I02-Macromolecular Crystallography
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Thomas J. M.
Beenakker
,
Dennis P. A.
Wander
,
Wendy
Offen
,
Marta
Artola
,
Lluís
Raich
,
Maria J.
Ferraz
,
Kah-yee
Li
,
Judith H. P. M.
Houben
,
Erwin R.
Van Rijssel
,
Thomas
Hansen
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Johannes M. F. G.
Aerts
,
Carme
Rovira
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587]
Abstract: The conformational analysis of glycosidases affords a route to their specific inhibition through transition-state mimicry. Inspired by the rapid reaction rates of cyclophellitol and cyclophellitol aziridine—both covalent retaining β-glucosidase inhibitors—we postulated that the corresponding carba “cyclopropyl” analogue would be a potent retaining β-glucosidase inhibitor for those enzymes reacting through the 4H3 transition-state conformation. Ab initio metadynamics simulations of the conformational free energy landscape for the cyclopropyl inhibitors show a strong bias for the 4H3 conformation, and carba-cyclophellitol, with an N-(4-azidobutyl)carboxamide moiety, proved to be a potent inhibitor (Ki = 8.2 nM) of the Thermotoga maritima TmGH1 β-glucosidase. 3-D structural analysis and comparison with unreacted epoxides show that this compound indeed binds in the 4H3 conformation, suggesting that conformational strain induced through a cyclopropyl unit may add to the armory of tight-binding inhibitor designs.
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May 2017
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I02-Macromolecular Crystallography
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Sybrin P.
Schröder
,
Jasper W.
Van De Sande
,
Wouter W.
Kallemeijn
,
Chi-lin
Kuo
,
Marta
Artola
,
Eva J.
Van Rooden
,
Jianbing
Jiang
,
Thomas J. M.
Beenakker
,
Bogdan I.
Florea
,
Wendy
Offen
,
Gideon
Davies
,
Adriaan J.
Minnaard
,
Johannes M. F. G.
Aerts
,
Jeroen D. C.
Codée
,
Gijsbert A.
Van Der Marel
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Activity-based protein profiling has emerged as a powerful tool for visualizing glycosidases in complex biological samples. Several configurational cyclophellitol isomers have been shown to display high selectivity as probes for glycosidases processing substrates featuring the same configuration. Here, a set of deoxygenated cyclophellitols are presented which enable inter-class profiling of β-glucosidases and β-galactosidases.
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Nov 2017
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Liang
Wu
,
Jianbing
Jiang
,
Yi
Jin
,
Wouter W.
Kallemeijn
,
Chi-lin
Kuo
,
Marta
Artola
,
Wei
Dai
,
Cas
Van Elk
,
Marco
Van Eijk
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codee
,
Bogdan I.
Florea
,
Johannes M. F. G.
Aerts
,
Herman S.
Overkleeft
,
Gideon J.
Davies
Diamond Proposal Number(s):
[9948, 13587]
Abstract: Humans express at least two distinct β-glucuronidase enzymes that are involved in disease: exo-acting β-glucuronidase (GUSB), whose deficiency gives rise to mucopolysaccharidosis type VII, and endo-acting heparanase (HPSE), whose overexpression is implicated in inflammation and cancers. The medical importance of these enzymes necessitates reliable methods to assay their activities in tissues. Herein, we present a set of β-glucuronidase-specific activity-based probes (ABPs) that allow rapid and quantitative visualization of GUSB and HPSE in biological samples, providing a powerful tool for dissecting their activities in normal and disease states. Unexpectedly, we find that the supposedly inactive HPSE proenzyme proHPSE is also labeled by our ABPs, leading to surprising insights regarding structural relationships between proHPSE, mature HPSE, and their bacterial homologs. Our results demonstrate the application of β-glucuronidase ABPs in tracking pathologically relevant enzymes and provide a case study of how ABP-driven approaches can lead to discovery of unanticipated structural and biochemical functionality.
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Jun 2017
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Marta
Artola
,
Liang
Wu
,
Maria J.
Ferraz
,
Chi-lin
Kuo
,
Lluís
Raich
,
Imogen Z.
Breen
,
Wendy A.
Offen
,
Jeroen D. C.
Codée
,
Gijsbert A.
Van Der Marel
,
Carme
Rovira
,
Johannes M. F. G.
Aerts
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: The essential biological roles played by glycosidases, coupled to the diverse therapeutic benefits of pharmacologically targeting these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivalents of epoxides in organic synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here we present the synthesis, conformational analysis, and application of novel 1,6-cyclophellitol cyclosulfates. We show that 1,6-epi-cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose 4C1 chair conformation matches that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (β-cyclosulfate) reacts more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions.
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Jul 2017
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Marta
Artola
,
Christinne
Hedberg
,
Rhianna J.
Rowland
,
Lluís
Raich
,
Kassiani
Kytidou
,
Liang
Wu
,
Amanda
Schaaf
,
Maria Joao
Ferraz
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Carme
Rovira
,
Johannes M. F. G.
Aerts
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Fabry disease is an inherited lysosomal storage disorder that is characterized by a deficiency in lysosomal α-D-galactosidase activity. One current therapeutic strategy involves enzyme replacement therapy, in which patients are treated with a recombinant enzyme. Co-treatment with enzyme active-site stabilizers is advocated to increase treatment efficacy, a strategy that requires effective and selective enzyme stabilizers. Here, we describe the design and development of an α-D-gal-cyclophellitol cyclosulfamidate as a new class of neutral, conformationally constrained competitive glycosidase inhibitors that act by mimicry of the Michaelis complex conformation. We found that D-galactose-configured α-cyclosulfamidate 4 effectively stabilizes recombinant human α-D-galactosidase (agalsidase beta, Fabrazyme®) both in vitro and in cellulo.
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Aug 2019
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Gaucher disease is caused by inherited deficiency in glucocerebrosidase (GBA, a retaining β-glucosidase), and deficiency in GBA constitutes the largest known genetic risk factor for Parkinson’s disease. In the past, animal models of Gaucher disease have been generated by treatment with the mechanism-based GBA inhibitors, conduritol B epoxide (CBE), and cyclophellitol. Both compounds, however, also target other retaining glycosidases, rendering generation and interpretation of such chemical knockout models complicated. Here we demonstrate that cyclophellitol derivatives carrying a bulky hydrophobic substituent at C8 are potent and selective GBA inhibitors and that an unambiguous Gaucher animal model can be readily generated by treatment of zebrafish with these.
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Mar 2019
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I03-Macromolecular Crystallography
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Jianbing
Jiang
,
Wouter W.
Kallemeijn
,
Daniel W.
Wright
,
Adrianus M. C. H.
Van Den Nieuwendijk
,
Veronica Coco
Rohde
,
Elisa Colomina
Folch
,
Hans
Van Den Elst
,
Bogdan I.
Florea
,
Saskia
Scheij
,
Wilma E.
Donker-koopman
,
Marri
Verhoek
,
Nan
Li
,
Martin
Schürmann
,
Daniel
Mink
,
Rolf G.
Boot
,
Jeroen D. C.
Codée
,
Gijsbert A.
Van Der Marel
,
Gideon J.
Davies
,
Johannes M. F. G.
Aerts
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[7864, 9948]
Abstract: GH29 a-L-fucosidases catalyze the hydrolysis of a-L-fucosidic linkages. Deficiency in human lysosomal a-Lfucosidase (FUCA1) leads to the recessively inherited disorder, fucosidosis. Herein we describe the development of fucopyranose-configured cyclophellitol aziridines as activity-based probes (ABPs) for selective in vitro and in vivo labeling of GH29 a-L-fucosidases from bacteria, mice and man. Crystallographic analysis on bacterial a-L-fucosidase confirms that the ABPs act by covalent modification f the active site nucleophile. Competitive activity-based protein profiling identified L-fuconojirimycin as the single GH29 a-L-fucosidase inhibitor from eight configurational isomers.
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Jun 2015
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I03-Macromolecular Crystallography
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Sybrin P.
Schröder
,
Liang
Wu
,
Marta
Artola
,
Thomas
Hansen
,
Wendy A.
Offen
,
Maria J.
Ferraz
,
Kah-yee
Li
,
Johannes M. F. G.
Aerts
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587]
Abstract: Gluco-azoles competitively inhibit glucosidases by transition-state mimicry and their ability to interact with catalytic acid residues in glucosidase active sites. We noted that no azole-type inhibitors described, to date, possess a protic nitrogen characteristic for 1H-imidazoles. Here, we present gluco-1H-imidazole, a gluco-azole bearing a 1H-imidazole fused to a glucopyranose-configured cyclitol core, and three close analogues as new glucosidase inhibitors. All compounds inhibit human retaining β-glucosidase, GBA1, with the most potent ones inhibiting this enzyme (deficient in Gaucher disease) on a par with glucoimidazole. None inhibit glucosylceramide synthase, cytosolic β-glucosidase GBA2 or α-glucosidase GAA. Structural, physical and computational studies provide first insights into the binding mode of this conceptually new class of retaining β-glucosidase inhibitors.
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Mar 2018
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I03-Macromolecular Crystallography
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Daniël
Lahav
,
Bing
Liu
,
Richard J. B. H. N.
Van Den Berg
,
Adrianus M. C. H.
Van Den Nieuwendijk
,
Tom
Wennekes
,
Amar T.
Ghisaidoobe
,
Imogen
Breen
,
Maria J.
Ferraz
,
Chi-lin
Kuo
,
Liang
Wu
,
Paul P.
Geurink
,
Huib
Ovaa
,
Gijsbert A.
Van Der Marel
,
Mario
Van Der Stelt
,
Rolf G.
Boot
,
Gideon J.
Davies
,
Johannes M. F. G.
Aerts
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chemical tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining β-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell extracts containing recombinant, overexpressed glycosidase as the easily accessible enzyme source.
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Sep 2017
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I03-Macromolecular Crystallography
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Casper
De Boer
,
Nicholas G. S.
Mcgregor
,
Evert
Peterse
,
Sybrin P.
Schröder
,
Bogdan I.
Florea
,
Jianbing
Jiang
,
Jos
Reijngoud
,
Arthur F. J.
Ram
,
Gilles P.
Van Wezel
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Herman S.
Overkleeft
,
Gideon
Davies
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Cellulases and related β-1,4-glucanases are essential components of lignocellulose-degrading enzyme mixtures. The detection of β-1,4-glucanase activity typically relies on monitoring the breakdown of purified lignocellulose-derived substrates or synthetic chromogenic substrates, limiting the activities which can be detected and complicating the tracing of activity back to specific components within complex enzyme mixtures. As a tool for the rapid detection and identification of β-1,4-glucanases, a series of glycosylated cyclophellitol inhibitors mimicking β-1,4-glucan oligosaccharides have been synthesised. These compounds are highly efficient inhibitors of HiCel7B, a well-known GH7 endo-β-1,4-glucanase. An elaborated activity-based probe facilitated the direct detection and identification of β-1,4-glucanases within a complex fungal secretome without any detectable cross-reactivity with β-D-glucosidases. These probes and inhibitors add valuable new capacity to the growing toolbox of cyclophellitol-derived probes for the activity-based profiling of biomass-degrading enzymes.
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Jul 2020
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