Peptides, Vol. 14, pp. 1259-1262, 1993 0196-9781/93 $6.00 + .00 Printed in the USA. Copyright © 1993 Pergamon Press Ltd.

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

C H R I S T O P H E R C A R D O Z O l A N D M A R I A N O R L O W S K I

Departments of Medicine and Pharmacology, Mount Sinai School of Medicine of the City University of New York, New York, N Y 10029

Received 23 March 1993

CARDOZO, C. AND M. ORLOWSKI. Evidence that enzymatic conversion of N-[ l (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 ofangiotensin converting enzyme. PEPTIDES 14(6) 1259-1262, 1993.--N-[ 1 (R,S)-Carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-ami- nobenzoate (cFP-AAF-pAB) is a potent, substrate-related, specific inhibitor of endopeptidase 24.15, an enzyme involved in the metabolism of bioactive peptides including bradykinin, neurotensin, and proenkephalin, and prodynorphin-derived enkephalin precursors. The observation that this inhibitor causes a pronounced decrease in blood pressure after intravenous infusion into normotensive rats posed the question of the mechanism of this hypotensive response. It was suggested previously that cFP-AAF- pAB is an inhibitor of angiotensin converting enzyme (ACE) and that this function can account for the hypotensive response to the inhibitor. We present here evidence that cFP-AAF-pAB has no intrinsic ACE-inhibitory activity. The previously observed inhibition is shown to be dependent on cleavage of the Ala-Phe bond in the inhibitor by endopeptidase 24,11 (enkephalinase, EC 3.4.24.11 ), a contaminant of some ACE preparations.

Metalloendopeptidases lnhibitors

ENDOPEPTIDASE 24.15 (EP 24.15), a zinc-metalloenzyme highly active and widely distributed in brain, occurs in two forms, one associated with the soluble protein fraction of homogenates, the other associated with membrane fractions (1,14,15). That EP 24.15 functions in the formation and degradation ofbioactive peptides was shown by its ability to generate Leu- and Met- enkephalin from opioid peptide precursors, and to rapidly cleave a number of peptides, including luteinizing hormone-releasing hormone (LHRH), bradykinin, and neurotensin ( 1,7,12,14,15). Specificity studies using natural peptides and synthetic substrates provided the basis for the rational design and synthesis of specific, substrate-based, active site-directed inhibitors of the enzyme (6,13). One of the most potent of these inhibitors, N-[I(R,S)-

carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP- AAF-pAB), has been used to probe the function of the enzyme in vivo. Intravascular injection of this inhibitor has been shown to prolong the half-life of circulating LHRH (11) and to cause a pronounced decrease in blood pressure in normotensive rats (9). Although the mechanism of this hypotensive response has not been established, a recent report that cFP-AAF-pAB inhibits angiotensin converting enzyme (ACE) with a Ki in the submi- cromolar range suggested the possibility that inhibition of this enzyme is the underlying cause of the in vivo effect of the in- hibitor on blood pressure (5). This finding was surprising in view of the known structural requirements for binding of ligands to the active site of ACE, which would make effective binding of

Requests for reprints should be addressed to Dr. Christopher Cardozo, Box 1215, Department of Pharmacology, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029.

1259

1260 CARDOZO AND ORLOWSKI

compounds similar to cFP-AAF-pAB unlikely (8). We report here that further studies of the effect of cFP-AAF-pAB on ACE activity showed that the inhibitor has no intrinsic inhibitory activity toward angiotensin converting enzyme and that endo- peptidase 24.11 (EP 24.11 ) contaminating the ACE preparation cleaves the inhibitor to cFP-Ala-Ala and Phe-pAB, the former having angiotensin converting enzyme-inhibiting activity.

METHOD

Angiotensin converting enzyme from rabbit lung and fura- noacroyl-l-phenylalanylglycylglycine (Fa-Phe-Gly-Gly) were obtained from Sigma Chemical Co. (St. Louis, MO). cFP-AAF- pAB and N-[l(R,S)-carboxy-3-phenylpropyl]-Phe-p-amino- benzoate (cFP-F-pAB) were synthesized as described previously (13,16). Endopeptidase 24.11 was isolated from rat kidney as described (2). Aminopeptidase N was isolated from hog kidney and freed of contaminating endopeptidase 24.11 as described (2).

The ACE activity was determined with Fa-Phe-Gly-Gly as substrate by following the decrease in absorbance of the reaction mixture at 328 nm as described previously (10). Reaction mix- tures contained 3 ag of ACE, substrate (50 aM), and inhibitors in 50 m M Tris-HC1 buffer (pH 7.5) containing 300 m M NaCI. The total volume of the reaction was 1 ml. Reactions were in- cubated at 26°C for 30 min.

Degradation of cFP-AAF-pAB was followed by HPLC. Re- action mixtures contained the inhibitor (100 aM), 50 ag of ACE, and 50 m M Tris-HC1 (pH 7.5) containing 300 m M NaCI in a final volume of 500 al. Some reaction mixtures also contained cFP-F-pAB ( 10 aM), an inhibitor of endopeptidase 24.11 (16). Reactions were incubated for 1 h at 37°C and then subjected to HPLC on a reverse-phase C 18 aBondapak column (Waters; 0.39 × 30 cm). Products were eluted with a linear gradient es- tablished between 10% and 60% acetonitrile, each containing 0.1% TFA. The flow rate was maintained at 1 ml/min, and emerging peaks were monitored by absorbance at 210 nm. Peaks were collected manually and their amino acid composition was determined after acid hydrolysis as described previously (4).

Inhibition of ACE by cFP-Ala-AIa was determined in reaction mixtures similar to those described above for measurement of ACE activity, cFP-Ala-AIa was prepared enzymatically by cleaving the Ala-Phe bond of cFP-AAF-pAB with a homoge- neous preparation of endopeptidase 24.11. Thus, cFP-AAF-pAB (86 aM) was incubated in 50 m M Tris-HC1 (37°C; pH 7.5) with endopeptidase 24.11 in the presence of aminopeptidase N. The progress of the reaction was monitored by subjecting aliquots of the reaction mixture to HPLC. After complete conversion of the inhibitor to cFP-Ala-Ala and Phe-pAB and degradation of the latter product by the aminopeptidase, the reaction was stopped by inactivating the enzymes by heating the reaction mixture at 100°C for 5 min. The concentration of the inhibitor necessary for reducing the rate of the ACE catalyzed reaction by 50% (IC50) was determined.

RESULTS

The effect of cFP-AAF-pAB on the activity of ACE is shown in Table 1. In agreement with the results of Chappell et al. (5), the activity of ACE was inhibited with an apparent ICso of about I to 2 aM. However, previous work in this laboratory has shown that cFP-AAF-pAB can be cleaved at the Aia-Phe bond by en- dopeptidase 24. l 1 ( l 1), yielding as one of the products of the reaction cFP-Ala-Ala, which is structurally quite similar to known inhibitors of ACE, such as enalaprilat (cFP-Ala-Pro). Because EP 24.11 is a membrane-bound peptidase found in large

TABLE 1 INHIBITION OF ACE BY cFP-AAF-pAB AND EFFECTS OF cFP-F-pAB,

AN INHIBITOR OF EP 24.11

Percent Inhibition in the Presence cFP-AAF-pAB Percent of cFP-F-pAB

(~M) Inhibition (10 #M)

None 0 9 1 ~M 52 24

10 ~tM 70 36

Activity was determined by following the change in absorbance of Fa- Phe-Gly-Gly at 328 nm. Reactions contained ACE (3 vg), substrate (50 tzM), and inhibitors as indicated, and were brought up to 1 ml with 0.05 MTris-HCI, pH 7.5, containing 300 mM NaCI. Reactions were performed at 26°C. Data represent the mean of three determinations.

amounts in the lung (l l), it was necessary to consider the pos- sibility that inhibition of ACE was due to cleavage of the inhibitor by EP 24.11 contaminating the ACE preparation. Indeed, the inhibitory activity ofcFP-AAF-pAB toward ACE was markedly reduced when cFP-F-pAB, a specific and potent inhibitor of EP 24.11 (16), was included in the reaction mixtures (Table l). Control experiments showed that cFP-F-pAB has essentially no inhibitory effect on ACE (Table l).

To confirm that cleavage of the inhibitor occurred at the Ala- Phe bond, reactions were subjected to HPLC and the products were analyzed for amino acid composition and pAB content. Two products were obtained (Fig. 1). The first, containing only alanine, was identified as cFP-Ala-Ala, since the only other pos- sible peptide, cFP-Ala, contains a bond that is resistant to acid hydrolysis, cFP-Ala-Ala eluted as a doublet due to the slightly different retention times of the R and S diastereomers. The sec- ond peak contained Phe and pAB, thus confirming that the cleavage occurred exclusively at the Aia-Phe bond. As shown in Fig. I(A), incubation of the inhibitor with 50 #g of the enzyme preparation for 1 h resulted in virtually complete degradation of the inhibitor. Formation of cFP-Ala-Ala was inhibited by including in the reaction mixture cFP-F-pAB (2 aM), an inhibitor of EP 24.11 [Fig. l(B)].

The inhibitory potency of cFP-Ala-Ala for ACE was deter- mined in studies of effects of the inhibitor on the degradation of Fa-Phe-Gly-Gly. The ICso was found to be approximately 43 nM (four determinations). Using the equation describing the relationship between ICso and Ki for a competitive inhibitor,

Ki = IC5o/(1 + [S]/K,,)

where [S] is the substrate concentration (50 aM) and Km for this substrate is 300 a M (10), yields a value of Ki of 37 nM for cFP- Ala-Ala. Thus, cFP-AIa-Ala is a rather potent ACE inhibitor.

DISCUSSION

The data presented here provide evidence that cFP-AAF- pAB does not directly inhibit ACE. This conclusion is based on the finding that inhibition is dependent on cleavage of the in- hibitor at the Ala-Phe bond by an endopeptidase contaminating the ACE preparation. That the contaminating enzyme is identical with EP 24.11 was confirmed in experiments showing that cFP- F-pAB, a potent and specific inhibitor of EP 24.11 (16), abolished both cleavage of the Ala-Phe bond in cFP-AAF-pAB and the

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FIG. 1. Degradation ofcFP-AAF-pAB by the angiotensin converting enzyme preparation. For identification of the products, peaks were collected manually and subjected to amino acid analysis after acid hydrolysis. The peaks comprising cFP-Ala-Ala and cFP-AAF-pAB are seen as doublets due to the slightly different retention times of the R and S diastereomers of these compounds. (A) HPLC chromatogram of a reaction mixture containing cFP-AAF-pAB and the ACE preparation after incubation for 60 min. (B) Chromatogram of a reaction mixture containing cFP-AAF-pAB, ACE, and an inhibitor of EP 24. l l, cFP-F-pAB ( l 0 ~M). The concentration of cFP-F-pAB in the incubation mixtures is tenfold less than that of cFP-AAF-pAB, and thus this inhibitor is not seen on the chromatograms.

inhibitory effect of the EP 24.15 inhibitor on ACE. Correct in- terpretation of enzymatic reactions is always dependent on the purity of the enzyme preparation, and it is not surprising that the ACE preparations used in the current study contain contam- inating EP 24.11 since both these enzymes are highly concen- trated in the lung and are both associated with membrane frac- tions. Since the source of our enzyme preparation was the same as that used by Chappell et al. (5), it is likely that the preparation used in their study was also contaminated by EP 24.11, and that the inhibition of ACE was also due to formation of cFP-Ala- Ala. The same interpretation must also be applied to the inhi- bition observed by these investigators in studies of ACE in pooled rat blood, since EP 24.11 has been found in significant amounts in serum from both rats and humans (3,11). In contrast to our findings, Chappell et al. reported that incubation of cFP-AAF- pAB with the ACE preparation did not result in degradation of the inhibitor. Although the reaction conditions were not speci- fied, it is likely that the amounts of enzyme and inhibitor used by these authors in the reaction mixture were insufficient to detect the degradation products of the inhibitor by HPLC. Also, since the Ki of cFP-AA toward ACE is about 37 nM, it would have been sufficient at a concentration of 1 #M of cFP-AAF- pAB to degrade less than 4% of the inhibitor (an amount almost impossible to detect) to obtain significant inhibition of ACE.

The finding that intravenous infusion of cFP-AAF-pAB into normotensive rats causes a profound decrease in blood pressure poses the question of the mechanism of this effect. Attenuation of this response by pretreatment of the animals with a B2 bra- dykinin receptor antagonist, and potentiation of the hypotensive effect of bradykinin by cFP-AAF-pAB, suggested that the inhib- itor prevents the in vivo degradation of bradykinin. However, since ACE, EP 24.11, and EP 24.15 are capable of degrading bradykinin, all of these enzymes must be considered as potential targets of inhibitor action. It is unlikely that the hypotensive response is due to inhibition by cFP-AAF-pAB of EP 24.11, since its K~ toward this enzyme is more than three orders of magnitude higher than toward EP 24.15. However, in vivo in- hibition of ACE must be considered since endogenous EP 24.11 is capable of converting cFP-AAF-pAB into an ACE inhibitor. That EP 24.11 cleaves cFP-AAF-pAB in vivo is indicated by findings that pretreatment of animals with an inhibitor of EP 24.11 before infusion ofcFP-AAF-pAB reduces urinary excretion ofpAB by one hundredfold and increases serum concentrations of cFP-AAF-pAB by more than tenfold (11). Moreover, studies by Yang and coworkers indicate that significant inhibition of ACE appears after injection of cFP-AAF-pAB (17). Further study of the possibility that the hypotensive response to cFP-AAF- pAB is due to conversion of the inhibitor to cFP-AIa-Ala will

1262 C A R D O Z O A N D ORLOWSKI

require additional experiments in which the effect of the inhibitor on blood pressure is studied under conditions in which its con- version to an ACE inhibitor is abolished by pretreatment of animals with an inhibitor of EP 24.11.

ACKNOWLEDGEMENTS

This work was supported by Grant DK 25377 (to M.O.) and by an NRSA fellowship (HL 08254) from the National Institutes of Health (to C.C.).

REFERENCES

1. Acker, G. R.; Molineaux, C.; Orlowski, M. Synaptosomal membrane- bound form of endopeptidase-24.15 generates leu-enkephalin from dynorphin ~-s, a- and ~-neoendorphin and met-enkephalin from met- enkephalin-Arg6-GlyLLeu s. J. Neurochem. 48:284-292; 1987.

2. Almenoff, J.; Orlowski, M. Membrane-bound kidney metalloen- dopeptidase: Interaction with synthetic substrates, natural peptides and inhibitors. Biochemistry 22:590-599; 1983.

3. Almenoff, J.; Teirstein, A. S.; Tfiornton, J. V.; Orlowski, M. Iden- tification ofa thermolysin-like metalloendopeptidase in serum: Ac- tivity in normal subjects and in patients with sarcoidosis. J. Lab. Clin. Med. 103:420-431; 1984.

4. Cardozo, C.; Vinitsky, A.; Hidalgo, M. C.; Michaud, C., Orlowski, M. A 3,4-dichloroisocoumarin resistant component of the multi- catalytic proteinase complex. Biochemistry 31:7373-7380; 1992.

5. Chappell, M. C.; Welches, W. R.; Brosnihan, K. B.; Ferrario, C. M. Inhibition of angiotensin converting enzyme by the metalloendo- peptidase 3.4.24.15 inhibitor c-phenylpropylalanylalanyl-phenyl- alanyl-p-aminobenzoate. Peptides 13:943-946; 1992.

6. Chu, T. G.; Orlowski, M. Active site directed N-carboxymethyl pep- tide inhibitors of a soluble metalloendopeptidase from rat brain. Biochemistry 23:3598-3603; 1984.

7. Chu, T. G.; Orlowski, M. Soluble metalloendopeptidase from rat brain: Action on enkephalin-containing peptides and other bioactive peptides. Endocrinology 116:1418-1425; 1985.

8. Ehlers, M. R. W.; Riordan, J. F. Angiotensin-converting enzyme: New concepts concerning its biological role. Biochemistry 28:5311- 5318; 1989.

9. Genden, E. M.; Molineaux, C. J. Inhibition ofendopeptidase-24.15 decreases blood pressure in normotensive rats. Hypertension 18: 360-365; 1991.

10. Holmquist, B.; Bunning, P.; Riordan, J. F. A continuous spectro- photometric assay for angiotensin converting enzyme. Anal. Biochem. 95:540-548; 1979.

1 I. Lasdun, A.; Reznik, S.; Molineaux, C. J.; Orlowski, M. Inhibition of endopeptidase 24.15 slows the in vivo degradation of LHRH. J. Pharmacol. Exp. Ther. 251:439-447; 1989.

12. Molineaux, C. J.; Lasdun, A.; Michaud, C.; Orlowski, M. Endopep- tidase-24.15 is the primary enzyme which degrades luteinizing hor- mone releasing hormone both in vitro and in vivo. J. Neurochem. 51:624-633; 1988.

13. Orlowski, M.; Michaud, C.; Molineaux, C. J. Substrate-related potent inhibitors of brain metalloendopeptidase. Biochemistry 27:597-602; 1988.

14. Orlowski, M.; Michaud, C.; Chu, T. G. A soluble metalloendopep- tidase from rat brain. Purification of the enzyme and determination of specificity with synthetic and natural peptides. Eur. J. Biochem. 135:81-88; 1983.

15. Orlowski, M.; Reznik, S.; Ayala, J.; Pierotti, A. R. Endopeptidase 24.15 from rat testis. Isolation of the enzyme and its specificity toward synthetic and natural peptides, including enkephalin-containing peptides. Biochem. J. 261:951-958; 1989.

16. Pozsgay, M.; Michaud, C.; Liebman, M.; Orlowski, M. Substrate and inhibitor studies of thermolysin-like neutral metalloendopep- tidase from kidney membrane fractions. Comparison with bacterial thermolysin. Biochemistry 25:1292-1299; 1986.

17. Yang, X.; Scicli, A. G.; Carretero, O. A. A metalloendopeptidase 24-15 inhibitor: Effect on blood pressure and renal function. Hy- pertension 21:549; 1993 (abstract).