Journal of Molecular Graphics and Modelling 53 (2014) 206–211 Contents lists available at ScienceDirect Journal of Molecular Graphics and Modelling j ourna l h om epa ge: www.elsevier.com/locate/JMGM Potent angiotensin-converting enzyme inhibitory tripeptides identified by a computer-based approach Tran Hai-Bang, Kuniyoshi Shimizu ∗ Faculty of Agriculture, Kyushu University, Fukuoka, Japan a r t i c l e i n f o Article history: Accepted 13 August 2014 Available online 23 August 2014 Keywords: ACE inhibition Hypotensive peptides Docking Rational design ADME prediction a b s t r a c t Currently, peptides and peptidomimetics are the main focus in attempts to identify inhibitors of angiotensin-converting enzyme (ACE), the dipeptidyl carboxypeptidase that causes blood vessels to con- strict and blood pressure to increase. This study was conducted to identify the most potent ACE-inhibitory tripeptides with a proline C-terminus, using a novel three-step (tautomerization-docking-ADME simu- lation) virtual screening process and in vitro assays. Sixteen candidates were identified, and their IC 50 values ranged from 5.6 to 274.4 M. ACE inhibition activity for 14 of the 16 tripeptides was reported for the first time. We also found that changing from the L-form to the D-form of the amino acid at the amino and carboxyl termini resulted in a decrease of inhibition, but a greater decrease was observed for C- terminal changes. With low IC 50 values and high-predicted bioavailability, the peptides identified by our protocol are comparable in terms of ACE-inhibition to those derived from costly and time-consuming wet screening. Our in vitro and docking results showed that the configuration of the C-terminus is a critical parameter contributing to the inhibitory activity of tripeptides with proline at this position. These find- ings will contribute to the use of simulation tools for rational drug design, especially for ACE inhibitors. © 2014 Elsevier Inc. All rights reserved. 1. Introduction One of the most commonly used antihypertensive therapies is the inhibition of angiotensin-converting enzyme (ACE), the dipep- tidyl carboxypeptidase that causes blood vessels to constrict and blood pressure to increase [1]. Since the discovery of the first ACE peptide inhibitor (from a viper’s venom), significant efforts have been made to develop ACE inhibitors by both synthetic and nature- derived approaches. The identification of ACE inhibitor candidates has involved a costly and time-consuming process of screening and activity testing. In this context, high-throughput screening (HTS), in which large libraries of synthetic and natural compounds are subjected to bioassays [2], seems to be a solution. However, the two drawbacks of the HTS approach are the low hit rates of active compounds and incomplete information about the biological target features [3]. Recently, the use of computers for evaluating the bioactivi- ties of large compound libraries against a target structure, known ∗ Corresponding author at: Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel.: +81 92 642 3002; fax: +81 92 642 3002. E-mail address: shimizu@agr.kyushu-u.ac.jp (K. Shimizu). as “virtual screening” [4], has been facilitated by the elucidation of protein structures by X-ray crystallography and the development of simulation software. In virtual screening, docking is a helpful tool for predicting the optimal pose and binding affinity of small molecules (ligands) when they bind to a target protein (receptor) to form a stable complex. Docking tools can also be used to iden- tify potential candidates at a specified activity from a library of thousands of (or even more) compounds or to determine a lead structure by optimizing the potential candidates in the library [5]. In the field of ACE inhibition, peptides and peptidomimetics are the dominant structures, as most of the drugs currently used for ACE inhibition therapy are peptide-like compounds. Peptides have been reported with low oral bioavailability and intestinal absorp- tion [6], but many studies (both in vivo and placebo-controlled trials) have shown the hypotensive effect of different peptides on rats and humans [7–13]. For hypotensive peptides, proline at the C-terminus of peptides was proven to be more potent than other naturally occurring amino acids [14]. The presence of proline and proline-like moieties in the structures of antihypertensive drugs and many ACE inhibitor peptides demonstrates the vital role of this moiety in ACE-inhibitory agents. There is virtually no absorption for peptide sequences with more than four amino acids, whereas di- and tripeptides have been demonstrated to be actively transported by many oligopeptide http://dx.doi.org/10.1016/j.jmgm.2014.08.002 1093-3263/© 2014 Elsevier Inc. All rights reserved.