Inhibition of angiotensin converting enzyme by phosphoramidates and polyphosphates

N alpha-Phosphoryl-L-alanyl-L-proline is a reversible competitive inhibitor of angiotensin converting enzyme with a Ki of 1.4 nM. Alkylation of one phosphate oxygen with methyl, ethyl, or benzyl does not change the Ki. The high activity of the O-alkylated inhibitors demonstrates that the two phosphate oxygen anions do not constitute a bidentate ligand of the active site zinc ion. Substitution of valyltryptophan, glycylglycine, or delta-aminovaleric acid for alanylproline in the phosphoramidate raises the Ki to 12 nM, 25 microM, and 178 microM, respectively. Methylation of the alanine nitrogen in phosphorylalanylproline raises the Ki to 29 microM. Polyphosphates inhibit converting enzyme with the following Ki's: phosphate, approximately 300 mM; pyrophosphate, 2 mM; tripolyphosphate, 18 microM; tetrapolyphosphate, 150 microM. The inhibition by tripolyphosphate appears to be competitive and is unaffected by the addition of excess zinc ion. Since the Ki of tripolyphosphate is nearly 10-fold lower than that of N-phosphoryl-delta-aminovaleric acid and is near that of N alpha-phosphorylglycylglycine, its terminal phosphates may bind the zinc site and the cationic site on the enzyme, thus spanning the S1' and S2' sites.     

Inhibition of angiotensin converting enzyme by aldehyde and ketone substrate analogues

Three classes of carbonyl-containing substrate analogues and partial substrate analogues have been tested for their ability to inhibit angiotensin converting enzyme. (4-Oxobutanoyl)-L-proline is proposed to occupy the S1' and S2' subsites on the enzyme, thus locating its aldehyde carbonyl group at the position of the active site zinc atom. This aldehyde is 70% hydrated in aqueous solution and could mimic a tetrahedral intermediate occurring during enzyme-catalyzed substrate hydrolysis, but its Ki is only 760 microM. Carbobenzoxy-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalaninal is proposed to occupy the S1 through S4 subsites on the other side of the zinc atom. Its weak Ki of 60 microM is nearly equipotent to its parent peptide terminating in phenylalanine. However, ketoace, (5RS)-(5-benzamido-4-oxo-6-phenylhexanoyl)-L-proline [Almquist, R.G., Chao, W.R., Ellis, M.E., & Johnson, H.L. (1980) J. Med. Chem. 23, 1392-1398], one of the third class of inhibitors proposed to occupy subsites S1 through S2' on both sides of the zinc atom, has a Ki of 0.0006 microM under our assay conditions, orders of magnitude more potent than its parent peptide. The carbonyl carbon of ketoace is less than 3% hydrated in aqueous solution as determined by carbon-13 nuclear magnetic resonance spectroscopy. If the hydrate is the species bound to converting enzyme, its Ki must be less than 18 pM. Ketoace is a slow-binding inhibitor of converting enzyme, but its overall Ki is dependent on its concentration and therefore prevents calculation of kinetic constants for slow binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endogenous inhibitor of angiotensin converting enzyme in the rat heart

We have identified a substance in the rat heart which inhibits ACE. This substance was characterized to be a sulfhydryl (SH) protein, the SH moiety being essential for the inhibitory activity. The inhibitory activity disappeared when the extract was boiled, or the ultrafiltrate(mol.wt.less than 10,000) was used, or when the extract was pretreated with the SH-blocking agent 5,5'-dithiobis-(2-nitro benzoic acid) at 0.5mM or the SH oxidizing agent diamide at 1mM. This substance was fractionated with Thiopropyl Sepharose affinity chromatography, precipitation with 40% ammonium sulfate saturation and high performance liquid chromatography. The mode of inhibition was competitive. In the presence of 20 micrograms/ml of this substance, the contraction of rat aortic strips induced by 5 x 10(-8)M ANG I was inhibited by 60%. This endogenous inhibitor of ACE may modulate the activity of ACE in the heart, in response to alterations in the oxidation-reduction balance in the tissue.     

Inhibition of angiotensin converting enzyme: dependence on chloride

In a previous report [Shapiro, R., Holmquist, B., & Riordan, J. F. (1983) Biochemistry 22, 3850], it was demonstrated that activation of angiotensin converting enzyme (ACE) by chloride is strongly dependent on substrate structure, and three substrate classes were identified on the basis of activation behavior. The present study examines the chloride dependence of the inhibition of ACE by nine inhibitors [(D-3-mercapto-2-methylpropanoyl)-L-Pro (captopril), N-[1(S)-carboxy-3-phenylpropyl]-L-Ala-L-Pro (MK-422), L-Ala-L-Pro, N-(phenylphosphoryl)-L-Phe-L-Phe, Gly-L-Trp, N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly, L-Phe-L-Arg, N alpha-(3-mercaptopropanoyl)-L-Arg, and N alpha-[1(S)-carboxy-3-phenylpropyl]-L-Ala-L-Lys] containing structural features characteristic of the three classes of substrates. Apparent Ki values for all inhibitors are markedly (70-250-fold) decreased by 300 mM chloride. However, the enhancement of inhibition is achieved at significantly lower chloride concentrations with those inhibitors having an ultimate arginine or lysine than with the remainder. This variability parallels that previously found for activation of substrate hydrolysis. The effect of chloride on the individual steps in the formation and dissociation of the steady-state enzyme-inhibitor complexes was determined with the slow-binding inhibitor MK-422. Pre-steady-state analysis indicates that binding of both MK-422 and captopril follows a (minimally) two-step mechanism: (formula; see text) in which rapid formation of an enzyme-inhibitor complex is followed by a slow isomerization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free radical reactions involving the angiotensin converting enzyme inhibitor captopril

Summary

Using the pulse radiolysis technique, absolute rate constants have been obtained for the reaction of captopril with several free radicals. The results demonstrate that although captopril reacts rapidly with a number of free radicals, such as the hydroxyl radical (k = 5.1 × 10⁹ dm^?3mol^?1s^?1) and the thiocyanate radical anion (k = 1.3 × 10⁷ dm^?3mol^?1s^?1), it is not exceptional in this ability. Similarly, the reactions with carbon centred radicals although rapid are an order of magnitude slower than those observed with glutathione. Additional lipid peroxidation studies further demonstrate that captopril is a much less effective antioxidant than glutathione. The data go some way to supporting the view that any attenuation of reperfusion injury by captopril is not through a direct free radical scavenging mechanism but may be afforded by other, non-radical-mediated mechanisms.     

Inhibition of angiotensin converting enzyme: mechanism and substrate dependence

The interaction of angiotensin converting enzyme with six metal-coordinating [(D-3-mercapto-2-methylpropanoyl)-L-Pro (captopril), N-[1(S)-carboxy-3-phenylpropyl]-L-Ala-L-Pro (MK-422), N-(phenylphosphoryl)-L-Phe-L-Phe, N alpha-(3-mercaptopropanoyl)-L-Arg, N alpha-[1(S)-carboxy-3-phenylpropyl]-Ala-L-Lys, and N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly] and three dipeptide inhibitors (Gly-L-Trp, L-Phe-L-Arg, and L-Ala-L-Pro) was examined at pH 7.5 in the presence of 300 mM NaCl. Inhibition modes, apparent Ki [Ki(app)] values, and shapes of 1/v vs. [I] plots were found to vary with the substrate employed. All inhibitors except Phe-Arg were competitive with the substrate furanacryloyl (Fa)-Phe-Gly-Gly, while five of seven tested with Fa-Phe-Phe-Arg as substrate produced mixed patterns. Ki-(app) values for N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly, N-(phenylphosphoryl)-L-Phe-L-Phe, Gly-Trp, and MK-422 were 8.3-, 5.5-, 4.7-, and 2.6-fold lower, respectively, when Fa-Phe-Gly-Gly was substrate, compared with values measured with Fa-Phe-Phe-Arg. In contrast, Ki(app) values for Phe-Arg and (3-mercaptopropanoyl)-Arg were lower (2.8- and 2.2-fold, respectively) when Fa-Phe-Phe-Arg was the substrate. Plots of 1/v vs. [I] for most of the inhibitors were nonlinear, to an extent which was also substrate dependent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition by angiotensin converting enzyme inhibitors of endothelin secretion from cultured human endothelial cells.

We conducted a study to determine whether angiotensin converting enzyme inhibitors (ACEIs) inhibit endothelin secretion from cultured human endothelial cells. Confluent umbilical vein endothelial cells were incubated in multi-well plates with culture medium containing either captopril (10(-6), 10(-5), 10(-4) M) or enalaprilat (10(-7), 10(-6), 10(-5) M) for 6 hours. Immunoreactive endothelin in the medium was measured by radioimmunoassay. Calf serum (CS) stimulated endothelin release in a concentration-dependent manner, and both ACEIs inhibited 5% CS-stimulated endothelin release in a concentration-dependent manner. To explore the mechanisms of ACEI-induced suppression of endothelin release, the effects of angiotensin II (10(-8), 10(-7), 10(-6) M), angiotensin converting enzyme (0.1, 1, 10 mU/ml), bradykinin (10(-8), 10(-7), 10(-6) M), and sodium nitroprusside (10(-6), 10(-5), 10(-4) M) on endothelin release were also examined. Although angiotensin II and angiotensin converting enzyme had no significant effect on endothelin release, concentration-dependent suppression occurred with bradykinin and sodium nitroprusside. These results indicate that ACEIs inhibit the stimulated release of endothelin from human endothelial cells, and provide indirect evidence that ACEI-induced ET suppression may be mediated via potentiation of autacoid formation from the cells.     

The angiotensin I converting enzyme.

The angiotensin I converting enzyme (kininase II; peptidyl dipeptidase; EC3.4.15.1) has a dual function: it converts angiotensin I to angiotensin II and it inactivates bradykinin. Lung, kidney, guinea pig plasma and testicles are among the richest sources of the enzyme. Vascular endothelial cells and bursh borders of renal proximal tubular cells contain high concentrations of the enzyme. The availability of synthetic peptide inhibitors was a great help in establishing the function of converting enzyme in normal and pathological conditions.     

Inhibition of canine lung angiotensin converting enzyme by ACTH and structurally related peptides

Human ACTH and structurally related peptides, such as ACTH 7–38, ACTH 4–11, ACTH 1–10 and ACTH 18–39, noncompetitively inhibited the activity of angiotensin I converting enzyme (dipeptidyl carboxypeptidase; E.C. 3.4.15.1) in the preparation from canine lung. The Ki values were 1.5 μM and 0.54 μM for ACTH and ACTH 7–38, respectively, using[¹⁴C] -Hip-his-leu as the substrate. These results suggest that ACTH and ACTH 7–38 are potent inhibitors of angiotensin I converting enzyme without being substrate for the enzyme.     

Direct scavenging of free radicals by captopril, an angiotensin converting enzyme inhibitor

Captopril, an angiotensin converting enzyme (ACE) inhibitor, was hypothesized to be a potential scavenger of free radicals because of the presence of a thiol group. The scavenging action of captopril was examined against superoxide anion (O2-), hydroxyl radical (OH.), hypohalite radical (HOCL) either generated biochemically, or derived from activated polymorphonuclear leukocytes (PMN). Our results indicate that captopril is an extremely potent free radical scavenger, scavenging power being as effective as superoxide dismutase (SOD) against O2-, or dimethylthiourea against OH., but better than allopurinol against OCL. plus HOCL. Free radical scavenging action of captopril against PMN-derived free radical is equivalent to the combined effects of SOD, catalase and allopurinol.     

Electronic spectroscopy of cobalt angiotensin converting enzyme and its inhibitor complexes

Zinc, the catalytically essential metal of angiotensin converting enzyme (ACE), has been replaced by cobalt(II) to give an active, chromophoric enzyme that is spectroscopically responsive to inhibitor binding. Visible absorption spectroscopy and magnetic circular dichroic spectropolarimetry have been used to characterize the catalytic metal binding site in both the cobalt enzyme and in several enzyme-inhibitor complexes. The visible absorption spectrum of cobalt ACE exhibits a single broad maximum (525 nm) of relatively low absorptivity (epsilon = 75 M-1 cm-1). In contrast, the spectra of enzyme-inhibitor complexes display more clearly defined maxima at longer wavelengths (525-637 nm) and of markedly higher absorptivities (130-560 M-1 cm-1). The large spectral response indicates that changes in the cobalt ion coordination sphere occur on inhibitor binding. Magnetic circular dichroic spectropolarimetry has shown that the metal coordination geometry in the inhibitor complexes is tetrahedral and of higher symmetry than in cobalt ACE alone. The presence of sulfur----cobalt charge-transfer bands in both the visible absorption and magnetic circular dichroic spectra of the cobalt ACE-Captopril complex confirm direct ligation of the thiol group of the inhibitor to the active-site metal.     

Discovery of a potent angiotensin converting enzyme inhibitor via virtual screening

Prompted by the prominent role of angiotensin converting enzyme (ACE) in hypertension, heart failures, myocardial infarction and diabetic nephropathy, we have attempted to discover novel ACE inhibitors through ligand-based virtual screening. Molecular docking method and rigorously validated model was utilized to search a natural compounds database. Finally, 36 compounds were randomly selected and subjected to in vitro ACE kinase inhibitory assay using fluorescence assays method. The results showed that three compounds (Licochalcone A, Echinatin and EGCG) have strong potential to be developed as a new class of ACE inhibitors. Further chemical modification via fragment modifications guided by structure and ligand-based computational methodologies can lead to discover better agents as potential clinical candidates.     

Secretion of metalloendopeptidase 24.15 (EC 3.4.24.15).

The metalloendopeptidase EP24.15 (EC3.4.24.15) is a neuropeptide-metabolizing enzyme present in neural and endocrine tissues, presumably functioning extracellularly. Because the majority of the EP24.15 activity is identified in the soluble fraction of cellular homogenates, suggesting that the enzyme is primarily an intracellular protein, we addressed the issue of how EP24.15 arrives in the extracellular environment. We utilized a model system of neuroendocrine secretion, the AtT20 cell. According to both enzymatic activity and immunologic assays, EP24.15 was synthesized in and released from AtT20 cells. Under basal conditions and after stimulation by corticotropin-releasing hormone or the calcium ionophore A23187, EP24.15 activity accumulated in the culture medium. This secretion was not attributable to cell damage, as judged by the absence of release of cytosolic enzyme markers and the ability to exclude trypan blue dye. Pulse-chase analysis and subcellular fractionation of AtT20 cell extracts suggested that the mechanism of EP24.15 secretion is not solely via classical secretory pathways. Additionally, drugs which disrupt the classical secretory pathway, such as Brefeldin A and nocodazole, blocked A23187-stimulated EP24.15 release yet had no effect on basal EP24.15 release, suggesting differences in the basal and stimulated pathways of secretion for EP24.15. In summary, EP24.15 appears to be secreted from AtT20 pituitary cells into the extracellular milieu, where the enzyme can participate in the physiologic metabolism of neuropeptides.     

Activation of angiotensin converting enzyme by monovalent anions

The angiotensin converting enzyme catalyzed hydrolysis of furanacryloyl-Phe-Gly-Gly is activated by monovalent anions in the order C1- greater than Br- greater than F- greater than NO3- greater than CH3COO-. In the alkaline pH region, increasing anion concentrations decrease the KM but do not change the kcat. This behavior is characteristic of an ordered bireactant mechanism in which the anion binds to the enzyme prior to the substrate. At acidic pH values, however, the anion activation is a result of both a decrease in KM and an increase in kcat, implying a bireactant mechanism in which anion and substrate bind randomly. For both the ordered and the bireactant mechanisms the anion serves as an essential activator. The effect of chloride on enzyme activity was studied over the pH range 5-10 under kcat/KM conditions and demonstrates that the apparent chloride binding constant increases from 3.3 mM at pH 6.0 to 190 mM at pH 9.0. The kcat vs. pH profile exhibits two pK values of 5.6 and 9.6, while the variation of KM with pH is characterized by a pK of 8.9 and a 2-fold increase between pH 6.5 and 7.5. The chloride activation of the hydrolysis of furanacryloyl-Phe-Gly-Gly is compared with that of the physiological substrates angiotensin I and bradykinin.     

Inactivation of angiotensin converting enzyme by sodium nitroprusside

Angiotensin I converting enzyme is rapidly inactivated by sodium nitroprusside and that inactivation is suppressed in the presence of chloride ion and by the presence of L-alanyl-L-proline or glycyl-L-tryptophan, which are both competitive inhibitors of its catalytic activity. The inactivation by sodium nitroprusside appears to result from the modification of an unusually reactive lysine residue in or near the active site.