Determination of kinetic and equilibrium parameters of three phenolic acids with CaSR using multiple affinity...

In our previous study, we found that three phenolic acids, including gallic acid, caffeic acid and paeonol, can act as the novel ligands towards human calcium sensing receptor (CaSR). To further develop drug candidates with ideal properties in the early discovery stage, we explored the binding characteristics of the three ones using multiple affinity chromatographic approaches and further verified them by Isothermal Titration Calorimetry. Compared with the equilibrium constant alone obtained by the injection amount-dependent method and the kinetic rate constant alone measured by the peak-decay method, nonlinear chromatography can simultaneously the determine association equilibrium constants of 2.13 × 10⁵, 1.87 × 10⁵, and 0.27 × 10⁵ M^-1 and dissociation rate constants of 2.16, 2.26, and 17.6 s^-1 for gallic acid, caffeic acid, and paeonol, respectively. Our findings indicated that nonlinear chromatography can effectively evaluate ligand-protein interaction in the early stage of drug discovery, and gallic acid, with the strongest binding strength and the slowest dissociation kinetics, represented a promising candidate in regard to the rationalization of drug pharmacokinetics.