Structural determinants of the domain-selectivity of novel inhibitors of human testis angiotensin-converting enzyme
Jean M. Watermeyer · Open University of Cape Town (University of Cape Town) · 2008
Angiotensin-converting enzyme (ACE) is a zinc metallopeptidase and a key drug target for the treatment of hypertension, due to its action on vasoregulatory peptides angiotensin I and bradykinin.ACE consists of two active domains, the N and C domains, having similar structures and activities.Each domain is globular and largely a-helical, with the active zinc ion buried in a cleft that divides the domain in half.Some substrates are cleaved preferentially by one domain, with the cleavage of angiotensin I depending largely on the C domain.It has been suggested that C-domain-selective inhibitors might not cause the side effects arising from current ACE inhibitor therapies.Here, X-ray co-crystal structures have been solved of three novel C-domain-selective inhibitors in the active site of a glycosylationdeficient mutant of human testis ACE, tACE-G 13, equivalent to the minimally-glycosylated C domain.This is the first successful application of this mutant in the drug design process.Inhibitors (5S)-5-[(N-benzoyl)amino]-4-oxo-6-phenyl-hexanoyl-L-tryptophan (kA W) and (5S)-5-[(N -benzoyl)amino ]-4-oxo-6-phenyl-hexanoyl-L-phenylalanine (kAF) were derived from keto-ACE by P 2 ' substitution.Their co-crystal structures, to 2.18 and 2.17 A, respectively, reveal the hydrated geminal diolate form of the ketone zinc-binding group, reminiscent of the tetrahedral transition state.Two enantiomers of inhibitor (SR)-I-(N(2)-(1carboxy-3-phenylpropyl)-L-Iysyl)-L-tryptophan (lisW) were derived from lisinopril by P 2 ' substitution, however structures solved to 2.40 and 2.30 A revealed the selection of the Lamino-acid form, lisW-S, by the active site.Inter-domain substitutions have been identified among the side chains interacting with these three inhibitors which account for their domainselectivity.Based on this work, several of these substitutions, E376D, V380T, Y391F and V518S, have subsequently been verified as important for the domain-selectivity ofkA Wand kAF.The P 2 ' Trp moieties ofkA Wand lisW-S take different conformations in the active site, indicating the large volume of the S2' pocket and cooperativity of binding between the P 2 ' and PI'moieties.Conserved active site water molecules were identified, including water 1165 of the kA W structure which may be important for the chloride activation of the C domain due to its proximity to the chloride ligand R522.These data provide a platform for further structurebased design of domain-selective inhibitors.