Topological effects and binding modes operating with multivalent iminosugar-based glycoclusters and mannosidases

Multivalent iminosugars have been recently explored for glycosidase inhibition. Affinity enhancements due to multivalency have been reported for specific targets, which are particularly appealing when a gain in enzyme selectivity is achieved but raise the question of the binding mode operating with this new class of inhibitors. Here we describe the development of a set of tetra- and octavalent iminosugar probes with specific topologies and an assessment of their binding affinities toward a panel of glycosidases including the Jack Bean alpha-mannosidase (JBaMan) and the biologically relevant class II alpha-mannosidases from Drosophila melanogaster belonging to glycohydrolase family 38, namely Golgi alpha-mannosidase ManIIb (GM) and lysosomal alpha-mannosidase LManII (LM). Very different inhibitory profiles were observed for compounds with identical valencies, indicating that the spatial distribution of the iminosugars is critical to fine-tune the enzymatic inhibitory activity. Compared to the monovalent reference, the best multivalent compound showed a dramatic 800-fold improvement in the inhibitory potency for JBaMan, which is outstanding for just a tetravalent ligand. The compound was also shown to increase both the inhibitory activity and the selectivity for GM over LM. This suggests that multivalency could be an alternative strategy in developing therapeutic GM inhibitors not affecting the lysosomal mannosidases. Dynamic light scattering experiments and atomic force microscopy performed with coincubated solutions of the compounds with JBaMan shed light on the multivalent binding mode. The multivalent compounds were shown to promote the formation of JBaMan aggregates with different sizes and shapes. The dimeric nature of the JBaMan allows such intermolecular cross-finking mechanisms to occur.