Novel mechanism of inhibition of human angiotensin-I-converting enzyme (ACE) by a highly specific phosphinic tripeptide

Abstract

Human ACE (angiotensin-I-converting enzyme) has long been regarded as an excellent target for the treatment of hypertension and related cardiovascular diseases. Highly potent inhibitors have been developed and are extensively used in the clinic. To develop inhibitors with higher therapeutic efficacy and reduced side effects, recent efforts have been directed towards the discovery of compounds able to simultaneously block more than one zinc metallopeptidase (apart from ACE) involved in blood pressure regulation in humans, such as neprilysin and ECE-1 (endothelin-converting enzyme-1). In the present paper, we show the first structures of testis ACE [C-ACE, which is identical with the C-domain of somatic ACE and the dominant domain responsible for blood pressure regulation, at 1.97Å (1 Å=0.1 nm)] and the N-domain of somatic ACE (N-ACE, at 2.15Å) in complex with a highly potent and selective dual ACE/ECE-1 inhibitor. The structural determinants revealed unique features of the binding of two molecules of the dual inhibitor in the active site of C-ACE. In both structures, the first molecule is positioned in the obligatory binding site and has a bulky bicyclic P(1)' residue with the unusual R configuration which, surprisingly, is accommodated by the large S(2)' pocket. In the C-ACE complex, the isoxazole phenyl group of the second molecule makes strong pi-pi stacking interactions with the amino benzoyl group of the first molecule locking them in a 'hand-shake' conformation. These features, for the first time, highlight the unusual architecture and flexibility of the active site of C-ACE, which could be further utilized for structure-based design of new C-ACE or vasopeptidase inhibitors.

Figure 1. Chemical structures of ACE/ECE-1 dual inhibitors FI and FII and their potency

Compound FI adopts the S configuration and compound FII adopts the R configuration.

Figure 2. Overall topology of C-ACE and N-ACE molecules

(A) A cartoon representation of the C-ACE structure with bound dual-inhibitor molecules at sites A and B (FII-A and FII-B) and the observed N-glycosylated sugars (green sticks) at the N-glycosylation sites (Asn⁷² and Asn¹⁰⁹). The bound inhibitor molecules at the two sites, FII-A and F II-B, are shown in stick representation, coloured in yellow and cyan respectively. The active-site Zn²⁺ ion and the location of the two Cl⁻ ions are shown in green and skyblue spheres respectively. (B) A cartoon representation of N-ACE in complex with the dual inhibitor. The bound dual inhibitor (FII) and the N-glycosylated sugars are shown as yellow and green sticks respectively. The Zn²⁺ ion and the bound Cl⁻ ion are also shown and coloured as in C-ACE.

Figure 3. Details of FII binding in C-ACE and N-ACE active sites

(A–C) Close-up views of the active site showing bound inhibitors. (A) Interaction for C-ACE with the dual inhibitor at the primary site FII-A. (B) Interaction for N-ACE with the dual inhibitor at the primary site FII-A. (C) Interaction of C-ACE with the dual inhibitor at the secondary binding site FII-B. The interacting residues in the active site are labelled. The electron density map shown is the F_o−F_c map contoured at 3σ level. The Zn²⁺ ion and water molecules are in green and sky-blue spheres, and the inhibitor molecules are shown in stick representation. (D and E) Surface diagram with inhibitors showing their potential arrangement in C-ACE and N-ACE. (D) C-ACE with two FII molecules (sites A and B) bound at the active-site cavity. The isoxazole phenyl group of the second molecule makes a strong pi–pi stacking interaction with the amino benzoyl group of the first molecule locking them in a ‘hand-shake’ conformation. (E) N-ACE with a single dual inhibitor in the active site at (site FII-A). (F) The orientation of dual inhibitor (FII) in comparison with other known inhibitors. Comparison of the orientation of FII binding (the present study) against previously reported lisinopril [27] and RXPA380 [30] in the active site of the C-ACE. FII (green sticks), lisinopril (PDB code 1O86; cyan sticks) and RXPA380 (PDB code 2CO2; magenta sticks) bound in the active site of co-crystal structures of C-ACE are superimposed. The Zn²⁺ ion and water molecules are shown as green and sky-blue spheres. Active-site residues of C-ACE interacting with FII are labelled and their hydrogen-bond interactions are shown as magenta dotted lines. (G). Comparison of the orientation of the dual inhibitor (S configuration; FI, yellow sticks) compared with lisinopril (cyan sticks) [27] and RXPA380 (green sticks) [30] from their respective complexes with C-ACE. (H and I) Comparison of dual-inhibitor-binding sites in C-ACE with N-ACE. (H) Superimposition of dual-inhibitor binding FII-A in C-ACE (magenta) with dual-inhibitor binding in N-ACE (cyan). The dual inhibitors are shown in stick representation, and differences in residues near the dual-inhibitor-binding site in both structures are labelled (magenta, C-ACE; cyan, N-ACE). (I) Superimposition of dual-inhibitor binding FII-B in C-ACE with N-ACE (purple). Residues from C-ACE and N-ACE are labelled in magenta and cyan respectively. The dual inhibitor FII-B is shown in yellow stick representation.