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. 1996 Jul;118(5):1269–1277. doi: 10.1111/j.1476-5381.1996.tb15533.x

Synthetic inhibitors of endopeptidase EC 3.4.24.15: potency and stability in vitro and in vivo.

R A Lew 1, F Tomoda 1, R G Evans 1, L Lakat 1, J H Boublik 1, L A Pipolo 1, A I Smith 1
PMCID: PMC1909604  PMID: 8818353

Abstract

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.

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Selected References

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  1. Barrett A. J., Brown M. A., Dando P. M., Knight C. G., McKie N., Rawlings N. D., Serizawa A. Thimet oligopeptidase and oligopeptidase M or neurolysin. Methods Enzymol. 1995;248:529–556. doi: 10.1016/0076-6879(95)48034-x. [DOI] [PubMed] [Google Scholar]
  2. Cardozo C., Orlowski M. Evidence that enzymatic conversion of N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate, a specific inhibitor of endopeptidase 24.15, to N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala is necessary for inhibition of angiotensin converting enzyme. Peptides. 1993 Nov-Dec;14(6):1259–1262. doi: 10.1016/0196-9781(93)90185-j. [DOI] [PubMed] [Google Scholar]
  3. Charles C. J., Espiner E. A., Richards A. M., Sybertz E. J. Endopeptidase inhibition in angiotensin-induced hypertension. Effect of SCH 39370 in sheep. Hypertension. 1995 Jul;26(1):89–94. doi: 10.1161/01.hyp.26.1.89. [DOI] [PubMed] [Google Scholar]
  4. Chu T. G., Orlowski M. Active site directed N-carboxymethyl peptide inhibitors of a soluble metalloendopeptidase from rat brain. Biochemistry. 1984 Jul 31;23(16):3598–3603. doi: 10.1021/bi00311a005. [DOI] [PubMed] [Google Scholar]
  5. Chu T. G., Orlowski M. Soluble metalloendopeptidase from rat brain: action on enkephalin-containing peptides and other bioactive peptides. Endocrinology. 1985 Apr;116(4):1418–1425. doi: 10.1210/endo-116-4-1418. [DOI] [PubMed] [Google Scholar]
  6. Genden E. M., Molineaux C. J. Inhibition of endopeptidase-24.15 decreases blood pressure in normotensive rats. Hypertension. 1991 Sep;18(3):360–365. doi: 10.1161/01.hyp.18.3.360. [DOI] [PubMed] [Google Scholar]
  7. Hersh L. B., Morihara K. Comparison of the subsite specificity of the mammalian neutral endopeptidase 24.11 (enkephalinase) to the bacterial neutral endopeptidase thermolysin. J Biol Chem. 1986 May 15;261(14):6433–6437. [PubMed] [Google Scholar]
  8. Jirácek J., Yiotakis A., Vincent B., Lecoq A., Nicolaou A., Checler F., Dive V. Development of highly potent and selective phosphinic peptide inhibitors of zinc endopeptidase 24-15 using combinatorial chemistry. J Biol Chem. 1995 Sep 15;270(37):21701–21706. doi: 10.1074/jbc.270.37.21701. [DOI] [PubMed] [Google Scholar]
  9. Lasdun A., Orlowski M. Inhibition of endopeptidase 24.15 greatly increases the release of luteinizing hormone and follicle stimulating hormone in response to luteinizing hormone/releasing hormone. J Pharmacol Exp Ther. 1990 Jun;253(3):1265–1271. [PubMed] [Google Scholar]
  10. Lasdun A., Reznik S., Molineaux C. J., Orlowski M. Inhibition of endopeptidase 24.15 slows the in vivo degradation of luteinizing hormone-releasing hormone. J Pharmacol Exp Ther. 1989 Nov;251(2):439–447. [PubMed] [Google Scholar]
  11. Lew R. A., Hey N. J., Tetaz T. J., Glucksman M. J., Roberts J. L., Smith A. I. Substrate specificity differences between recombinant rat testes endopeptidase EC 3.4.24.15 and the native brain enzyme. Biochem Biophys Res Commun. 1995 Apr 26;209(3):788–795. doi: 10.1006/bbrc.1995.1569. [DOI] [PubMed] [Google Scholar]
  12. Lew R. A., Tetaz T. J., Glucksman M. J., Roberts J. L., Smith A. I. Evidence for a two-step mechanism of gonadotropin-releasing hormone metabolism by prolyl endopeptidase and metalloendopeptidase EC 3.4.24.15 in ovine hypothalamic extracts. J Biol Chem. 1994 Apr 29;269(17):12626–12632. [PubMed] [Google Scholar]
  13. Ludbrook J. On making multiple comparisons in clinical and experimental pharmacology and physiology. Clin Exp Pharmacol Physiol. 1991 Jun;18(6):379–392. doi: 10.1111/j.1440-1681.1991.tb01468.x. [DOI] [PubMed] [Google Scholar]
  14. Ludbrook J. Repeated measurements and multiple comparisons in cardiovascular research. Cardiovasc Res. 1994 Mar;28(3):303–311. doi: 10.1093/cvr/28.3.303. [DOI] [PubMed] [Google Scholar]
  15. Molineaux C. J., Lasdun A., Michaud C., Orlowski M. Endopeptidase-24.15 is the primary enzyme that degrades luteinizing hormone releasing hormone both in vitro and in vivo. J Neurochem. 1988 Aug;51(2):624–633. doi: 10.1111/j.1471-4159.1988.tb01084.x. [DOI] [PubMed] [Google Scholar]
  16. Orlowski M., Michaud C., Chu T. G. A soluble metalloendopeptidase from rat brain. Purification of the enzyme and determination of specificity with synthetic and natural peptides. Eur J Biochem. 1983 Sep 1;135(1):81–88. doi: 10.1111/j.1432-1033.1983.tb07620.x. [DOI] [PubMed] [Google Scholar]
  17. Orlowski M., Michaud C., Molineaux C. J. Substrate-related potent inhibitors of brain metalloendopeptidase. Biochemistry. 1988 Jan 26;27(2):597–602. doi: 10.1021/bi00402a015. [DOI] [PubMed] [Google Scholar]
  18. Pozsgay M., Michaud C., Liebman M., Orlowski M. Substrate and inhibitor studies of thermolysin-like neutral metalloendopeptidase from kidney membrane fractions. Comparison with bacterial thermolysin. Biochemistry. 1986 Mar 25;25(6):1292–1299. doi: 10.1021/bi00354a015. [DOI] [PubMed] [Google Scholar]
  19. Salacinski P. R., McLean C., Sykes J. E., Clement-Jones V. V., Lowry P. J. Iodination of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenyl glycoluril (Iodogen). Anal Biochem. 1981 Oct;117(1):136–146. doi: 10.1016/0003-2697(81)90703-x. [DOI] [PubMed] [Google Scholar]
  20. Schwartz J. C., Gros C., Lecomte J. M., Bralet J. Enkephalinase (EC 3.4.24.11) inhibitors: protection of endogenous ANF against inactivation and potential therapeutic applications. Life Sci. 1990;47(15):1279–1297. doi: 10.1016/0024-3205(90)90192-t. [DOI] [PubMed] [Google Scholar]
  21. Skidgel R. A., Erdös E. G. Novel activity of human angiotensin I converting enzyme: release of the NH2- and COOH-terminal tripeptides from the luteinizing hormone-releasing hormone. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1025–1029. doi: 10.1073/pnas.82.4.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sybertz E. J., Chiu P. J., Vemulapalli S., Pitts B., Foster C. J., Watkins R. W., Barnett A., Haslanger M. F. SCH 39370, a neutral metalloendopeptidase inhibitor, potentiates biological responses to atrial natriuretic factor and lowers blood pressure in desoxycorticosterone acetate-sodium hypertensive rats. J Pharmacol Exp Ther. 1989 Aug;250(2):624–631. [PubMed] [Google Scholar]
  23. Sybertz E. J., Jr, Chiu P. J., Watkins R. W., Vemulapalli S. Neutral metalloendopeptidase inhibition: a novel means of circulatory modulation. J Hypertens Suppl. 1990 Dec;8(7):S161–S167. [PubMed] [Google Scholar]
  24. Telford S. E., Smith A. I., Lew R. A., Perich R. B., Madden A. C., Evans R. G. Role of angiotensin converting enzyme in the vascular effects of an endopeptidase 24.15 inhibitor. Br J Pharmacol. 1995 Mar;114(6):1185–1192. doi: 10.1111/j.1476-5381.1995.tb13332.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tisljar U., Knight C. G., Barrett A. J. An alternative quenched fluorescence substrate for Pz-peptidase. Anal Biochem. 1990 Apr;186(1):112–115. doi: 10.1016/0003-2697(90)90582-t. [DOI] [PubMed] [Google Scholar]
  26. Williams C. H., Yamamoto T., Walsh D. M., Allsop D. Endopeptidase 3.4.24.11 converts N-1-(R,S)carboxy-3-phenylpropyl-Ala-Ala-Phe-p-carboxyanilide into a potent inhibitor of angiotensin-converting enzyme. Biochem J. 1993 Sep 15;294(Pt 3):681–684. doi: 10.1042/bj2940681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yamamoto K., Chappell M. C., Brosnihan K. B., Ferrario C. M. In vivo metabolism of angiotensin I by neutral endopeptidase (EC 3.4.24.11) in spontaneously hypertensive rats. Hypertension. 1992 Jun;19(6 Pt 2):692–696. doi: 10.1161/01.hyp.19.6.692. [DOI] [PubMed] [Google Scholar]

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