Immobilized angiotensin II type I receptor: A powerful method of high throughput screening for antihypertensive...

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 (k_d) 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 (K_A: 0.12 × 10⁴ and 1.5 × 10⁴/M) and slower kinetics (k_d: 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.