I04-Macromolecular Crystallography
|
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.
|
Sep 2024
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Tim P.
Ofman
,
Jurriaan J. A.
Heming
,
Alba
Nin-Hill
,
Florian
Küllmer
,
Elisha
Moran
,
Megan
Bennett
,
Roy
Steneker
,
Anne-Mei
Klein
,
Gijs
Ruijgrok
,
Ken
Kok
,
Zach W. B.
Armstrong
,
Johannes M. F. G.
Aerts
,
Gijsbert A.
Van Der Marel
,
Carme
Rovira
,
Gideon J.
Davies
,
Marta
Artola
,
Jeroen D. C.
Codée
,
Hermen S.
Overkleeft
Diamond Proposal Number(s):
[32736, 24948]
Open Access
Abstract: Glycoside hydrolases (glycosidases) take part in myriad biological processes and are important therapeutic targets. Competitive and mechanism-based inhibitors are useful tools to dissect their biological role and comprise a good starting point for drug discovery. The natural product, cyclophellitol, a mechanism-based, covalent and irreversible retaining β-glucosidase inhibitor has inspired the design of diverse α- and β-glycosidase inhibitor and activity-based probe scaffolds. Here, we sought to deepen our understanding of the structural and functional requirements of cyclophellitol-type compounds for effective human α-glucosidase inhibition. We synthesized a comprehensive set of α-configured 1,2- and 1,6-cyclophellitol analogues bearing a variety of electrophilic traps. The inhibitory potency of these compounds was assessed towards both lysosomal and ER retaining α-glucosidases. These studies revealed the 1,6-cyclophellitols to be the most potent retaining α-glucosidase inhibitors, with the nature of the electrophile determining inhibitory mode of action (covalent or non-covalent). DFT calculations support the ability of the 1,6-cyclophellitols, but not the 1,2-congeners, to adopt conformations that mimic either the Michaelis complex or transition state of α-glucosidases.
|
Apr 2024
|
|
I03-Macromolecular Crystallography
|
Alexandra
Males
,
Ken
Kok
,
Alba
Nin-Hill
,
Nicky
De Koster
,
Sija
Van Den Beukel
,
Thomas J. M.
Beenakker
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Johannes M. F. G.
Aerts
,
Herman S.
Overkleeft
,
Carme
Rovira
,
Gideon J.
Davies
,
Marta
Artola
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Class I inverting exo-acting α-1,2-mannosidases (CAZY family GH47) display an unusual catalytic itinerary featuring ring-flipped mannosides, 3S1 → 3H4‡ → 1C4. Conformationally locked 1C4 compounds, such as kifunensine, display nanomolar inhibition but large multigene GH47 mannosidase families render specific “isoform-dependent” inhibition impossible. Here we develop a bump-and-hole strategy in which a new mannose-configured 1,6-trans-cyclic sulfamidate inhibits α-D-mannosidases by virtue of its 1C4 conformation. This compound does not inhibit the wild-type GH47 model enzyme by virtue of a steric clash, a “bump”, in the active site. An L310S (a conserved residue amongst human GH47 enzymes) mutant of the model Caulobacter GH47 awoke 574 nM inhibition of the previously dormant inhibitor, confirmed by structural analysis of a 0.97 Å structure. Considering that L310 is a conserved residue amongst human GH47 enzymes, this work provides a unique framework for future biotechnological studies on N-glycan maturation and ER associated degradation by isoform-specific GH47 α-D-mannosidase inhibition through a bump-and-hole approach.
|
Nov 2023
|
|
I03-Macromolecular Crystallography
|
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.
|
Oct 2023
|
|
I03-Macromolecular Crystallography
|
Daniël
Van Der Gracht
,
Rhianna J.
Rowland
,
Véronique
Roig-Zamboni
,
Maria J.
Ferraz
,
Max
Louwerse
,
Paul P.
Geurink
,
Johannes M. F. G.
Aerts
,
Gerlind
Sulzenbacher
,
Gideon J.
Davies
,
Herman S.
Overkleeft
,
Marta
Artola
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Lysosomal exoglycosidases are responsible for processing endocytosed glycans from the non-reducing end to produce the corresponding monosaccharides. Genetic mutations in a particular lysosomal glycosidase may result in accumulation of its particular substrate, which may cause diverse lysosomal storage disorders. The identification of effective therapeutic modalities to treat these diseases is a major yet poorly realised objective in biomedicine. One common strategy comprises the identification of effective and selective competitive inhibitors that may serve to stabilize the proper folding of the mutated enzyme, either during maturation and trafficking to, or residence in, endo-lysosomal compartments. The discovery of such inhibitors is greatly aided by effective screening assays, the development of which is the focus of the here-presented work. We developed and applied fluorescent activity-based probes reporting on either human GH30 lysosomal glucosylceramidase (GBA1, a retaining β-glucosidase) or GH31 lysosomal retaining α-glucosidase (GAA). FluoPol-ABPP screening of our in-house 358-member iminosugar library yielded compound classes selective for either of these enzymes. In particular, we identified a class of N-alkyldeoxynojirimycins that inhibit GAA, but not GBA1, and that may form the starting point for the development of pharmacological chaperone therapeutics for the lysosomal glycogen storage disease that results from genetic deficiency in GAA: Pompe disease.
|
Aug 2023
|
|
I04-Macromolecular Crystallography
|
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.
|
Aug 2022
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
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.
|
Sep 2021
|
|
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]
Open Access
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
|
|
I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
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.
|
Aug 2019
|
|
