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 (AT 1 R) based affinity chromatography in antihypertensive compound identification. We fused haloalkane dehalogenase at C-terminus of AT 1 R 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 AT 1 R 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 AT 1 R 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 AT 1 R 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 × 10 4 and 1.5 × 10 4 /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 AT 1 R has the capacity to probe antihypertensive compounds.
The protein tyrosine phosphatases(PTPs) comprise a family of enzymes that specifically dephosphorylate tyrosyl residues. Among them, SHP-1 has been regarded as one of the best validated intracellular tyrosine phosphatases. Downregulation of SHP-1 has shown remarkable efficacy in improving insulin sensitivity in vivo in insulin signaling pathway. In this study, we found the role of Candesartan cilexetil targeting at SHP-1. The results indicate that Candesartan cilexetil was a competitive inhibitor to SHP-1(IC 50 =85.6 μmol/L and K
i =24 μmol/L). We also found that Candesartan cilexetil was more sensitive towards SHP-1 compared with other PTPs. Through the consequence of Western blotting, it showed that Candesartan cilexetil can strengthen the level of tyrosine phosphorylation of several key cellular proteins[such as insulin receptor(IR), insulin receptor substrate(IRS) and ERK]_in insulin signaling pathway in HepG 2 cells and improve the insulin sensitivity through inhibiting the protein phosphorylation of SHP-1. These findings showed that Candesartan cilexetil might be an important inhibitor of SHP-1 and had a great application potential in the treatment of diabetes through inhibiting the level of SHP-1 in insulin signaling pathway.
危险品化学品经营许可证（带存储）