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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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]
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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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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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
I04-Macromolecular Crystallography
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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.
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Nov 2018
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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]
Open Access
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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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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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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