moiety such as deoxygenation and replacement by fluorine of a “strategic” hydroxyl group. New compounds have revealed highly promising activities with a range of β-galactosidase-compromised human cell lines and may serve as leads towards new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.
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Sep 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Tessa
Keenan
,
Fabio
Parmeggiani
,
Julien
Malassis
,
Clement Q.
Fontenelle
,
Jean-Baptiste
Vendeville
,
Wendy
Offen
,
Peter
Both
,
Kun
Huang
,
Andrea
Marchesi
,
Alex
Heyam
,
Carl
Young
,
Simon J.
Charnock
,
Gideon J.
Davies
,
Bruno
Linclau
,
Sabine L.
Flitsch
,
Martin A.
Fascione
Diamond Proposal Number(s):
[13587, 18598]
Abstract: Fluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluorogalactose, giving insight into changes in the active site that may account for the specificity of the enzyme toward certain substrate analogs.
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Jul 2020
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