Microwave Synthesis,Biological Evaluation And Molecular Docking Of Angiotensin Ⅰ-Converting Enzyme Inhibitiry Peptides

AngiotensinⅠ-converting enzyme(ACE) plays a pivotal role in the regulation of blood pressure. It increases blood pressure by promoting the generation of angiotensin II and the degradation of bradykinin. So angiotensinⅠ-converting enzyme inhibitors are very important drugs for clinical treatment of hypertension.The tripetpides Leu-Arg-Pro(LRP, isolated from a-zein) and Leu-Lys-Pro (LKP, isolated from fish protein) have IC50 values of 0.27μmol/Land 0.32μmol/L, respectively. In this paper, in order to investigate the structure-activity relationship of ACE inhibitory peptides and design high activity ACE inhibitory peptides, our work focused on the influence of amino acid composition and molecular conformation changes on ACE inhibitory activity. Based on the structures of LRP and LKP, we designed and synthesized 14 new ACE peptide inhibitors, and studied the relationship between ACE inhibitory activity and structure, the binding site between inhibitors and ACE have been analysed by molecular docking.In this paper, microwave-assisted solid phase peptide synthesis method and Fmoc orthogonal protected strategy were used with Trt(2-Cl) resin or Wang resin as solid carrier,HBTU-HOBt as coupling reagents and 20%piperidine in DMF as deprotecting reagents. LRP,LKP and their analogues have been synthesized. The peptides were then precipitated by ice ether. After centrifugation, the purity and mass of each end-product was analyzed by RP-HPLC and ESI-MS, respectively.ACE inhibitory activity in vitro was assayed. The result showed that among the 14 new ACE peptides, Leu-Arg-Pro-Phe-Phe showed the strongest inhibition against ACE with an IC50 value of 0.26μmol/L in vitro. The presence of Pro in the middle of the peptide made it very resistant to hydrolysis by digestive enzymes in vivo. For the first series based on LRP, we could see that two identical residues (-Phe-Phe) at the C-terminus yielded the most inhibition, and changing one of the two Phe residues to other amino acid caused a reduction in the inhibitory activity toward ACE; for the second series based on LKP, pepides having L-Pro at the C-termini displayed the highest inhibitory activities, secondly it was D-Pro, and the weakest was Ac6c. ACE therefore appeared to show high stereospecificity with respect to the amino acid residues at the C-termini of these peptide inhibitors. Molecular docking was performed with the designed peptides as well as the two model peptides (Leu-Arg-Pro and Leu-Arg-Pro), in order to study the binding interaction. The result showed that except peptides 12 and 14, the rest of the synthetic peptides mainly occupied the S1 subsite, forming H-bonds with residues such as Tyr523, Ala356 and Glu403, and also accommodated other interactions with residues such as His353, Phe391 and Val518. The sequences which had the highest and the lowest ACE-inhibitory activities were chose to analyze in details, and the formation of H-bonding was consistent with the result obtained for in vitro activity assay.