Binding kinetics of five drugs to beta2-adrenoceptor using peak profiling method and nonlinear chromatography

Investigations of drug-protein interactions have advanced our knowledge of ways to design more rational drugs. In addition to extensive thermodynamic studies, ongoing works are needed to enhance the exploration of drug-protein binding kinetics. In this work, the beta2-adrenoceptor (β₂-AR) was immobilized on N, N'-carbonyldiimidazole activated amino polystyrene microspheres to prepare an affinity column (4.6 mm × 5.0 cm, 8 μm). The β₂-AR column was utilized to determine the binding kinetics of five drugs to the receptor. Introducing peak profiling method into this receptor chromatographic analysis, we determined the dissociation rate constants (k_d) of salbutamol, terbutaline, methoxyphenamine, isoprenaline hydrochloride and ephedrine hydrochloride to β₂-AR to be 15 (±1), 22 (±1), 3.3 (±0.2), 2.3 (±0.2) and 2.1 (±0.1) s^-1, respectively. The employment of nonlinear chromatography (NLC) in this case exhibited the same rank order of k_d values for the five drugs bound to β₂-AR. We confirmed that both the peak profiling method and NLC were capable of routine measurement of receptor-drug binding kinetics. Compared with the peak profiling method, NLC was advantageous in the simultaneous assessment of the kinetic and apparent thermodynamic parameters. It will become a powerful method for high throughput drug-receptor interaction analysis.