Synthetic inhibitors of endopeptidase EC 3.4.24.15: potency and stability in vitro and in vivo.
1. The role of the metalloendopeptidase EC 3.4.24.15 (EP 24.15) in peptide metabolism in vivo is unknown, in part reflecting the lack of a stable enzyme inhibitor. The most commonly used inhibitor, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP-AAY-pAB, Ki = 16 nM), although selective in vitro, is rapidly degraded in the circulation to cFP-Ala-Ala, an angiotensin converting enzyme (ACE) inhibitor. This metabolite is thought to be generated by neutral endopeptidase (NEP; EC 3.4.24.11), as the Ala-Tyr bond of cFP-AAY-pAB is cleaved by NEP in vitro. In the present study, we have examined the role of NEP in the metabolism of cFP-AAY-pAB in vivo, and have tested a series of inhibitor analogues, substituted at the second alanine, for both potency and stability relative to the parent compound. 2. Analogues were screened for inhibition of fluorescent substrate cleavage by recombinant rat testes EP 24.15. D-Ala or Asp substitution abolished inhibitory activity, while Val-, Ser- and Leu-substituted analogues retained activity, albeit at a reduced potency. A relative potency order of Ala (1) > Val (0.3) > Ser (0.16) > Leu (0.06) was observed. Resistance to cleavage by NEP was assessed by incubation of the analogues with rabbit kidney membranes. The parent compound was readily degraded, but the analogues were twice (Ser) and greater than 10 fold (Leu and Val) more resistant to cleavage. 3. Metabolism of cFP-AAY-pAB and the Val-substituted analogue was also examined in conscious rabbits. A bolus injection of cFP-AAY-pAB (5 mg kg-1, i.v.) significantly reduced the blood pressure response to angiotensin I, indicating ACE inhibition. Pretreatment with NEP inhibitors, SCH 39370 or phosphoramidon, slowed the loss of cFP-AAY-pAB from the plasma, but did not prevent inhibition of ACE. Injection of 1 mg kg-1 inhibitor resulted in plasma concentrations at 10 s of 23.5 microM (cFP-AAY-pAB) and 18.0 microM (cFP-AVY-pAB), which fell 100 fold over 5 min. Co-injection of 125I-labelled inhibitor revealed that 80-85% of the radioactivity had disappeared from the circulation within 5 min, and h.p.l.c. analysis demonstrated that only 25-30% of the radiolabel remained as intact inhibitor at this time. Both analogues were cleared from the circulation at the same rate, and both inhibitors blunted the pressor response to angiotensin I, indicative of ACE inhibition. 4. These results suggest that both NEP and other clearance/degradation mechanisms severely limit the usefulness of peptide-based inhibitors such as cFP-AAY-pAB. To examine further EP 24.15 function in vivo, more stable inhibitors, preferably non-peptide, must be developed, for which these peptide-based inhibitors may serve as useful molecular templates.
Lew
R A
RA
Baker Medical Research Institute, Prahran, Victoria, Australia.
Tomoda
F
F
Evans
R G
RG
Lakat
L
L
Boublik
J H
JH
Pipolo
L A
LA
Smith
A I
AI
D000704:Analysis of Variance; D000804:Angiotensin II / Q000494:pharmacology; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000378:metabolism*; D000818:Animals; D001794:Blood Pressure / Q000187:drug effects; D001920:Bradykinin / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006020:Glycopeptides / Q000494:pharmacology*; D007987:Gonadotropin-Releasing Hormone; D066298:In Vitro Techniques; D007668:Kidney / Q000166:cytology / Q000201:enzymology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000097:blood / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology*; D011817:Rabbits; D051381:Rats; D013237:Stereoisomerism; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D000806:Angiotensin-Converting Enzyme Inhibitors; D004151:Dipeptides; D006020:Glycopeptides; D011480:Protease Inhibitors; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; C060631:Sch 39370; D007987:Gonadotropin-Releasing Hormone; D008666:Metalloendopeptidases; D001920:Bradykinin; C008890:phosphoramidon