AspA rgVal TyrlleHi sPto PheHi sLeu AspArg VaiTyr fleHis Pro

Angiotensin 1 Angiolcnsion II

During the initial stages of development of ACE inhibitors, little was known concerning its structure and characteristics other than that it was a zinc-dependent enzyme. In the early work a model of the active site of ACE was tentatively constructed, based on information from the well known structure of carboxypeptidase A, where a zinc moiety at the active site forms a complex with the scissile amide bond of the peptide substrate and a positively charged residue binds to the negatively charged C-terminal carboxyl group of the substrate. Unlike Carboxypeptidase A which cleaves a single amino acid from the C-terminus, ACE cleaves off a dipeptide. ACE does not show specificity for C-terminal hydrophobic amino acids, indicating that its active site does not have a hydrophobic pocket corresponding to that possessed by carboxypeptidase A. Two sites, corresponding to side chain substituents of the two terminal amino acid residues of the substrate (AAi and AA2, see 8.48) were included in the model and have since been validated by structure-activity studies.

D-Benzylsuccinic acid, a biproduct inhibitor of carboxypeptidase A, was introduced into the structure of a dipeptide and became the starting point for the design of ACE inhibitors. Proline, the C-terminus amino acid in naturally occurring peptide inhibitors such as the nonapeptide SQ 20881 (Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro), became the natural choice as the C-terminal amino acid in synthetic inhibitors. Structural requirements for binding to the active site were determined by using simple peptide inhibitors.

Zinc-binding ligand. Succiny-L-proline, the first bi-product inhibitor, has a weak inhibitory activity for ACE. Replacement of the carboxyl group of succinyl-L-proline by a sulphydryl function as zinc chelating group produced an inhibitor, captopril (8.48), which was 1650-fold more potent and orally active. An alkyl chain length of 2 carbons, between the amide carbonyl and zinc-complexing ligand, is optimum for efficent binding. Other zinc-binding ligands such as N-carboxylate and phosphate ions also effective.

AA1 substituent. The presence of an a-substituent contributes to inhibitory potency but is not essential. Methyl or benzyl substituents increase the inhibitory potency of succinyl-L-proline, but a cyclohexyl residue results in loss of potency. These substituents may contribute to increased binding, but the increase in inhibitory potency is more likely to be due to the restriction in conformation introduced at the chiral centre.

AA2 substituent. Binding is enhanced by a free C-terminal carboxyl group and esterification reduces inhibitory potency. Succinyl-L-proline is the most effective, although other C-terminal aromatic amino acids and leucine residues are also acceptable but are less efficent than proline. The superiority of L-proline is probably due to its rigid structure which may lock the carboxyl group into a favourable conformation for interaction with the positively charged residue (probably protonated arginine) at the active site of the enzyme.

AA1-AA2 amide bond. The presence of an amide bond in the correct position is critical for binding of the inhibitor. Analogues which either lack the amide bond or in which the amide group is displaced have significantly reduced inhibitory potency.

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