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

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, K,= 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. ACE. Injection of 1 mg kg-' inhibitor resulted in plasma concentrations at 10 s of 23.5 ,uM (cFP-AAYpAB) and 18.0 giM (cFP-AVY-pAB), which fell 100 fold over 5 min. Co-injection of 1251I-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.

Role of bradykinin receptors in the renal effects of inhibition of angiotensin converting enzyme and endopeptidases 24.11 and 24.15 in conscious rabbits

“…The treatments and P values are the same as for Figure 2. rabbits. However, these effects are entirely attributable to inhibition of ACE by the metabolite cFP-AA (Telford et al, 1995;Lew et al, 1996). Similar observations have been made in anaesthetized rats in which the phenylalanine substituted analogue cFP-AAF-pAB was used (Yang et al, 1994).…”

“…Although both cFP-AAY-pAB and cFP-AAF-pAB have low affinity for EP 24.11, they can inhibit this enzyme at higher concentrations (Ki values of 35 and 17 gM respectively). We have found previous in studies in conscious rabbits that a bolus dose of cFP-AAY-pAB of 5 mg kg-1, although rapidly cleared from the circulation (100 fold lower levels in 5 min), results in peak blood levels over 100 gM (Lew et al, 1996), which would be expected to inhibit EP 24.11. We chose to use a dose of 0.1 mg kg-' Hoe 140 in our subsequent experiment, and unexpectedly found that it increased Na+ excretion.…”

“…These studies did not supported by the results of our own experiments in conscious rabbits, in which we found that a similar i.v. dose of cFP-AAY-pAB (8.3 jimol kg-'/5 mg kg-'), although rapidly cleared from the circulation, resulted in peak plasma levels of 137 + 31 gM (Lew et al, 1996). Therefore, in the present study we have tested the renal effects of cFP-AAY-pAB in rabbits pretreated with both the ACE-inhibitor, captopril and the EP 24.11 inhibitor, SCH 39370 (Sybertz et al, 1989), both of which have negligible activity as inhibitors of EP 24.15 (Acker et al, 1987;R.A.…”

Development and characterization of novel potent and stable inhibitors of endopeptidase EC 3.4.24.15

“…Biological stability was enhanced 20-fold (Ser) and 10-fold (Val, Leu) in comparison with cFP. However, the Val-substituted analogue was cleared from the circulation of conscious rabbits at the same rate as cFP and, like cFP, blunted the pressor response to angiotensin I, again indicative of ACE inhibition [13].…”

“…l K i (1j[S]\K m ). The K m had been determined as 19.8 µM by a Lineweaver-Burk analysis of initial velocities measured in triplicate at a range of QFS concentrations (1.1-140 mM) [13]. ACE (human plasma) was assayed by using the fluorimetric ACE method, as described by Friedland and Silverstein [14], which measures the degradation of hippuryl-His-Leu.…”