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

Siastatin B is a potent and effective iminosugar inhibitor of three diverse glycosidase classes, namely, sialidases, ß-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 ß-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-ß-d-glucuronidases and the anticancer target human heparanase. This revealed submicromolar inhibition of exo-ß-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 broad-spectrum glycosidase inhibitor, identify a new class of glycosidase inhibitor, and suggest new functionalities that can be incorporated into future generations of glycosidase inhibitors.

Conformational and Electronic Variations in 1,2- and 1,5a-Cyclophellitols and their Impact on Retaining α-Glucosidase Inhibition.

Glycoside hydrolases (glycosidases) take part in myriad biological processes and are important therapeutic targets. Competitive and mechanism-based inhibitors are useful tools to dissect their biological role and comprise a good starting point for drug discovery. The natural product, cyclophellitol, a mechanism-based, covalent and irreversible retaining ß-glucosidase inhibitor has inspired the design of diverse α- and ß-glycosidase inhibitor and activity-based probe scaffolds. Here, we sought to deepen our understanding of the structural and functional requirements of cyclophellitol-type compounds for effective human α-glucosidase inhibition. We synthesized a comprehensive set of α-configured 1,2- and 1,5a-cyclophellitol analogues bearing a variety of electrophilic traps. The inhibitory potency of these compounds was assessed towards both lysosomal and ER retaining α-glucosidases. These studies revealed the 1,5a-cyclophellitols to be the most potent retaining α-glucosidase inhibitors, with the nature of the electrophile determining inhibitory mode of action (covalent or non-covalent). DFT calculations support the ability of the 1,5a-cyclophellitols, but not the 1,2-congeners, to adopt conformations that mimic either the Michaelis complex or transition state of α-glucosidases.