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Glucocerebrosidase (GBA), a lysosomal retaining β‐d‐glucosidase, has recently been shown to hydrolyze β‐d‐xylosides and to transxylosylate cholesterol. Genetic defects in GBA cause the lysosomal storage disorder Gaucher disease (GD), and also constitute a risk factor for developing Parkinson's disease. GBA and other retaining glycosidases can be selectively visualized by activity‐based protein profiling (ABPP) using fluorescent probes composed of a cyclophellitol scaffold having a configuration tailored to the targeted glycosidase family. GBA processes β‐d‐xylosides in addition to β‐d‐glucosides, this in contrast to the other two mammalian cellular retaining β‐d‐glucosidases, GBA2 and GBA3. Here we show that the xylopyranose preference also holds up for covalent inhibitors: xylose‐configured cyclophellitol and cyclophellitol aziridines selectively react with GBA over GBA2 and GBA3 in vitro and in vivo, and that the xylose‐configured cyclophellitol is more potent and more selective for GBA than the classical GBA inhibitor, conduritol B‐epoxide (CBE). Both xylose‐configured cyclophellitol and cyclophellitol aziridine cause accumulation of glucosylsphingosine in zebrafish embryo, a characteristic hallmark of GD, and we conclude that these compounds are well suited for creating such chemically induced GD models., New selective GBA inhibitor: We show that the β‐d‐xylose configured epoxide and aziridine are potent selective inhibitors towards GBA in vitro and in vivo, and their selectivity and inhibitory effect towards GBA are superior to the widely used GBA inhibitor Conduritol B‐epoxide (CBE).

Glycosphingolipids are important building blocks of the outer leaflet of the cell membrane. They are continuously recycled, involving fragmentation inside lysosomes by glycosidases. Inherited defects in degradation cause lysosomal glycosphingolipid storage disorders. The relatively common glycosphingolipidosis Gaucher disease is highlighted here to discuss new insights in the molecular basis and pathophysiology of glycosphingolipidoses reached by fundamental research increasingly using chemical biology tools. We discuss improvements in the detection of glycosphingolipid metabolites by mass spectrometry and review new developments in laboratory diagnosis and disease monitoring as well as therapeutic interventions.

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.

Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GIcNAc-Man(5)GlcNAc(2) to produce GlcNAcMan(3) GlcNAc(2), 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 alpha-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 alpha-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.

We thank The Netherlands Organization for Scientific Research (NWO-CW, ChemThem grant to J.M.F.G.A. and H.S.O.), the European Research Council (ERC-2011-AdG290836 “Chembiosphing”, to H.S.O.) and Sanofi Genzyme (research grant to J.M.F.G.A. and H.S.O. and postdoctoral contract to M.A.). We acknowledge ChemAxon for kindly providing the Instant JChem software to manage our compound library. T.M.G., V.O. and S.A.M. are supported by a Wellcome Trust Research Career Development Fellowship and Institutional Strategic Support Funding. 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 ʟ‐ido‐configured cyclohexene has enabled the synthesis of new covalent inhibitors and ABPs of α‐ʟ‐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‐ʟ‐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. Publisher PDF

The cellular recycling of glycosphingolipids (GSLs) is mediated by specific lysosomal glycosidases. Inherited deficiencies in these enzymes cause lysosomal storage disorders. Some of the common disorders are Gaucher disease (GD) and Fabry disease (FD) resulting from the defects in lysosomal glucocerebrosidase (GBA) degrading glucosylceramide and α‐galactosidase A (GLA) degrading globotriaosylceramide. Here, GSL accumulation in tissues slows down with age despite ongoing lysosomal turnover of endogenous and endocytosed GSLs. Biochemical adaptations might explain this phenomenon. One crucial adaptation is the deacylation of accumulating GSLs in lysosomes by acid ceramidase. The soluble bases glucosylsphingosine in GD and globotriaosylsphingosine in FD are capable of leaving lysosomes and cells. In the case of GD, a further adaptation involves the cytosol‐faced enzyme GBA2. This enzyme allows extra‐lysosomal degradation of GlcCer while possibly generating glucosylated cholesterol. The beneficial and harmful effects of these adaptations are discussed. Key concepts: Glycosphingolipids (GSLs) are membrane constituents composed of a ceramide with one or more sugars. The simplest GSL is glucosylceramide (GlcCer). Ongoing recycling of GSLs in cells includes lysosomal degradation by the sequential action of glycosidases and acid ceramidase. Deficiency of lysosomal glycosidase leads to lysosomal storage diseases caused by accumulation of the corresponding substrate in lysosomes. The most common glycosphingolipidoses are Gaucher disease (GD) and Fabry disease (FD). GD is an autosomal recessive disorder caused by deficient activity of the lysosomal enzyme acid β‐glucosidase (glucocerebrosidase; GBA) resulting in lysosomal accumulation of GlcCer. FD is an X‐linked disorder caused by deficient activity of the lysosomal enzyme α‐galactosidase A (GLA) resulting in lysosomal accumulation of globotriaosylceramide (Gb3). Accumulation of storage lipids during GBA and GLA tends to slow down with age, likely partly due to poorly appreciated biochemical adaptations. Active conversion of accumulating GlcCer in lysosomes of GBA‐deficient cells is mediated by acid ceramidase, resulting in the formation of water‐soluble glucosylsphingosine (GlcSph). Likewise, globotriaosylsphingosine (lysoGb3) is formed from accumulating in lysosomes of GLA‐deficient cells. Elevated plasma GlcSph and lysoGb3 levels can be sensitively measured LC–MS and may assist in diagnosing and monitoring of the disease and response to treatment in GD and FD patients, respectively. Increased GlcSph level in GD patients acts as an autoantigen, causing ongoing B‐cell proliferation, leading to multiple myeloma. Increased lysoGb3 level in FD patients is thought to cause damage to nociceptive neurons and podocytes, thus contributing to pain and renal failure. In GD, the cytosol‐faced enzyme β‐glucosidase GBA2 allows degradation of GlcCer outside lysosomes. Through transglycosylation, GBA2 may generate glucosylcholesterol and ceramide from GlcCer and cholesterol. The toxic effects of secondary metabolites such as glycosphingoid bases (GlcSph in GD and lysoGb3 in FD) and glucosylated metabolites (GlcChol in GD) warrant further investigations. info:eu-repo/semantics/publishedVersion

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., 1,6-epi-Cyclophellitol cyclosulfate (“α-cyclosulfate”) is a conceptually new, potent and selective irreversible α-glucosidase inhibitor that acts through mimicry of the α-glucosidase Michaelis complex 4C1 chair conformation.

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., α-Glucosidases control food digestion, protein folding, and glycogen turnover. Designer probes report on tissue- and pH-specific α-glucosidase activity and reveal loss of α-glucosidase activity in Pompe disease patient material.

The title linker is stable under various biological conditions and can be cleaved chemoselectively with hydrazine. Its use is demonstrated in the activity-based enrichment and identification of proteasome active subunits from cell extracts.