Structural basis of the inhibition of GH1 β-glucosidases by multivalent pyrrolidine iminosugars

The synthesis of multivalent pyrrolidine iminosugars via CuAAC click reaction between different pyrrolidineazide derivatives and tri- or hexavalent alkynyl scaffolds is reported. The new multimeric compounds, together with the monomeric reference, were evaluated as inhibitors against two homologous GH1 beta-glucosidases (BglA and BglB from Paenibacillus polymyxa). The multivalent inhibitors containing an aromatic moiety in the linker between the pyrrolidine and the scaffold inhibited the octameric BglA (mu M range) but did not show affinity against the monomeric BglB, despite the similarity between the active site of both enzymes. A modest multivalent effect (rp/n = 12) was detected for the hexavalent inhibitor 12. Structural analysis of the complexes between the monomeric and the trimeric iminosugar inhibitors (4 and 10) and BglA showed the insertion of the inhibitors at the active site of BglA, confirming a competitive mode of inhibition as indicated by enzyme kinetics. Additionally, structural comparison of the BglA/4 complex with the reported BglB/2F-glucose complex illustrates the key determinants responsible for the inhibitory effect and explains the reasons of the inhibition of BglA and the no inhibition of BglB. Potential inhibition of other beta-glucosidases with therapeutic relevance is discussed under the light of these observations.