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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Investigation on the Inhibition of the Two Active Sites ofAngiotensin I Converting (ACE) Enzyme by Modified ProlylPeptidesBoryana K. Yakimova 1 , Bozhidarka Pandova 2 , Stanislav G.Yanev 2 ,Bozhidar B. Tchorbanov 1 , and Ivanka B. Stoineva 11 Institute of Organic Chemistry with Centre of Phytochemistry; 2 Dept.Drug Toxicology,Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia, 1113, BulgariaIntroductionMany bioactive peptides derived from food proteins or artificial synthetic products inhibitAngiotensin I converting enzyme (ACE) in the cardiovascular system and contribute to theprevention and treatment of hypertension. Angiotensin I converting enzyme (ACE) belongsto the class of zinc proteases and has two distinct active catalytic sites, called the N- and C-domains. The relative potencies of competitive artificial inhibitors are different for the Cdomain (Lisinopril > Enalapril > Captopril) and the N domain (Captopril > Enalapril >Lisinopril) [1]. In this study we investigate the inhibitory potency of prolyl peptides incomparison with Lisinopril which prefer to bind to the C domain.Results and DiscussionThree different proline peptides derivatives, two-, three- and tetra-peptides and threeaminoacyl carbohydrates were tested for their inhibitory potency compared with the wellknown ACE inhibitor - Lisinopril (pyrrolidine derivative which is not metabolized and isexcreted unchanged in the urine). An original HPLC method was applied for ACE activitydetermination. Rabbit serum was used as enzyme source. Serum aliquot was incubated inbuffered medium with the ACE substrate analogue Hippuryl-Histidyl-Leucine (HHL). TheIC 50 values were determined by non-linear regression analysis of enzyme activity/inhibitorconcentration curves using software package GraphPad Prism 5.0.From the six compounds studied, the highest inhibitory potency possessed H-Val-Pro-Pro-OH with IC 50 around 26 µM, followed by H-Val-Pro-Pro-Pro-OH (IC 50 around 0.51mM), and H-Val-Pro-OH (IC 50 around 0.9 mM) (Figure 1). Sucrose-O-Pro, Glucose-O-Proand Sucrose-O-Pro-NBoc were ineffective up to 1 mM concentration. The inhibitory effectof Lisinopril was much greater than that of H-Val-Pro-Pro-OH. The enzyme activity was100% inhibited after 0.8 µM.1,0VPP0,80,60,40,20,0VPVPPP2 3 4Fig. 1. Relative inhibitory potency of prolyl peptides.Using the mapping of subside structures based on Lisinopril binding to tACE [2], where thetripeptide interacted with Ala354, His353, His513, Lys511 and Tyr520 and basing on ourtheoretical calculations [3], it is possible to conclude that the structures of the tripeptidesVal-Pro-Pro with highest inhibitory potency allow only the NH 2 …O=C and C=O…HOinteractions with Ala354 and Tyr520 in the cavity of the enzyme (Figure 2).346