The inhibition of carboxypeptidase A and angiotensin-converting enzyme by target enzyme-activated inhibitors and N-acylhydrazones

Abstract

Zinc metallo-peptidases such as carboxypeptidase A (CPA), angiotensin converting enzyme (ACE), matrix metalloenzymes and enkephalinase, play important roles in human biology and are therefore of interest in fundamental and practical research. In each of these enzymes a Zn^2+ ion, which is catalytically essential, is bound in the active site. Among the most effective inhibitors for these enzymes are compounds designed to bind competitively to the active site through strong metal-inhibitor interactions. The use of metal binding inhibitors as therapeutic agents against specific enzymes may suffer from the problem of undesirable side affects resulting from non-specific interactions with other metalloenzymes (e.g., Captopril for inhibition of ACE). Incorporation of the metal binding moiety within a substrate analogue such that the metal binding group will be released only upon interaction with the target enzyme may be a solution. In this study it has been found that thioester substrate analogues can act as mechanism-based inhibitors of carboxypeptidase A and angiotensin converting enzyme. Enzymatic hydrolysis of the thioester group releases a free thiol inhibitor which binds tightly to the active site through a strong ZN^2+-sulfur interaction and non-covalent interactions with the active site region.

The thioester (S)-2-(S-benzoylthio)-3-phenylpropanoic acid (50) was designed as a mechanism-based inhibitor of CPA, where the CPA-catalyzed hydrolysis would result in the release of thiol inhibitor (S)-2-mercapto-3-phenylpropanoic acid (35). Examination of the potency of 35 as an inhibitor of the peptidase activity of CPA has revealed that it is a much more potent inhibitor (Ki=12.6 nM) than previously reported (Ki=1.2uM) and that previous studies had likely examined inhibition by the disulfide formed by an unusually facile air oxidation of 35.

The hydrolysis of 50 by CPA was found to proceed with burst kinetics at room temperature. The pre-steady state phase of the process was accompanied by a burst of proton release and a lag in thiol release. The similarity of the kinetic parameters found for CPA-catalyzed hydrolysis of 50 and those found for hydrolysis of O-benzoyl-(S)-phenyllactate suggest that the rate limiting step in each case may involve the release of benzoate. A molecular mechanisms consistent with these observations is proposed.

The thioester 50 has been shown to be an effective time-dependent inhibitor of the peptidase activity of CPA thus demonstrating the feasibility of target enzyme-activated inhibition by a latent metal binding agent. Similarly, CBZ-Phe-Y[CO-S]-Ala-Phe (86) has been synthesized and shown to be an effective enzyme-activated inhibitor of the dipeptidase activity of ACE.

In addition, N-acylhydrazones, which are known metal binding agents currently being explored as potential therapeutic agents in several areas including as potential anti-HIV agents through inhibition of HIV-1 reverse transcriptase (RT), have been studied as potential inhibitors of ACE and CPA. Although none of these compounds was found to be an effective inhibitor of ACE, one such compound, N-(4-t-butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone (NAH306), which is a potent inhibitor of RT, was found to inhibit CPA. The discovery that this compound inhibits CPA, not by removal of the active site metal ion, but by binding to the enzyme in a mixed uncompetitive-competitive mode has led to the proposal of a molecular mechanism involving binding to the S1 and S2 subsites of free CPA as well as to the binary complex of CPA with phenylalanine, the hydrolysis product of the peptidase substrate hippuryl-L-phenylaline.