Glycosphingoid bases are elevated in inherited lysosomal storage disorders with deficient activity of glycosphingolipid catabolizing glycosidases. We investigated the molecular basis of the formation of glucosylsphingosine and globotriaosylsphingosine during deficiency of glucocerebrosidase (Gaucher disease) and α‐galactosidase A (Fabry disease). Independent genetic and pharmacological evidence is presented pointing to an active role of acid ceramidase in both processes through deacylation of lysosomal glycosphingolipids. The potential pathophysiological relevance of elevated glycosphingoid bases generated through this alternative metabolism in patients suffering from lysosomal glycosidase defects is discussed.
Human
nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that
controls levels of glycolipids and whose activity is linked to several
human disease states. There is a major need to design or discover
selective GBA2 inhibitors both as chemical tools and as potential
therapeutic agents. Here, we describe the development of a fluorescence
polarization activity-based protein profiling (FluoPol-ABPP) assay
for the rapid identification, from a 350+ library of iminosugars,
of GBA2 inhibitors. A focused library is generated based on leads
from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset
against the other glucosylceramide metabolizing enzymes, glucosylceramide
synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic
retaining β-glucosidase, GBA3. Our work, yielding potent and
selective GBA2 inhibitors, also provides a roadmap for the development
of high-throughput assays for identifying retaining glycosidase inhibitors
by FluoPol-ABPP on cell extracts containing recombinant, overexpressed
glycosidase as the easily accessible enzyme source.
Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAc-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 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-epicyclophellitol 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.
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