Confirming therapeutic target of protopine using immobilized β₂ -adrenoceptor coupled with site-directed molecular...

Drug-protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized β₂ -adrenoceptor (β₂ -AR) by linkage of the receptor on macroporous silica gel surface through N,N'-carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site-directed molecular docking. Subsequent application of immobilized β₂ -AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount-dependent method. The association constants of protopine to β₂ -AR by the 2 methods were (1.00 ± 0.06) × 10⁵ M^-1 and (1.52 ± 0.14) × 10⁴ M^-1 . The numbers of binding sites were (1.23 ± 0.07) × 10^-7 M and (9.09 ± 0.06) × 10^-7 M, respectively. These results indicated that β₂ -AR is the specific target for therapeutic action of protopine in vivo. The target-drug binding occurred on Ser¹⁶⁹ in crystal structure of the receptor. Compared with frontal analysis, injection amount-dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high-throughput drug-receptor interaction analysis.