On the meaning of affinity: Cluster glycoside effects and concanavalin A

The inhibition of protein-carbohydrate interaction provides a powerful ther apeutic strategy for the treatment of myriad human diseases. To date, appli cation of such approaches have been frustrated by the inherent low affinity of carbohydrate ligands for their protein receptors. Because lectins typic ally exist in multimeric assemblies, a variety of polyvalent saccharide lig ands have been prepared in the search for high affinity. The cluster glycos ide effect, or the observation of high affinity derived from multivalency i n oligosaccharide Ligands, apparently represents the best strategy for over coming the "weak binding" problem. Here we report the synthesis of a series of multivalent dendritic saccharides and a biophysical evaluation of their interaction with the plant lectin concanavalin A. Although a 30-fold enhan cement in affinity on a valence-corrected basis is observed by agglutinatio n assay, calorimetric titration of soluble protein with a range of multival ent ligands reveals no enhancement in binding free energies. Rather, IC50 v alues from agglutination measurements correlate well with entropies of bind ing. This observation suggests that hemagglutination measures a phenomenon distinct from binding that is typified by a large favorable entropy and an unfavorable enthalpy: this process is almost certainly aggregation. Support ing this assertion, we report crystal structures of multivalent ligands cro ss-linking concanavalin A dimers. To the best of our knowledge, these struc tures are the first reported of their kind. Our results indicate that hemag glutination assays evaluate the ability of ligands to inhibit the formation of cross-linked lattices, a process only tangentially related to reversibl e ligand binding. Cluster glycoside effects observed in agglutination assay s must, therefore, be viewed with caution. Such effects may or may not be r elevant to the design of therapeutically useful saccharides.