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Confirming therapeutic target of protopine using immobilized β2‐adrenoceptor coupled with site‐directed molecular docking and the target‐drug interaction by frontal analysis and injection amount–dependent method

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) × 105M−1 and (1.52 ± 0.14) × 104M−1. The numbers of binding sites were (1.23 ± 0.07) × 10−7M and (9.09 ± 0.06) × 10−7M, 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.