Potent Angiotensin Converting Enzyme Inhibitor Research Articles

Ace inhibitors as a template for the design of bradykinin B 2 receptor antagonists

Angiotensin converting enzyme (ACE) degrades both angiotensin II and bradykinin. Accordingly, we hypothesize that ACE inhibitors can serve as models to design antagonists for the bradykinin receptor. The potent ACE inhibitor Quinapril was modified to serve as a spacer separating two lipophilic positive charges required for bradykinin binding. The synthesis and bradykinin receptor activity of a series of antagonists 2–10 based on this hypothesis is described in this report.

Identification and properties of a peptidyl dipeptidase in the housefly, Musca domestica, that resembles mammalian angiotensin-converting enzyme.

[D-Ala2,Leu5]Enkephalin was readily metabolized by membranes (40,000 g pellet) prepared from heads of the housefly, Musca domestica, with Gly3-Phe4 being the major site of cleavage. This hydrolysis was only partially inhibited (40%) by 10 microM phosphoramidon, an inhibitor of endopeptidase-24.11, but was almost totally abolished in the presence of a mixture of 10 microM phosphoramidon and 10 microM captopril, a potent inhibitor of mammalian angiotensin-converting enzyme (ACE). An assay for ACE employing Bz-Gly-His-Leu as the substrate was used to confirm the presence of an ACE-like peptidyl dipeptidase activity in fly head membranes. The peptidase had a Km of 1.91 mM for Bz-Gly-His-Leu and a pH optimum of 8.2. The activity was inhibited by 100 microM EDTA and was greatly activated by ZnCl2 but not other bivalent metal ions. Captopril, lisinopril, fosinoprilat and enalaprilat, all selective inhibitors of mammalian ACE, were also good inhibitors of the insect enzyme with IC50 values of 400 nM, 130 nM, 16 nM and 290 nM respectively. An M(r) value of around 87,000 was obtained for this enzyme from gel-filtration chromatography, indicating that the insect enzyme is similar in size to mammalian testicular ACE (M(r) = 90,000-110,000) and not the larger form of the enzyme (M(r) = 150,000-180,000) found in mammalian somatic tissues. The fly peptidyl dipeptidase was released from membranes into a soluble fraction by incubating the head membranes at 37 degrees C but not at 0 degree C, suggesting that the insect ACE-like enzyme can be solubilized from cell surfaces through the activity of a membrane-bound enzyme activity. In conclusion, we have shown the existence of a peptidyl dipeptidase in membranes from the heads of M. domestica, which has similar properties to those of mammalian ACE.

A quantum mechanical conformational search of captopril, a potent inhibitor of the angiotensin-converting enzyme

A full conformational search of the potent angiotensin-converting enzyme (ACE) inhibitor captopril is determined at the semiempirical AM1 level with a modified version of ampac (version 2.2). In particular, 216 initial conformations of captopril are optimized, generating 123 unique structures within 10.8 kcal mol −1 of the global minima. The lowest energy conformation is two-fold degenerate, containing both a trans-syn and a cis-syn structure. The lowest trans-anti conformation is 1.2 kcal mol −1 higher and the lowest energy cis-anti structure is 4.3 kcal mol − above the global minima. These results are compared to previous calculated and experimental results.

Potentiation by ACE inhibitors of the dilator response to bradykinin in the coronary microcirculation: interaction at the receptor level.

1. To examine the possibility that angiotensin-converting enzyme (ACE) inhibitors modulate the action of bradykinin at the receptor level, their effect on the dilator response to bradykinin was studied in the isolated saline-perfused heart of the rabbit. 2. Continuous infusion of bradykinin (10 nM) elicited a transient decrease in coronary perfusion pressure (CPP) and increased prostacyclin (PGI2) release which returned to baseline values within 30 min. 3. Subsequent co-infusion of ramiprilat (> or = 10 nM) or moexiprilat, but not of the less potent ACE inhibitor n-octyl-ramipril (RA-octyl), caused another fall in CPP and an increase in PGI2 release, the magnitude and time course of which were almost identical to the first response to bradykinin. No change in CPP or PGI2 release was observed when the ACE inhibitors were administered in the absence of exogenous bradykinin. 4. Infusion of D-Arg[Hyp3]-bradykinin (10 nM), a specific B2-receptor agonist which was significantly more resistant to degradation by ACE than bradykinin, produced virtually identical changes in CPP and PGI2 release when compared to bradykinin. Subsequent co-infusion of ramiprilat was similarly effective in restoring the fall in CPP and increase in PGI2 release elicited by D-Arg[Hyp3]-bradykinin as in the presence of bradykinin. 5. In concentrations which should block the degradation of bradykinin by ACE in the coronary vascular bed, two ACE substrates, hippuryl-L-histidyl-L-leucine (0.2 mM) and angiotensin I (0.3 microM), were unable to elicit a significant change in CPP or PGI2 release while ramiprilat and another ACE inhibitor, quinaprilat, were still active in the presence of these substrates. 6. To reveal the potential B2-receptor action of ramiprilat, its effect on the constrictor response to bradykinin was studied in the rabbit isolated jugular vein. Ramiprilat (0.1 MicroM), but not RA-octyl (1 MicroM),potentiated the endothelium-independent, B2-receptor-mediated constrictor response to bradykinin, but not that to the thromboxane-mimetic U46619 (9,11-dideoxy-ll alpha,9 alpha-epoxymethano-prostaglandin F2.).Moreover, ramiprilat but not RA-octyl caused a concentration-dependent, B2-receptor antagonist sensitive increase in tone when administered alone.7. These findings suggest that an interaction of ACE inhibitors with the B2-receptor or its signal transduction pathway rather than an accumulation of bradykinin within the vascular wall is responsible for the restoration of the endothelial response to bradykinin (dilatation, PGI2 release) in the coronary vascular bed of the rabbit.

Trandolapril Dose-Response in Spontaneously Hypertensive Rats

The dose-related effects of trandolapril on serum angiotensin-converting enzyme (ACE) activity, blood pressure and cardiac hypertrophy were studied after 2-week oral treatment in adult spontaneously hypertensive rats. Trandolapril caused a dose-dependent decrease in mean blood pressure at doses of 0.03-3 mg/kg. Inhibition of serum ACE was demonstrated by even the lowest dose (-9% at 0.003 mg/kg), was about 40% at 0.03 mg/kg, and rose to 84% at 3 mg/kg. Regression of cardiac hypertrophy (heart:body weight) was seen at doses of trandolapril as low as 0.03 mg/kg (-5.1%), which was also the minimal effective antihypertensive dose. Clear dose-response curves were observed for trandolapril from 0.03 mg/kg upwards with respect to hypotensive effect and regression of cardiac hypertrophy, which were not seen with enalapril. Enalapril had no significant effect on plasma ACE activity except at the highest dose (10 mg/kg), despite demonstrating hypotensive effects with smaller doses. These results indicate that trandolapril is a more potent (by a factor of about 30) inhibitor of ACE than enalapril (only 34% inhibition at 10 mg/kg) and causes a greater degree of regression of cardiac hypertrophy.

Cough induced activity of spirapril in rats

1. 1. We examined the effect of spirapril, a potent angiotensin converting enzyme (ACE) inhibitor, on the number of capsaicin-induced coughs in rats and compared with that of enalapril. 2. 2. Chronic treatment with enalapril, at doses of 1 and 3 mg/kg, p.o., significantly and dose-dependently enhanced the number of capsaicin-induced coughs. 3. 3. Chronic treatment with higher dose of spirapril (3 mg/kg, p.o.) also significantly enhanced the number of capsaicin-induced coughs. However, lower dose (1 mg/kg, p.o.) of spirapril had no significant effect on the number of capsaicin-induced coughs. 4. 4. These results suggest that cough induced activity, one of the most serious side effects associated with chronic treatment with ACE inhibitors, of spirapril is relatively lower than that of enalapril.

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.

It was reported recently that N-1-(R,S)carboxy-3-phenylpropyl-Ala-Ala-Phe-p-carboxyanilide (CPP-A-A-F-pAB), an inhibitor of endopeptidase 3.4.24.15 (E-24.15), also inhibits angiotensin-converting enzyme (ACE) from rabbit lung. We have found that this compound is without effect on ACE purified from pig kidney, at a concentration some 1000-fold greater than the Ki reported for inhibition of the enzyme from lung. However, preincubation of CPP-A-A-F-pAB with neutral endopeptidase 3.4.24.11 (E-24.11) does result in potent inhibitory effects on ACE. We have shown this to be due to formation of a fragment, CPP-A-A, the structure of which is closely related to ACE inhibitors such as enalaprilat. CPP-A-A was found to be a potent inhibitor of pig ACE. Under the conditions used it had an IC50 value of 1.6 x 10(-8) M, compared with the value obtained for captopril of 7.5 x 10(-10) M. These results have important implications for studies of E-24.15 when using CPP-A-A-F-pAB in vivo or in crude tissue extracts where E-24.11 might also be present.

Stereochemistry-activity relationships of ACE inhibitors. Conformational studies by1H and13C NMR of perindopril and selected stereoisomers

Perindopril, the tert-butylamine salt of 1-{(2S)-2-[(1S)-(1-carbethoxybutyl)amino]-1-oxopropyl}-(2S,3aS,7aS)-perhydroindole-2-carboxylic acid, is an inhibitor of angiotensin-converting enzyme (ACE) and a new drug for the treatment of hypertension. The interaction between the inhibitor and the enzyme was investigated by studying the active diacid metabolite of perindopril, its stereoisomers and a desmethyl analogue. The pharmacological study allowed the measurement of the in vitro activities of the different compounds. The 1H and 13C NMR studies have shown that the cis-trans equilibrium about the amide bond is strongly dependent on the configuration of the chiral centres and on the pH of the solution. The pKa of the different acid-base species were measured. The results show that perhydroindole derivatives are potent inhibitors of ACE as long as they fulfil the following basic requirements: (1) an S configuration of the carbon bearing the terminal carboxy group; (2) an S methyl substituent in the alanine residue; however, the inhibitor potency is not modified on the replacement of the alanine residue of the perindopril by a glycine residue; and (3) less stringently, an S configuration of the C-1 butyl carbon. Under these conditions the Zn binding ligand (chain carboxylate group) is devoid of steric hindrance in the trans conformers. No direct relationship appeared between the relative amount of the trans form and the activity. The cyclic skeleton of the perhydroindole derivatives provided a strong hydrophobic interaction with the active enzymatic site, whatever the configurations at C-3a and C-7a. Lipophilic interactions involving the different parts of the inhibitor are not independent of each other.

Lisinopril, A New Angiotensin Converting Enzyme Inhibitor

The synthesis and biological activity of functionalized lisinopril, a potent angiotensin converting enzyme (ACE) inhibitor is described. Selective functionalization of lisinopril is achieved at the secondary amino position by a photochemical method, whereas esterfication of the carboxylic groups and modification at the primary amino group is achieved by chemical methods. Autoradiographic investigations using competitive125I radioactive binding assays with the modified lisinopril reveal that the terminal amino group modification enhanced the binding to ACE, whereas the secondary amino group functionalization did not differ significantly from the binding properties of native lisinopril. However, esterification of the carboxyl groups reduced the inhibitory potentency from nMto μM. These results suggest that lisinopril can be derivatized with preservation of inhibition potency toward ACE. These modifications may find utility in the development of photoaffinity labeling agents for ACE or to incorporate bifunctional chelating agents carrying diagnostic radiometals for the development of cardiac imaging agents.

Atrial and brain natriuretic peptides: secretion during exercise in patients with essential hypertension and modulation by acute angiotensin-converting enzyme inhibition.

1. This study examined whether brain and atrial natriuretic peptides (BNP, ANP) are secreted together through the coronary sinus from the heart, and whether plasma concentrations of BNP and ANP were affected by ergometric exercise in patients with essential hypertension. The effects of temocapril, a potent angiotensin-converting enzyme (ACE) inhibitor, on plasma concentrations of these peptides was also examined. 2. The plasma concentrations of immunoreactive (ir) BNP and ir-ANP in the coronary sinus in seven patients with ischaemic heart disease during cardiac catheterization were far greater than values with plasma obtained at the same time from the femoral artery. 3. The plasma concentrations of ir-BNP and ir-ANP increased with exercise and were correlated with each other. Temocapril reduced the blood pressure and slightly (but significantly) decreased the levels of both peptides at rest and during exercise. 4. The results suggest that BNP and ANP were secreted together through the coronary sinus from the heart. The secretion was increased by exercise and suppressed by acute ACE inhibition. The increase in these peptides during exercise may reflect a compensatory mechanism against further elevation of blood pressure.

ANGIOTENSIN CONVERTING ENZYME INHIBITOR IMPROVES CARDIAC HYPERTROPHY AND FAILURE IN A RAT MODEL OF MYOCARDIAL INFARCTION

The usefulness of angiotensin converting enzyme (ACE) inhibitor in the treatment of congestive heart failure is well known. In order to study the effect of ACE inhibitor on compensatory cardiac hypertrophy and failure, captopril, a potent ACE inhibitor, was administered to experimental myocardial infarction (MI) rats.The systolic blood pressure was significantly lowered and the left ventricular end-diastolic pressure was lowered in captopril-treated MI rats. Both the left and right ventricular weight/body weight ratios were significantly greater in non-treated MI rats than in controls and captopril-treated MI rats, while there were no significant differences between the latter two groups.Percent V3 myosin heavy chain was increased in non-treated MI rats, but this increase was obviously suppressed by administration of captopril for 4 weeks. β-MHC mRNA expression determined by northern blotting analysis was also distinctly lower in 1 week treated rats than in non-treated MI rats. The plasma atrial natriuretic peptide (ANP) concentration andγ-rANP mRNA expression were significantly lower in captopril-treated MI rats than in non-treated MI rats.Thus, captopril induced regression of cardiac hypertrophy, reversed the cardiac MHC profile and improved hemodynamic alterations in experimental MI rats. The present results further suggest that not only afterload reduction but also preload reduction may be responsible for the beneficial effects of captopril in cardiac hypertrophy and failure.

Effect of chronic hypertension on the blood-brain barrier permeability of libenzapril.

Very little information is available on the permeability of the blood-brain barrier (BBB) to small polar drugs in chronic hypertension. The blood and cerebrospinal fluid (CSF) pharmacokinetics of libenzapril (LZP), a potent angiotensin converting enzyme inhibitor, were investigated in hypertensive (SH) and normotensive (SD) rats. Following intravenous bolus administration of this hydrophilic drug, the terminal rate constant for elimination (beta), steady-state volume of distribution (Vdss), and systemic clearance (CL) were similar in these two animal groups. Other pharmacokinetic parameters (Cpo, alpha, k12, and k21) were significantly (P less than 0.05) greater in the hypertensive group, except for the volume of the central compartment (Vc) and ratio of Vc to Vdss, which were smaller in SH rats. The ratio of area under the concentration-time curve (AUC) in CSF to blood was about twofold higher in SH rats compared to normotensive rats, showing increased BBB permeability in hypertensive rats. An acute brain uptake study was also performed in SH, SD, and WK rats by intracarotid administration of 14C-LZP along with 3H2O as a reference marker. Both LZP and water transport was found to be significantly higher (about two- to five-fold) in six of the seven different brain regions in SH rats as compared to the normotensive (SD and WK) controls. Because of this simultaneous increase in concentrations of both the drug and the reference marker, BUI values were not affected. Regional brain concentrations in SH rats were also linearly correlated with the mean arterial pressure (MAP) values, providing further evidence of the systemic pressure related increase in BBB permeability.

Evidence for the involvement of sperm angiotensin converting enzyme in fertilization.

Recently it has been observed that ejaculated human sperm possess high angiotensin converting enzyme (ACE) activity and that this enzyme is released during the process of capacitation. This observation raises the possibility that ACE may be involved in the fertilization process. To verify this hypothesis, we tested the effects of a potent ACE inhibitor, Captopril, on acrosome reaction induced by capacitating medium (3.5% HSA-added BWW) and on ability of human capacitated spermatozoa to penetrate zona-free hamster oocytes. Addition of Captopril (100 nmol l-1) modified neither sperm motility nor viability at any time considered, but significantly reduced the acrosome reaction percentages of sperm incubated in capacitating medium. Furthermore, Captopril significantly reduced the percentage of penetrated oocytes. The mean penetration rates both in the absence and presence of Captopril were 65.5 +/- 4.9% and 26.9 +/- 2.3% (P less than 0.001) respectively. These findings provide evidence that sperm release of ACE during capacitation may have a physiological role in the regulation of the mechanisms that allow sperm acrosome reaction and thus fertilizability.

The pharmacokinetics of benazepril relative to other ace inhibitors

Benazepril is a prodrug that, following rapid conversion to benazeprilat, is a potent nonsulfhydryl inhibitor of angiotensin-converting enzyme. The absorption, bioactivation, distribution, and elimination of benazepril and benazeprilat have been evaluated in healthy subjects, hypertensive patients, and patients with characteristics known to alter the pharmacokinetic disposition of ACE inhibitors, such as renal impairment, hepatic impairment, and advanced age. Following oral administration, benazepril is absorbed and transformed into benazeprilat in the liver. Coadministration of benazepril with food delays absorption slightly but does not affect the ultimate bioavailability of benazeprilat. Severe hepatic impairment slows conversion of benazepril to benazeprilat but does not affect the overall bioavailability of benazeprilat; thus dosage adjustment is not necessary in the hepatically impaired population. Mild-to-moderate renal impairment (creatinine clearance greater than 30 ml/min) slightly increases benazeprilat concentrations; severe renal impairment (creatinine clearance less than 30 ml/min) reduces benazeprilat elimination and requires dosage reduction. In elderly patients, benazepril disposition is the same as in younger patients, although benazeprilat clearance is slightly reduced. No clinically significant drug-drug interactions occur with benazepril and many other medications commonly prescribed to elderly hypertensive patients. The pharmacokinetic characteristics of benazepril are stable over a wide range of conditions, and dosage adjustments for pharmacokinetic reasons are required infrequently.

Quinapril

Quinapril is a monoethyl ester which is hydrolysed after absorption to form an active metabolite, quinaprilat, which is a more potent angiotensin converting enzyme (ACE) inhibitor than the parent drug. Quinaprilat has a short elimination half-life but a potent binding affinity for ACE which enables once daily administration. Data from clinical studies indicate that quinapril 10 to 40 mg daily, given as a single dose, is an effective antihypertensive agent, suitable as monotherapy for reducing high blood pressure and maintaining satisfactory control during long term treatment of mild to severe hypertension. Dosages of 80 mg daily have been used in some patients. Concomitant diuretic therapy usually elicits a response in patients who fail to respond adequately to monotherapy. Initial studies suggest that quinapril also has a role in the treatment of mild to severe congestive heart failure. In the few long term studies conducted the beneficial acute haemodynamic effects were maintained during long term treatment and were accompanied by symptomatic and functional improvement. The majority of these patients responded to twice daily administration. Adverse effects associated with the antihypertensive action of quinapril are generally mild, well tolerated and are similar to those of other ACE inhibitors. Thus, quinapril appears to be a useful alternative ACE inhibitor for the treatment of mild to severe hypertension and congestive heart failure.

Molecular and crystal structures of MDL27,467A hydrochloride and quinapril hydrochloride, two ester derivatives of potent angiotensin converting enzyme inhibitors

The molecular structures of MDL27,467A hydrochloride, [4 alpha,7 alpha(R*),12b beta]-7-[[1-(ethoxycarbonyl)-3-phenyl-propyl] amino]-1,2,3,4,6,7,12a,12b-octahydro-6-oxopyrido[2,1- a][2]benzazepine-4-carboxylic acid diphenylmethyl ester hydrochloride, and quinapril hydrochloride, [3S-[2[R*(R*)],3R]]-2-[2[[1- (ethoxycarbonyl)-3-phenylpropyl]-amino]-1-oxopropyl]-1,2,3,4- tetrahydro-3-isoquinolinecarboxylic acid hydrochloride, were determined by X-ray diffraction methods. The modified, C-terminal dipeptide portions and the phenylpropyl fragments in both crystal structures adopt similar conformations. The binding positions for several pharmacophores are defined by the constraint of the tricyclic system in the crystallographic structure of MDL27,467A hydrochloride. Conformational energy calculations show that the phenyl ring of the tetrahydro-3-isoquinoline system of quinapril does not fit into the S2 hydrophobic pocket of angiotensin converting enzyme.

Vascular Effects of Perindopril

Vascular remodeling is central to the pathophysiology of hypertension and atherosclerosis. The effects of antihypertensive drugs on this process are important to consider from a mechanistic and a pathogenetic point of view in relation to vascular complications of hypertension, e.g., decrease in vascular reserves, shift in cerebral blood flow autoregulation and atherosclerosis development. There is now evidence that, in addition to several other growth factors, vasoactive peptides such as angiotensin II may act as vascular smooth muscle growth promoting substances. Based on these data, the effects of perindopril, a potent and long-lasting angiotensin-converting enzyme (ACE) inhibitor, on structural and mechanical properties of the arterial wall, have been studied in animal models of hypertension as well as in humans. Perindopril completely reversed aortic medial hypertrophy and arterial stiffening observed in renovascular hypertensive rats. Similar benefits were reported in mesenteric resistance vessels of spontaneously hypertensive rats. The effect of perindopril was totally in keeping with potent inhibition of vascular ACE and emphasized the potential role of angiotensin II as a vascular growth modulator. Clinical studies confirmed animal experiments; both suggest that increases in arterial compliance and distensibility following perindopril is likely to be related to drug-induced modification of the arterial wall, at least partially independently of blood pressure reduction. The increase in arterial compliance was associated with a selective decrease in pulse pressure, a finding that is important, not only for the arterial wall, but also for the structure and function of the hypertensive heart.

Bradykinin-Induced Contractions of Canine Prostate and Bladder: Effect of Angiotensin-Converting Enzyme Inhibition

Alpha-adrenergic, cholinergic, and serotonergic receptor-mediated contractile responses have been well characterized in the genitourinary tissues of several mammalian species. The present study characterizes the in vitro contractile responsiveness of canine bladder and prostate to the peptides, bradykinin, angiotensin I, and angiotensin II. All preparations contracted to 0.15M KCl. Bradykinin elicited contractile responses in both prostate (10−10 to 10−7 M) and bladder (10−10 to 10−6 M). In both tissues, angiotensin II produced minimal responses and angiotensin I failed to elicit contractions. The potent angiotensin converting enzyme (ACE) inhibitor, enalaprilic acid [MK-422] (10−6 M) increased the contractile response of the prostate to bradykinin two-fold while having no effect on bradykinin-induced contractions in the bladder. Enalaprilic acid did not affect the contractile responses of the two tissues to angiotensin I or angiotensin II. The canine urogenital tissue contractile responses to bradykinin, angiotensin I, and angiotensin II may have relevance to human physiology. Previous studies have demonstrated that human prostatic tissue, specifically benign prostatic hyperplasia (BPH), has the highest concentration of ACE activity of tissues evaluated. Bradykinin is a potent peptidergic contractile agent in canine bladder and prostate. The activity of enalaprilic acid to amplify the bradykinin-induced contractions in the canine prostate is consistent with high levels of ACE in the tissue. These data confirm the sensitivity of the canine prostate to bradykinin and report for the first time, the ability of bradykinin to induce contractions in the prostate. These studies support the possibility that bradykinin may be involved in mediating micturition under normal and pathological states such as infravesical obstruction secondary to BPH. Furthermore, the results from these investigations in canine urogenital tissues, if applicable to humans, suggest that urinary function be closely monitored in patients receiving ACE inhibitor therapy. (J. Urol, 144: 390-392, 1990)

A deeply recessed active site in angiotensin-converting enzyme is indicated from the binding characteristics of biotin-spacer-inhibitor reagents

Two biotinylated derivatives of the angiotensin-converting enzyme (ACE) inhibitor, lisinopril, were synthesized. Compounds BL11 (ε-biotinamidocaproyl-lisinopril) and BL19 (ε-biotinamidocaproyl-β-alanyl-β-alanyl-lisinopril) have, respectively, 11 and 19 atoms of spacing structure between the biotin and the inhibitor moieties. Both compounds were found to be potent inhibitors of mouse kidney ACE, but they lost this ability in the presence of streptavidin in free solution. However, BL19 (but not BL11), when complexed to ACE, retained enough residual binding strength for streptavidin to allow the complex to be specifically removed from solution by streptavidin-agarose beads. It was thus possible to employ BL19 for the affinity isolation of ACE from crude mixtures. These results indicate that the bound determinant of lisinopril must lie at least 11 Å below the outer surface of the ACE molecule.

Production of angiotensin-converting enzyme inhibitors from baker's yeast glyceraldehyde-3-phosphate dehydrogenase.

Angiotensin-converting enzyme (ACE) inhibitors were excised from the molecule of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) preparation of baker's yeast by heating at 120 degrees C in 1 M AcOH-20 mM HCl. Three inhibitors were then purified by gel-permeation and reverse-phase chromatographies. One of the yeast ACE inhibitors, YG-3, was GAPDH peptide 79-89 (Pro-Ala-Asn-Leu-Pro-Trp-Gly-Ser-Ser-Asn-Val, IC50:18 microM), and contained the sequence homologous to vertebrate ACE inhibitors (GAPDH peptides 79-86 or 81-88). Other inhibitors, YG-1 (Gly-His-Lys-Ile-Ala-Thr-Phe-Gln-Glu-Arg, IC50: 0.4 microM) and YG-2 (Gly-Lys-Lys-Ile-Ala-Thr-Tyr-Gln-Glu-Arg, IC50: 2 microM), corresponded to amino acid residues 68-77 in two different forms of yeast GAPDH, respectively. Their sequences were quite different from those of the venom peptide family. YG-1 was the most potent ACE inhibitor among yeast and vertebrate GAPDH peptides excised by acid-limited proteolysis. Thus, yeast GAPDH seems to be an excellent source of naturally occurring ACE inhibitors.