Precipitation-free high-affinity multivalent binding by inline lectin ligands.

Multivalent ligand-protein interactions are a key concept in biology mediating, for example, signalling and adhesion. Multivalent ligands often have tremendously increased binding affinities. However, they also can cause crosslinking of receptor molecules leading to precipitation of ligand-receptor complexes. Plaque formation due to precipitation is a known characteristic of numerous fatal diseases limiting a potential medical application of multivalent ligands with a precipitating binding mode. Here, we present a new design of high-potency multivalent ligands featuring an inline arrangement of ligand epitopes with exceptionally high binding affinities in the low nanomolar range. At the same time, we show with a multi-methodological approach that precipitation of the receptor is prevented. We distinguish distinct binding modes of the ligands, in particular we elucidate a unique chelating binding mode, where four receptor binding sites are simultaneously bridged by one multivalent ligand molecule. The new design concept of inline multivalent ligands, which we established for the well-investigated model lectin wheat germ agglutinin, has great potential for the development of high-potency multivalent inhibitors as future therapeutics.

Mechanistic Insight into Nanomolar Binding of Multivalent Neoglycopeptides to Wheat Germ Agglutinin.

Multivalent carbohydrate-protein interactions are frequently involved in essential biological recognition processes. Accordingly, multivalency is often also exploited for the design of high-affinity lectin ligands aimed at the inhibition of such processes. In a previous study (D. Schwefel et al., J. Am. Chem. Soc. 2010, 132, 8704-8719) we identified a tetravalent cyclopeptide-based ligand with nanomolar affinity to the model lectin wheat germ agglutinin (WGA). To unravel the structural features of this ligand required for high-affinity binding to WGA, we synthesized a series of cyclic and linear neoglycopeptides that differ in their conformational freedom as well as the number of GlcNAc residues. Combined evidence from isothermal titration calorimetry (ITC), enzyme-linked lectin assays (ELLA), and dynamic light scattering (DLS) revealed different binding modes of tetra- and divalent ligands and that conformational preorganization of the ligands by cyclization is not a prerequisite for achieving high binding affinities. The high affinities of the tetravalent ligands rather stem from their ability to form crosslinks between several WGA molecules. The results illustrate that binding affinities and mechanisms are strongly dependent on the used multivalent system which offers opportunities to tune and control binding processes.