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.
As an important protective mechanism against cerebral ischemia, angiogenesis has become a topic of interest in the treatment of ischemic stroke with the challenge that few drugs promote angiogenesis. Previous studies of the identification of drug–target interactions mainly focused on the overall structures of drugs and proteins, which limited the discovery of novel structure drugs. In this article, we proposed a new strategy for discovering proangiogenic drugs based on the assumption that drug–protein interaction is mediated by substructure and domain. First, we identified substructure–domain relationships according to the known drug–protein interactions and established the drug–substructure–domain–protein relationships of genes that are proangiogenic in brain tissue and expressed significantly during ischemic stroke. Then we quantified the intensity of interaction between each drug and each protein. Finally, we obtained 540 interactive relationships between 238 drugs and 54 genes, establishing a drug–gene network with two patterns of independent and complex drug–gene interactions. Both of the patterns facilitated finding not only drugs with the same overall structure but also drugs with a different overall structure based on the same or a similar protein spectrum. In addition, we analyzed the mechanism of action of each predicted drug and extracted drugs with similar mechanisms. In vitro, our results showed that azelnidipine, azilsartan, lercanidipine, nafcillin, and vortioxetine enhanced bEnd.3 cell proliferation, migration, tube formation, and the expression of angiogenic marker PCNA. Azelnidipine, azilsartan, lercanidipine, and nafcillin increased the level of expression of proangiogenic factor VEGF. Unlike previous studies focusing on the overall structures of drugs, our research highlighted local structural similarity, which has great potential in the search for more proangiogenic drugs with novel structures.
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