The enormous growth in drug discovery paradigm has necessitated continuous exploration of new methods for drug-protein interaction analysis. To enhance the role of these methodologies in designing rational drugs, this work extended an immobilized angiotensin II type I receptor (AT1R) based affinity chromatography in antihypertensive compound identification. We fused haloalkane dehalogenase at C-terminus of AT1R and expressed the fusion receptor in E. coli. The expressed receptor was covalently immobilized onto 8.0 μm microspheres by mixing the cell lysate with 6-chlorocaproic acid-modified amino polystyrene microspheres. The immobilized AT1R was utilized for thermodynamic and kinetic interaction analysis between the receptor and four specific ligands. Following confirmation of these interactions by molecular docking, we identified puerarin and rosmarinic acid by determining their binding to the receptor. Azilsartan, candesartan, valsartan and olmesartan displayed two kinds of binding sites to AT1R by injection amount-dependent method. By molecular docking, we recognize the driving forces of the interaction as electrostatic interaction, hydrogen bonds and van der Waals force. The dissociation rate constants (kd) of azilsartan, candesartan, valsartan and olmesartan to AT1R were 0.01138 ± 0.003, 0.05142 ± 0.003, 0.07547 ± 0.004 and 0.01310 ± 0.005 min-1 by peak profiling assay. Comparing with these parameters, puerarin and rosmarinic acid presented lower affinity (KA: 0.12 × 104 and 1.5 × 104/M) and slower kinetics (kd: 0.6864 ± 0.03 and 0.3005 ± 0.01 min-1) to the receptor. These results, taking together, indicated that the immobilized AT1R has the capacity to probe antihypertensive compounds.

中文翻译：

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固定的I型血管紧张素II受体：通过结合相互作用分析对高通量化合物进行鉴定的高通量筛选方法。

药物发现范式的迅猛发展，需要不断探索药物-蛋白质相互作用分析的新方法。为了增强这些方法在设计合理药物中的作用，这项工作扩展了基于固定化血管紧张素II型I受体（AT1R）的亲和色谱法在降压化合物鉴定中的作用。我们在AT1R的C末端融合了卤代烷脱卤酶，并在大肠杆菌中表达了融合受体。通过将细胞裂解液与6-氯己酸改性的氨基聚苯乙烯微球混合，将表达的受体共价固定在8.0μm微球上。固定的AT1R用于受体和四个特定配体之间的热力学和动力学相互作用分析。通过分子对接确认这些相互作用后，我们通过确定它们与受体的结合来鉴定葛根素和迷迭香酸。通过注射量依赖性方法，阿齐沙坦，坎地沙坦，缬沙坦和奥美沙坦显示出与AT1R的两种结合位点。通过分子对接，我们将相互作用的驱动力识别为静电相互作用，氢键和范德华力。通过峰分布分析，阿齐沙坦，坎地沙坦，缬沙坦和奥美沙坦对AT1R的解离速率常数（kd）为0.01138±0.003、0.05142±0.003、0.07547±0.004和0.01310±0.005min-1。与这些参数相比，葛根素和迷迭香酸对受体的亲和力较低（KA：0.12×104和1.5×104 / M），动力学较慢（kd：0.6864±0.03和0.3005±0.01 min-1）。这些结果加在一起