Investigating the Domain Specificity of Phosphinic Inhibitors RXPA380 and RXP407 in Angiotensin-Converting Enzyme

Abstract

Human somatic angiotensin-converting enzyme (ACE) is a membrane-bound dipeptidyl carboxypeptidase that contains two extracellular domains (N and C). Although highly homologous, they exhibit different substrate and inhibition profiles. The phosphinic inhibitors RXPA380 and RXP407 are highly selective for the C- and N-domains, respectively. A number of residues, implicated by structural data, are likely to contribute to this selectivity. However, the extent to which these different interactions are responsible for domain selectivity is unclear. In this study, a series of C- and N-domain mutants containing conversions to corresponding domain residues were used to scrutinize the contribution of these residues to selective inhibitor binding. Enzyme kinetic analyses of the purified mutants indicated that the RXPA380 C-selectivity is particularly reliant on the interaction between the P2 substituent and Phe 391 (testis ACE numbering). Moreover, a C-domain mutant in which Phe 391 has been changed to a Tyr residue, in addition to containing an N-domain S2' pocket (S2'F/Y), displayed the greatest shift toward a more N-domain-like Ki. None of the single mutations within the N-domain caused a large shift in RXP407's affinity for these enzymes. However, the double mutant containing the Tyr 369 to Phe change as well as Arg 381 to Glu displayed a 100-fold decrease in binding affinity, confirming that the S2 pocket plays a major role in RXP407 selectivity. Taken together, these data advance our understanding regarding the molecular basis for the remarkable ACE domain selectivity exhibited by these inhibitors.

A Modern Understanding of the Traditional and Nontraditional Biological Functions of Angiotensin-Converting Enzyme

“…In terms of how domain specificity is achieved, data from X-ray crystal analysis, the study of chimeric ACE domains, and the results of other RXP-like compounds suggest a key feature of specificity is the ACE S 2 pocket, the portion of ACE that binds the amino part of an inhibitor. In particular, Tyr369 and Arg381 have been implicated in N-domain selectivity for RXP 407 (Kröger et al, 2009;Anthony et al, 2010Anthony et al, , 2012. These two S 2 pocket residues appear critical in domain specificity since the equivalent amino acids in the C-domain, Phe967 and Glu979, have been determined by X-ray crystal analysis as being critical for the specificity of RXP A380 for the ACE C-domain.…”

Molecular recognition and regulation of human angiotensin-I converting enzyme (ACE) activity by natural inhibitory peptides

“…Based on the crystal structure of C-domain in complex with RXPA380 inhibitor and mutagenesis studies, the potency of this inhibitor has been attributed largely to hydrophobic interactions between the P2 and P2′ groups with Phe391 and Val380, respectively1827. In the case of the former Phe 391 is replaced by the more polar Tyr369 in the N-domain, which may present a steric clash with the P2 Phe of RXPA380.…”

High-Resolution Crystal Structures of Drosophila melanogaster Angiotensin-Converting Enzyme in Complex with Novel Inhibitors and Antihypertensive Drugs

“…This aromatic interaction is conserved in both AnCE and tACE in complex with RXPA380 and has been suggested to be a crucial residue for Cdomain selectivity. 17,34 A network of water-molecule-mediated interactions is also observed at the S 1 ′ pocket of the protein.…”

“…In support of this, it has been reported recently that a double mutant containing the Tyr369-to-Phe change as well as Arg381-to-Glu displayed a 100-fold decrease in binding affinity, confirming that the S 2 pocket plays a major role in RXP407 selectivity in the N-domain protein. 34 In addition, the single Arg381-to-Glu mutation only caused a 6-fold difference in the K i of the N-domain for RXP407 and the Tyr369-to-Phe mutation had little effect on binding to this inhibitor. 34 …”

“…34 In addition, the single Arg381-to-Glu mutation only caused a 6-fold difference in the K i of the N-domain for RXP407 and the Tyr369-to-Phe mutation had little effect on binding to this inhibitor. 34 …”