I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Casper
De Boer
,
Zachary
Armstrong
,
Vincent A. J.
Lit
,
Uri
Barash
,
Gijs
Ruijgrok
,
Ilanit
Boyango
,
Merle M.
Weitzenberg
,
Sybrin P.
Schröder
,
Alexi J. C.
Sarris
,
Nico J.
Meeuwenoord
,
Pedro
Bule
,
Yasmine
Kayal
,
Neta
Ilan
,
Jeroen D. C.
Codée
,
Israel
Vlodavsky
,
Herman S.
Overkleeft
,
Gideon J.
Davies
,
Liang
Wu
Diamond Proposal Number(s):
[13587, 18598]
Open Access
Abstract: Heparan sulfate proteoglycans (HSPGs) mediate essential interactions throughout the extracellular matrix (ECM), providing signals that regulate cellular growth and development. Altered HSPG composition during tumorigenesis strongly aids cancer progression. Heparanase (HPSE) is the principal enzyme responsible for extracellular heparan sulfate catabolism and is markedly up-regulated in aggressive cancers. HPSE overactivity degrades HSPGs within the ECM, facilitating metastatic dissemination and releasing mitogens that drive cellular proliferation. Reducing extracellular HPSE activity reduces cancer growth, but few effective inhibitors are known, and none are clinically approved. Inspired by the natural glycosidase inhibitor cyclophellitol, we developed nanomolar mechanism-based, irreversible HPSE inhibitors that are effective within physiological environments. Application of cyclophellitol-derived HPSE inhibitors reduces cancer aggression in cellulo and significantly ameliorates murine metastasis. Mechanism-based irreversible HPSE inhibition is an unexplored anticancer strategy. We demonstrate the feasibility of such compounds to control pathological HPSE-driven malignancies.
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Aug 2022
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I04-Macromolecular Crystallography
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Ken
Kok
,
Chi-Lin
Kuo
,
Rebecca E.
Katzy
,
Lindsey T.
Lelieveld
,
Liang
Wu
,
Véronique
Roig-Zamboni
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Gerlind
Sulzenbacher
,
Gideon J.
Davies
,
Herman S.
Overkleeft
,
Johannes M. F. G.
Aerts
,
Marta
Artola
Open Access
Abstract: α-Glucosidase inhibitors are potential therapeutics for the treatment of diabetes, viral infections, and Pompe disease. Herein, we report a 1,6-epi-cyclophellitol cyclosulfamidate as a new class of reversible α-glucosidase inhibitors that displays enzyme inhibitory activity by virtue of its conformational mimicry of the substrate when bound in the Michaelis complex. The α-d-glc-configured cyclophellitol cyclosulfamidate 4 binds in a competitive manner the human lysosomal acid α-glucosidase (GAA), ER α-glucosidases, and, at higher concentrations, intestinal α-glucosidases, displaying an excellent selectivity over the human β-glucosidases GBA and GBA2 and glucosylceramide synthase (GCS). Cyclosulfamidate 4 stabilizes recombinant human GAA (rhGAA, alglucosidase alfa, Myozyme) in cell medium and plasma and facilitates enzyme trafficking to lysosomes. It stabilizes rhGAA more effectively than existing small-molecule chaperones and does so in vitro, in cellulo, and in vivo in zebrafish, thus representing a promising therapeutic alternative to Miglustat for Pompe disease.
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Aug 2022
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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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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]
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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I03-Macromolecular Crystallography
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Nicholas G. S.
Mcgregor
,
Joan
Coines
,
Valentina
Borlandelli
,
Satoko
Amaki
,
Marta
Artola
,
Alba
Nin‐hill
,
Daniël
Linzel
,
Chihaya
Yamada
,
Takatoshi
Arakawa
,
Akihiro
Ishiwata
,
Yukishige
Ito
,
Gijsbert A.
Marel
,
Jeroen D. C.
Codée
,
Shinya
Fushinobu
,
Herman S.
Overkleeft
,
Carme
Rovira
,
Gideon J.
Davies
Diamond Proposal Number(s):
[18598]
Abstract: The recent discovery of zinc‐dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)3(Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn‐coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C−S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol‐derived β‐l‐arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non‐hydrolysable adduct analogous to the mechanistic covalent intermediate. This β‐l‐arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X‐ray crystal structures determined for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chemistry of cyclophellitol derivatives, the structures and simulations presented here support the assignment of a zinc‐coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.
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Feb 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Yurong
Chen
,
Zachary
Armstrong
,
Marta
Artola
,
Bogdan I.
Florea
,
Chi-Lin
Kuo
,
Casper
De Boer
,
Mikkel S.
Rasmussen
,
Maher
Abou Hachem
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Johannes M. F. G.
Aerts
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[18598]
Abstract: Amylases are key enzymes in the processing of starch in many kingdoms of life. They are important catalysts in industrial biotechnology where they are applied in, among others, food processing and the production of detergents. In man amylases are the first enzymes in the digestion of starch to glucose and arguably also the preferred target in therapeutic strategies aimed at the treatment of type 2 diabetes patients through down-tuning glucose assimilation. Efficient and sensitive assays that report selectively on retaining amylase activities irrespective of the nature and complexity of the biomaterial studied are of great value both in finding new and effective human amylase inhibitors and in the discovery of new microbial amylases with potentially advantageous features for biotechnological application. Activity-based protein profiling (ABPP) of retaining glycosidases is inherently suited for the development of such an assay format. We here report on the design and synthesis of 1,6-epi-cyclophellitol-based pseudodisaccharides equipped with a suite of reporter entities and their use in ABPP of retaining amylases from human saliva, murine tissue as well as secretomes from fungi grown on starch. The activity and efficiency of the inhibitors and probes are substantiated by extensive biochemical analysis, and the selectivity for amylases over related retaining endoglycosidases is validated by structural studies.
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Jan 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Zachary
Armstrong
,
Chi-Lin
Kuo
,
Daniël
Lahav
,
Bing
Liu
,
Rachel
Johnson
,
Thomas J. M.
Beenakker
,
Casper
De Boer
,
Chung-Sing
Wong
,
Erwin R.
Van Rijssel
,
Marjoke F.
Debets
,
Bogdan I.
Florea
,
Colin
Hissink
,
Rolf G.
Boot
,
Paul P.
Geurink
,
Huib
Ovaa
,
Mario
Van Der Stelt
,
Gijsbert M.
Van Der Marel
,
Jeroen D. C.
Codée
,
Johannes M. F. G.
Aerts
,
Liang
Wu
,
Herman S.
Overkleeft
,
Gideon
Davies
Diamond Proposal Number(s):
[18598]
Abstract: Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcMan5GlcNAc2 to produce GlcNAcMan3GlcNAc2, the precursor for all complex N-glycans, including the branched N-glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitol aziridine derivatives enabled activity-based protein profiling of α-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.
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Jul 2020
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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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I04-Macromolecular Crystallography
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Nicholas G. S.
Mcgregor
,
Marta
Artola
,
Alba
Nin-Hill
,
Daniel
Linzel
,
Mireille
Haon
,
Jos
Reijngoud
,
Arthur F. J.
Ram
,
Marie-Noelle
Rosso
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Gilles P.
Van Wezel
,
Jean-Guy
Berrin
,
Carme
Rovira
,
Herman S.
Overkleeft
,
Gideon J.
Davies
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-L-arabinofuranosidases among a wide variety of co-expressed carbohydrate-active enzymes. To selectively detect and identify α-L-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-L-arabinofuranosides were sought. The conformational free energy landscapes of free α-L-arabinofuranose and several rationally designed covalent α-L-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-L-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-L-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-L-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-L-arabinofuranosidases.
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Feb 2020
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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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