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Chemical biology of sphingolipids: fundamental studies and clinical applications

Final Report Summary - CHEMBIOSPHING (Chemical biology of sphingolipids: fundamental studies and clinical applications)

The ChemBioSphing program has delivered a wealth of tools and reagents for monitoring glycosidase activity – both the enzymes themselves and their metabolites – in their natural environment: the cell. Such tools are indispensable for understanding the impact of glycosidase malfunctioning on human disorders, including the inherited disorders that are characterised by mutations in lysosomal glycosidases, Gaucher disease and Fabry disease. Understanding these diseases in molecular detail has been the driving force behind the ChemBioSphing proposal and execution of the ChemBioSphing research has yielded activity-based probes highy specific for each of the glycosidases involved in these diseases: lysosomal glucosylceramidase (GBA, Gaucher) and lysosomal alpha-galactosidase (GAA, Fabry). These probes, that can be applied in living cells and animal models and thta with high accuracy and sensitivity mark their target enzymes (and in case of disease states, the relative absence of these activities), are accompanied by neutron-encoded sphingolipids. With the latter, (relative) lack of enzyme activity can be directly correlated to altered lipid pools, with as remarkable charateristic in both diseases the emergence, or drastic increase, of lysosphingolipids not (or hardly) encountered in healthy individuals. The glycosidase probes themselves are not just research tools but can actually be applied in a diagnostic setting, and one of the major (though yet unpublished) outcomes of the ChemBioSphing program is our development of a diagnostic assay in which urine from Gaucher or Fabry patients is collected, treated with the probes and the protein content separated om SDS PAGE. Disease states are directly correlated to enzyme activity, or lack thereof (in comparison with urine from healthy individuals) in an assay that should be easily translated to the clinic.
While the focus of the ChemBioSphing program has been on the glycosidases underlying Gaucher and Fabry disease, the program has in fact yielded a general probe design by means of which retaining exoglycosidases in general can be targeted. Published probes include those targeting alpha-fucosidases, beta-galactosidases, beta-glucuronidases and alpha-glucosidases, and unpublished probes that work equally well have been developed that modify alpha-mannosidases, beta-mannosidases and alpha-iduronidases. Each of these glycosidases are of fundamental and applied biological and biomedical relevance in their own right: they are genetically deficient in human lysosomal storage disorders and/or are relevant clinical targets for diseases ranging from diabetes to cancer. Thus the major conceptual breakthrough of the ChemBioSphing program has been the development of activity-based glycosidase probes.