Molecular Basis for Inhibition of Heparanases and β-Glucuronidases by Siastatin B

Abstract

Siastatin B is a potent and effective iminosugar inhibitor of three diverse glycosidase classes, namely, sialidases, β-D-glucuronidases, and N-acetyl-glucosaminidases. The mode of inhibition of glucuronidases, in contrast to sialidases, has long been enigmatic as siastatin B appears too bulky and incorrectly substituted to be accommodated within a β-D-glucuronidase active site pocket. Herein, we show through crystallographic analysis of protein-inhibitor complexes that siastatin B generates both a hemiaminal and a 3-geminal diol iminosugar (3-GDI) that are, rather than the parent compound, directly responsible for enzyme inhibition. The hemiaminal product is the first observation of a natural product that belongs to the noeuromycin class of inhibitors. Additionally, the 3-GDI represents a new and potent class of the iminosugar glycosidase inhibitor. To substantiate our findings, we synthesized both the gluco-and galacto-configured 3-GDIs and characterized their binding both structurally and kinetically to exo-β-D-glucuronidases and the anticancer target human heparanase. This revealed submicromolar inhibition of exo-β-D-glucuronidases and an unprecedented binding mode by this new class of inhibitor. Our results reveal the mechanism by which siastatin B acts as a broadspectrum glycosidase inhibitor, identify a new class of glycosidase inhibitor, and suggest new functionalities that can be incorporated into future generations of glycosidase inhibitors.

The Uptake of Heparanase into Mast Cells Is Regulated by Its Enzymatic Activity to Degrade Heparan Sulfate

“…Heparastatin (SF4), a compound optimized for inhibition of Hpse enzymatic activity, is a derivative of a bacterial product siastatin B that can inhibit the enzymatic activity of Hpse. The complex structure of human Hpse and siastatin B was recently demonstrated using 3D crystallography [ 55 ]. That study identified amino acid residues that are involved in the interaction of Hpse with siastatin B.…”