KinITC—One Method Supports both Thermodynamic and Kinetic SARs as Exemplified on FimH Antagonists

Abstract

Affinity data, such as dissociation constants (K ) or inhibitory concentrations (IC ), 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 k ) and (ii) a stabilization of the transition state or a destabilization of the unbound state (increased on-rate constant k ). Based on congeneric ligand modifications and structural input from co-crystal structures, a strong relationship between the formed hydrogen-bond network and k could be concluded, whereas electrostatic interactions and conformational restrictions upon binding were found to have mainly an impact on k .

Enhancing the enthalpic contribution of hydrogen bonds by solvent shielding

“…Data for the interactions FimH LD -2/3/4 and FimH FL -2/4 have been published before. 24,30,31 (C) Difference in the thermodynamic profiles between ligand binding to FimH FL and FimH LD . Error bars represent (propagated) 68% confidence intervals.…”

“…S5-S8, ESI †) for the interaction of deoxygenated n-heptyl a-D-mannoside derivatives 5-8 with FimH LD (A) and FimH FL (B). Data for the interactions of FimH LD with compounds 5-7 have been published elsewhere 30. (C) Difference in the thermodynamic profiles between ligand binding to FimH FL and FimH LD .…”

The Role of Fluorine in Glycomimetic Drug Design

“…Polar solvent molecules are much better at participating in the latter, and therefore, the C−F bonds in ligands are unable to compete with bulk solvent interactions [17b] . This suggests that fluorination will retain binding site interactions much better if they are of a time‐independent nature, and as they are less likely to be outcompeted by solvent, can improve drug–target residence times [28] . The concept of drug–target residence time, first proposed in 2006, [29] argues that the half‐life of the drug–target complex is a better determinant of biological activity than K d or IC 50 , as the latter are based on equilibrium scenarios, which are often never reached in vivo.…”

A Potent Mimetic of the Siglec‐8 Ligand 6’‐Sulfo‐Sialyl Lewisx

“…We determined binding kinetic data for the interactions of the disaccharide mimetics 2, 29, and 50 with Siglec-8 from ITC data using the kinITC technology. [54,55] This method analyses the equilibration time of each injection during a titration and fits this information to a kinetic model to derive rate constants. The binding kinetics of disaccharide 2 are characterized by a very short residence time of 1.2 s. For the mimetic 29, the residence time is increased by a factor of 5, probably mainly based on the reduced polarity.…”