I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
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.
|
Jan 2021
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
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.
|
Jul 2020
|
|
I03-Macromolecular Crystallography
|
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.
|
Jul 2020
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Sybrin P.
Schröder
,
Casper
De Boer
,
Nicholas G. S.
Mcgregor
,
Rhianna J.
Rowland
,
Olga
Moroz
,
Elena
Blagova
,
Jos
Reijngoud
,
Mark
Arentshorst
,
David
Osborn
,
Marc D.
Morant
,
Eric
Abbate
,
Mary A.
Stringer
,
Kristian B. R. M.
Krogh
,
Lluís
Raich
,
Carme
Rovira
,
Jean-Guy
Berrin
,
Gilles P.
Van Wezel
,
Arthur F. J.
Ram
,
Bogdan I.
Florea
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Keith S.
Wilson
,
Liang
Wu
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587]
Abstract: Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and endo-β-1,4-xylanases in the secretomes of Aspergillus niger, by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme–substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.
|
May 2019
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Marta
Artola
,
Chi-Lin
Kuo
,
Stephen
Mcmahon
,
Verena
Oehler
,
Thomas
Hansen
,
Martijn
Van der lienden
,
Xu
He
,
Hans
Van den elst
,
Bogdan I.
Florea
,
Allison R.
Kermode
,
Gijsbert A.
Van der marel
,
Tracey M.
Gloster
,
Jeroen D. C.
Codée
,
Herman S.
Overkleeft
,
Johannes M. F. G.
Aerts
Diamond Proposal Number(s):
[14980]
Open Access
Abstract: Cyclophellitol aziridines are potent irreversible inhibitors of retaining glycosidases and versatile intermediates in the synthesis of activity‐based glycosidase probes (ABPs). Direct 3‐amino‐2‐(trifluoromethyl)quinazolin‐4(3H)‐one‐mediated aziridination of l‐ido‐configured cyclohexene has enabled the synthesis of new covalent inhibitors and ABPs of α‐l‐iduronidase, deficiency of which underlies the lysosomal storage disorder mucopolysaccharidosis type I (MPS I). The iduronidase ABPs react covalently and irreversibly in an activity‐based manner with human recombinant α‐l‐iduronidase (rIDUA, Aldurazyme®). The structures of IDUA when complexed with the inhibitors in a non‐covalent transition state mimicking form and a covalent enzyme‐bound form provide insights into its conformational itinerary. Inhibitors 1–3 adopt a half‐chair conformation in solution (4H3 and 3H4), as predicted by DFT calculations, which is different from the conformation of the Michaelis complex observed by crystallographic studies. Consequently, 1–3 may need to overcome an energy barrier in order to switch from the 4H3 conformation to the transition state (2, 5B) binding conformation before reacting and adopting a covalent 5S1 conformation. rIDUA can be labeled with fluorescent Cy5 ABP 2, which allows monitoring of the delivery of therapeutic recombinant enzyme to lysosomes, as is intended in enzyme replacement therapy for the treatment of MPS I patients.
|
Nov 2018
|
|
I02-Macromolecular Crystallography
|
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.
|
Nov 2017
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
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.
|
Jun 2017
|
|
I04-Macromolecular Crystallography
|
Jianbing
Jiang
,
Chi-Lin
Kuo
,
Liang
Wu
,
Christian
Franke
,
Wouter W.
Kallemeijn
,
Bogdan I.
Florea
,
Eline
Van Meel
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Rolf G.
Boot
,
Gideon J.
Davies
,
Herman S.
Overkleeft
,
Johannes M. F. G.
Aerts
Diamond Proposal Number(s):
[9948]
Open Access
Abstract: The development of small molecule activity-based probes (ABPs) is an evolving and powerful area of chemistry. There is a major need for synthetically accessible and specific ABPs to advance our understanding of enzymes in health and disease. α-Glucosidases are involved in diverse physiological processes including carbohydrate assimilation in the gastrointestinal tract, glycoprotein processing in the endoplasmic reticulum (ER), and intralysosomal glycogen catabolism. Inherited deficiency of the lysosomal acid α-glucosidase (GAA) causes the lysosomal glycogen storage disorder, Pompe disease. Here, we design a synthetic route for fluorescent and biotin-modified ABPs for in vitro and in situ monitoring of α-glucosidases. We show, through mass spectrometry, gel electrophoresis, and X-ray crystallography, that α-glucopyranose configured cyclophellitol aziridines label distinct retaining α-glucosidases including GAA and ER α-glucosidase II, and that this labeling can be tuned by pH. We illustrate a direct diagnostic application in Pompe disease patient cells, and discuss how the probes may be further exploited for diverse applications.
|
May 2016
|
|
I03-Macromolecular Crystallography
|
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]
Open Access
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.
|
Jun 2015
|
|
