Angiotensin converting enzyme (ACE) is a zinc metallopeptidase that cleaves a wide variety of physiologically relevant substrates; including vasoregulatory peptides angiotensin I and bradykinin, the anti-fibrotic agent N-acetyl-SDKP, the neurotransmitter substance P and the adhesion molecule amyloid β peptide. Since ACE is primarily involved in the production of vasoconstrictor angiotensin II, ACE is a well-validated therapeutic target for hypertension, congestive heart failure, myocardial infarction and renal disease (including diabetic nephropathy). There are two forms of the enzyme: a somatic form of 150-180 kDa and a smaller germinal isoform of 100-110 kDa which is restricted to the testes. However, unlike nearly all zinc metallopeptidases the somatic enzyme has two catalytic sites that have subtle differences. Thus the structures of the ACE C- and N-terminal domains complexed with various clinically important, as well as domain selective, inhibitors reported by our group open the door to: 1) a better understanding of the structure-function relationships of each domain; and 2) the structure-based design of the next generation of domain specific ACE inhibitors. It is within this field that we are making a contribution to understanding the substrate and inhibitor selectivity of these homologous domains. We shall extend this work further by elucidating the 3D structure of the two-domain or full-length somatic enzyme and also the minimum glycosylation requirements of the N domain, allowing high throughput crystallisation studies. In parallel we are using mutagenesis, heterologous protein expression and kinetic analyses to investigate the effect discrete regions and amino acids of each domain have on conferring N- or C-domain specificity.

The crystal structure of a novel C-domain-specific inhibitor bound to the enzyme’s active site (residues unique to the C domain are in green, zinc ion in magenta, and  keto-ACE analogue kAW blue backbone) (Watermeyer et al., 2008)

Domain-selective ACE inhibitors have been identified by screening phosphinic peptide libraries (Dive et al. Proc Natl Acad Sci U S A. 96, 4330-5, 1999 ; Georgiadis et al., Biochemistry 43, 8048-54, 2004). However, no structure-based design of ACE inhibitors has been reported using the C and N domain crystal structures. Empirical investigations have shown that C-domain selectivity of inhibitors is influenced, by amongst other factors, the interaction of the inhibitor with certain residues in the S2' pocket of the active site. Thus we have introduced alternative functionalities onto the P2' residue of various well established ACE inhibitors and have observed improved C domain specificity. Furthermore, we have synthesised derivatives of the moderately C-domain selective inhibitor keto-ACE that have different P2' substituents. These inhibitors have displayed marked inhibitory potential using fluorogenic substrates and promising candidates have been co-crystallised with ACE for further X-ray crystallographic analyses.

The iterative drug design process whereby compounds generated by the molecular modelling are synthesized and then co-crystallized with ACE to generate new ACE-inhibitor crystal structures, which in turn are fed back to the molecular modelling group.

The C-domain activity of ACE and to a far lesser extent the N domain depend on the presence of chloride ions; in this regard, chloride dependence of ACE activity is unique among the metallopeptidase family. The molecular mechanism of chloride activation is not readily apparent from the structure; however, the primary ligand for the second chloride ion (Cl2), R522, lies on the same helix (a17) as Y520 and Y523 which interact with lisinopril and, presumably, also the substrate. We are using mutagenesis to investigate the role of key residues that interact with Cl2 as well as residues that may be responsible for the gating of a channel allowing the anion access to the chloride cavity.

Another area of interest to our group is the proteolytic release of membrane-bound proteins referred to as ectodomain shedding. Protein ectodomain shedding affects the function of a variety of structurally and functionally diverse molecules on the cell surface, including cytokines and growth factors, their receptors, adhesion proteins, and other molecules, such as the amyloid precursor protein, Notch and Delta. We are presently investigating the shedding of the LDL receptor and ACE. Future work involves the identification and isolation of the sheddases responsible for the release of these important ectoproteins.

Current Funding:

• Wellcome Trust
  
  
• National Research Foundation
  
  
• Cape Biotech Trust
  
  
• UCT

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This page last updated on 25 February, 2009