Competitive and Covalent Inhibitors of Human Lysosomal Retaining Exoglucosidases


Deficiency in human acid glucosylceramidase (GBA1, a retaining β‐glucosidase) causes the lysosomal sphingolipid storage disorder Gaucher disease, whereas deficiency in human acid α‐glucosidase (GAA, a retaining α‐glucosidase) triggers the lysosomal glycogen storage disorder Pompe disease. Both enzymes process their substrate following a two‐step double‐displacement mechanism involving a covalent enzyme–substrate intermediate. Structural analysis of glycosidases complexed to substrates and inhibitors has provided insight into the reaction coordinates followed by glycosidases during catalytic hydrolysis and has assisted in the design of potent and selective inhibitors. Competitive and covalent inhibitors of both GBA1 and GAA have been developed in the past decades, for fundamental studies, as diagnostics tools, leads for drug development and therapeutic drugs for the clinical treatment of lysosomal storage diseases.

Key Concepts

  • The structural and functional diversity of glycoconjugates is reflected by the vast number of glycoprocessing enzymes encountered in all domains of life.
  • Retaining glycosidases form a covalent intermediate during substrate processing, whereas inverting glycosidases do not.
  • Competitive and covalent retaining glycosidase inhibitors exist, whereas inverting glycosidases can normally only be blocked by competitive inhibitors.
  • Both covalent and competitive glycosidase inhibitors are found in nature and serve as inspiration for the design of synthetic inhibitors.
  • Structural analysis of glycosidases complexed to substrates and inhibitors provides insight into the reaction coordinates followed by glycosidases during the catalytic cycle.
  • Conformational analysis of the reaction coordinate of glycosidases is key in the design of potent and selective inhibitors.
  • Glycosidase inhibitors may serve as leads for drug development.
  • Activity‐based glycosidase probes can be applied in the discovery of glycosidase activities.
  • Activity‐based probes are useful starting points for the development of diagnostics assays in disease areas in which glycosidase activities are involved.
  • Genetic deficiency in glycosidases is the basis of numerous inherited disorders including Gaucher disease and Pompe disease.

Keywords: glycosidase; inhibitor; inherited disease; lysosomal storage disorder; enzyme; mechanism; suicide substrate; activity‐based protein profiling; glycobiology; chemical biology

Figure 1. (a) Retaining and inverting glycosidase mechanisms. Retaining β‐glucosidases (left) produce β‐glucopyranose from β‐glucosides in a two‐step double displacement mechanism. Inverting β‐glucosidases (right) produce α‐glucopyranose from β‐glucosides in a single step. (b) Conformational itineraries of retaining β‐glucosidase (left) and retaining α‐glucosidase (right). Only the first half of the two‐step mechanisms are shown.
Figure 2. Substrates (a), inhibitors (b) and activity‐based probes (c) of human lysosomal glucosylceramidase (GBA1, left) and human lysosomal α‐glucosidase (GAA, right).
Figure 3. Crystal structures of GBA1, GAA and related enzymes. (a) Three‐dimensional structure of human GBA1, showing the overall fold architecture of the enzyme. A molecule of 2 (green) can be observed bound to the enzyme. PDB: 2V3D; (b) three‐dimensional structure of human GAA, showing the fold architecture of the enzyme. A molecule of glucose (green) can be observed bound to the enzyme. PDB: 5KZW; (c) active site of GBA1 in complex with 2. Direct interactions between the ligand and protein residues are shown and side‐chain numbers are annotated (nuc., nucleophile; a./b., acid/base). PDB: 2V3D; (d) active site of GBA1 in complex with 4. PDB: 3GXF; (e) active site of GBA1 in covalent complex with 7. PDB: 2VT0; (f) active site of the GAA homologue α‐GluII from mouse, in complex with 2. Sidechain numbers are annotated. PDB: 5IEF; (g) active site of the GAA homologue CjAgd31B from Cellvibrio japonicus in covalent complex with 12. Sidechain numbers are annotated. PDB: 5I24; (h) active site of CjAgd31B in covalent complex with 9. PDB: 5NPB.
Figure 4. Reaction itineraries of cyclophellitol aziridine and cyclosulfate inhibitors as revealed by X‐ray crystallography. For clarity, only nucleophile (nuc.) and acid base (a./b.) residues have been shown in these figures. (a) Reaction itinerary of β‐glucosidase configured cyclophellitol probes with the β‐glucosidase TmGH1. (left) The sugar analogue of the nonhydrolysable carba‐cyclophellitol 10 sits in a 4H3 conformation above the TmGH1 nucleophile, in a position primed for nucleophilic attack. (right) Reaction of TmGH1 with 5 produces a covalent adduct bound to the enzyme nucleophile in a 4C1 conformation. PDB: 5N6S and 2JAL; (b) reaction itinerary of α‐glucosidase configured epi‐cyclophellitol aziridine 12 with the α‐glucosidase CjAgd31B. (left) 12 in complex with an inactive CjAgd31B D412N nucleophile mutant sits in a 4H3 conformation within the enzyme active site. (right) Reaction of wild type CjAgd31B with 12 gives a covalent adduct in a 1S3 conformation. PDB: 5NPD and 5I24; (c) reaction itinerary of cyclophellitol cyclosulfate 8 with the β‐glucosidase TxGH116 from Thermoanaerobacterium xylanolyticum. The 4C1 conformation of this probe does not match the typical β‐glucosidase Michaelis complex conformation, hence 8 reacts slowly with β‐glucosidases. (left) Unreacted 8 in complex with wild type TxGH116 is observed after short crystal soaking times. (right) Extended soaking times are required to observe TxGH116 reacted with 8, which forms a covalent adduct in a 4C1 conformation with the enzyme nucleophile. PDB: 5O0S and 5NPF; (d) reaction itinerary of epi‐cyclophellitol cyclosulfate 9 with the α‐glucosidase CjAgd31B. The 4C1 conformation of this probe matches the typical α‐glucosidase Michaelis complex conformation, and reacts rapidly. (left) Unreacted 9 in complex with an inactive CjAgd31B D412N nucleophile mutant. (right) Reacted 9 in complex with wild type CjAgd31B forms a covalent adduct on the nucleophile in a 1S3 conformation. PDB: 5NPC and 5NPB.
Figure 5. Activity‐based protein profiling of β‐ and α‐glucosidases in healthy and diseased (Gaucher and Pompe, respectively) individuals. (a) In vivo labelling of endogenous retaining β‐exoglucosidases in kidney tissue homogenates of mice using broad spectrum ABP 13 (which labels GBA1, GBA2 and GBA3) and specific GBA1 ABP 14. (b) Detection of GBA1 in Gaucher fibroblasts by labelling wild‐type and homozygous N370S, L444P and RecNCI collodion fibroblast with 10 nM of ABP 14 for 60 min. GBA1 was visualised by in‐gel fluorescence scanning (top panel) and by western blotting with 8E4 antibody (bottom panel). (c) In vitro GAA labelling at pH 4.0 with ABP 15 visualised by in‐gel fluorescence scanning (top panel), followed by Western blot detection (bottom panel) of GAA in various fibroblast lysates, containing wild‐type (Ctrls) or mutant (Pompe) GAA.


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Further Reading

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Breen, Imogen Z, Artola, Marta, Wu, Liang, Beenakker, Thomas JM, Offen, Wendy A, Aerts, Johannes MFG, Davies, Gideon J, and Overkleeft, Herman S(Jan 2018) Competitive and Covalent Inhibitors of Human Lysosomal Retaining Exoglucosidases. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0027591]