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Carbohydrate‐binding proteins are generally characterized by poor affinities for their natural glycan ligands, predominantly due to the shallow and solvent‐exposed binding sites. To overcome this drawback, nature has exploited multivalency to strengthen the binding by establishing multiple interactions simultaneously. The development of oligovalent structures frequently proved to be successful, not only for proteins with multiple binding sites, but also for proteins that possess a single recognition domain. Herein we present the syntheses of a number of oligovalent ligands for Siglec‐8, a monomeric I‐type lectin found on eosinophils and mast cells, alongside the thermodynamic characterization of their binding. While the enthalpic contribution of each binding epitope was within a narrow range to that of the monomeric ligand, the entropy penalty increased steadily with growing valency. Additionally, we observed a successful agonistic binding of the tetra‐ and hexavalent and, to an even larger extent, multivalent ligands to Siglec‐8 on immune cells and modulation of immune cell activation. Thus, triggering a biological effect is not restricted to multivalent ligands but could be induced by low oligovalent ligands as well, whereas a monovalent ligand, despite binding with similar affinity, showed an antagonistic effect. [ABSTRACT FROM AUTHOR]

Abstract: Affinity data, such as dissociation constants (KD) or inhibitory concentrations (IC50), are widely used in drug discovery. However, these parameters describe an equilibrium state, which is often not established in vivo due to pharmacokinetic effects and they are therefore not necessarily sufficient for evaluating drug efficacy. More accurate indicators for pharmacological activity are the kinetics of binding processes, as they shed light on the rate of formation of protein–ligand complexes and their half‐life. Nonetheless, although highly desirable for medicinal chemistry programs, studies on structure–kinetic relationships (SKR) are still rare. With the recently introduced analytical tool kinITC this situation may change, since not only thermodynamic but also kinetic information of the binding process can be deduced from isothermal titration calorimetry (ITC) experiments. Using kinITC, ITC data of 29 mannosides binding to the bacterial adhesin FimH were re‐analyzed to make their binding kinetics accessible. To validate these kinetic data, surface plasmon resonance (SPR) experiments were conducted. The kinetic analysis by kinITC revealed that the nanomolar affinities of the FimH antagonists arise from both (i) an optimized interaction between protein and ligand in the bound state (reduced off‐rate constant koff) and (ii) a stabilization of the transition state or a destabilization of the unbound state (increased on‐rate constant kon). Based on congeneric ligand modifications and structural input from co‐crystal structures, a strong relationship between the formed hydrogen‐bond network and koff could be concluded, whereas electrostatic interactions and conformational restrictions upon binding were found to have mainly an impact on kon. [ABSTRACT FROM AUTHOR]