Gaucher disease (GD) is a lysosomal storage disorder caused by inherited deficiencies in beta-glucocerebrosidase (GBA). Current treatments require rapid disease diagnosis and a means of monitoring... Show moreGaucher disease (GD) is a lysosomal storage disorder caused by inherited deficiencies in beta-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, a bi-functional aziridine ABP was synthesized as a potentially 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. Show less
Chen, Y.; Armstrong, Z.; Artola, M.; Florea, B.I.; Kuo, C.L.; Boer, C. de; ... ; Overkleeft, H.S. 2021
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... Show moreAmylases 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. Show less
