Browsing by Author "O'Neill, Hester G"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Restricted
    Characterization of domain-selective inhibitor binding in angiotensin-converting enzyme using a novel derivative of lisinopril
    (Portland Press, 2010) Watermeyer, Jean M; Kröger, Wendy L; O'Neill, Hester G; Sewell, Trevor B; Sturrock, Edward D
    Human ACE (angiotensin-converting enzyme) (EC 3.4.15.1) is an important drug target because of its role in the regulation of blood pressure via the renin–angiotensin–aldosterone system. Somatic ACE comprises two homologous domains, the differing substrate preferences of which present a new avenue for domainselective inhibitor design. We have co-crystallized lisW-S, a Cdomain-selective derivative of the drug lisinopril, with human testis ACE and determined a structure using X-ray crystallography to a resolution of 2.30 Å (1 Å = 0.1 nm). In this structure, lisW-S is seen to have a similar binding mode to its parent compound lisinopril, but the P2 tryptophan moiety takes a different conformation to that seen in other inhibitors having a tryptophan residue in this position. We have examined further the domain-specific interactions of this inhibitor by mutating Cdomain-specific active-site residues to their N domain equivalents, then assessing the effect of the mutation on inhibition by lisWS using a fluorescence-based assay. Kinetics analysis shows a 258-fold domain-selectivity that is largely due to the co-operative effect of C-domain-specific residues in the S2 subsite. The high affinity and selectivity of this inhibitor make it a good lead candidate for cardiovascular drug development.
  • Thumbnail Image
    Item
    Restricted
    Probing the basis of domain-dependent inhibition using novel ketone inhibitors of angiotensin-converting enzyme
    (American Chemical Society, 2008) Watermeyer, Jean M; Kroger, Wendy L; O'Neill, Hester G; Sewell, B Trevor; Sturrock, Edward D
    Human angiotensin-converting enzyme (ACE) has two homologous domains, the N and C domains, with differing substrate preferences. X-ray crystal structures of the C and N domains complexed with various inhibitors have allowed identification of active site residues that might be important for the molecular basis of this selectivity. However, it is unclear to what extent the different residues contribute to substrate domain selectivity. Here, cocrystal structures of human testis ACE, equivalent to the C domain, have been determined with two novel C domain-selective ketomethylene inhibitors, (5S)-5-[(N-benzoyl)amino]-4-oxo-6-phenylhexanoyl-l-tryptophan (kAW) and (5S)-5-[(N-benzoyl)amino]-4-oxo-6-phenylhexanoyl-l-phenylalanine (kAF). The ketone groups of both inhibitors bind to the zinc ion as a hydrated geminal diolate, demonstrating the ability of the active site to catalyze the formation of the transition state. Moreover, active site residues involved in inhibitor binding have been mutated to their N domain counterparts, and the effect of the mutations on inhibitor binding has been determined. The C domain selectivity of these inhibitors was found to result from interactions between bulky hydrophobic side chain moieties and C domain-specific residues F391, V518, E376, and V380 (numbering of testis ACE). Mutation of these residues decreased the affinity for the inhibitors 4−20-fold. T282, V379, E403, D453, and S516 did not contribute individually to C domain-selective inhibitor binding. Further domain-selective inhibitor design should focus on increasing both the affinity and selectivity of the side chain moieties.