Vol. 305, Issue 1, 205-211, April 2003

Structure-Activity Study of LVV-Hemorphin-7: Angiotensin AT₄ Receptor Ligand and Inhibitor of Insulin-Regulated Aminopeptidase

Joohyung Lee¹ , Tomris Mustafa¹ , Sharon G. McDowall, Frederick A. O. Mendelsohn, Michelle Brennan, Rebecca A. Lew, Anthony L. Albiston and Siew Yeen Chai

Howard Florey Institute (J.L., T.M., S.G.M., F.A.O.M., M.B., A.L.A., S.Y.C.) and Department of Pharmacology (J.L.), University of Melbourne, Parkville, Victoria, Australia; and Baker Heart Research Institute (R.A.L.), Melbourne, Australia

The decapeptide LVV-hemorphin-7 binds with high affinity to the angiotensin IV (Ang IV) receptor (AT₄ receptor), eliciting a number of physiological effects, including cellular proliferation and memory enhancement. We have recently shown that the AT₄ receptor is identical to insulin-regulated aminopeptidase (IRAP) and that both LVV-hemorphin-7 and Ang IV inhibit the catalytic activity of IRAP. In the current study, a series of alanine-substituted and N- or C-terminally modified analogs of LVV-hemorphin-7 were evaluated for their abilities to compete for ¹²⁵I-Ang IV binding in sheep adrenal and cerebellar membranes. Selected analogs were also analyzed for binding to recombinant human IRAP and inhibition of IRAP aminopeptidase activity. C-Terminal deletions of LVV-hemorphin-7 resulted in modest changes in affinity for IRAP, whereas deletion of the first three N-terminal residues abolished binding. Monosubstitutions of Tyr⁴ and Trp⁶ with alanine resulted in a 10-fold reduction in affinity. Competition binding studies using recombinant human IRAP demonstrated the same rank order of affinity as obtained for the ovine tissues. All LVV-hemorphin-7 analogs tested, except for Leu-Val-Val-Tyr, inhibit the cleavage of the synthetic substrate, leucine -naphthylamide, by IRAP, with K_i values between 56 and 620 nM. We find that the Val³ residue is crucial for LVV-hemorphin-7 binding to IRAP, whereas the C-terminal domain seems to play a minor role. The current study highlights the minimal residues necessary for binding and inhibition of IRAP and provides a basis to design peptidomimetic analogs for experimental and potentially clinical use.