Effect of •OH scavenging action by non-SH-containing angiotensin converting enzyme inhibitor imidaprilat using microdialysis

We examined the effect of non-SH-containing angiotensin converting enzyme (ACE) inhibitor imidaprilat on hydroxyl radical (•OH) generation using microdialysis. Salicylic acid in Ringer's solution containing sodium salicylate (0.5 n mol μL⁻¹ min⁻¹) was infused directly through a microdialysis probe to detect the generation of •OH as reflected by the formation of 2,3-dihydroxybenzoic acid (DHBA) in the myocardium of anesthetized rats. We compared the ability of two non-SH-containing ACE inhibitors (imidaprilat and enalaprilat) with an -SH-containing ACE inhibitor (captopril) to scavenge the •OH. When iron (II) was administered to animals pretreated with these three ACE inhibitors, a decrease in 2,3-DHBA of all three compounds was observed, as compared with the iron (II) only-treated group. All three ACE inhibitors were able to scavenge •OH generated by the action of iron (II). However, imidaprilat is a free radical scavenger more potent than enalaprilat. These results suggested that ACE inhibitors are probably not only related to the presence of the SH radical.     

A calorimetric study of the binding of lisinopril, enalaprilat and captopril to angiotensin-converting enzyme

The angiotensin I-converting enzyme (ACE; EC.3.4.15.1) is a dipeptidyl carboxypeptidase that plays a central role in blood pressure regulation. The somatic form of the enzyme is composed of two highly similar domains, usually referred to as N and C domains, each containing one active site. Nevertheless, a 1:1 stoichiometry for the binding of lisinopril, captopril or enalaprilat to somatic pig lung ACE is shown by isothermal titration calorimetry (ITC) and enzymatic assays. The binding of the three inhibitors at neutral pH is very tight and the enthalpy changes are positive, indicating that the binding is entropically driven. The origin of this thermodynamic signature is discussed under the new structural information available.     

Captopril and enalaprilat decrease antioxidant defences in human endothelial cells and are unable to protect against apoptosis

Angiotensin-converting enzyme (ACE) inhibitors were shown to improve endothelial dysfunction in various human diseases and some of these inhibitors have been proposed as enhancers of antioxidant defences. We measured glutathione peroxidase (GPX), superoxide dismutase (SOD) and malondialdehyde (MDA) in human endothelial cells treated with captopril or enalaprilat, two ACE inhibitors, and we showed that both inhibitors decreased GPX and SOD activities but not MDA, the end-product of lipoperoxidation. Captopril and enalaprilat were also unable to protect against etoposide-induced apoptosis in endothelial cells, indicating that they cannot be considered as protective drugs for the endothelium, in particular in clinical situations involving oxidative stress or apoptosis. Moreover, when used at high concentration captopril, but not enalaprilat, was toxic for endothelial cells with both necrotic and apoptotic effects.     

Role of intracellular antioxidant enzymes after in vivo myocardial ischemia and reperfusion

It has been recently claimed that the human B1 receptors for kinins bind angiotensin-converting enzyme (ACE) inhibitors via a potential zinc-binding domain and are pharmacologically stimulated by these drugs. We verified whether ACE inhibitors stimulate B1 receptors in vitro. The isolated rabbit aorta or mouse stomach responded by negligible contractions to the application of captopril, enalaprilat, or zofenoprilat. The human isolated umbilical vein also failed to respond to enalaprilat. All of these preparations were responsive to the B1 receptor agonists des-Arg9-bradykinin (BK) or Lys-des-Arg9-BK. Furthermore, enalaprilat applied continuously had no significant interaction with the effects of Lys-des-Arg9-BK on the rabbit aorta. Enalaprilat failed to stimulate [3H]arachidonate release, translocate the receptors (confocal microscopy), or stimulate ERK1/2 phosphorylation (immunoblot) in HEK-293 cells stably expressing the rabbit B1 receptor conjugated to yellow fluorescent protein. The phospho-ERK1/2 content of arterial smooth muscle cells of human or rabbit origin was increased by treatment with Lys-des-Arg9-BK but not with enalaprilat. ACE inhibitors do not act as bona fide agonists of the kinin B1 receptors.     

Angiotensin Converting Enzyme Inhibitors as Oxygen Free Radical Scavengers

The authors have compared the ability of two non-SH-containing angiotensin converting enzyme (ACE) inhibitors (enalaprilat and lisinopril) with an -SH containing ACE inhibitor (captopril) to scavenge the hydroxyl radical (OH). All three compounds were able to scavenge -OH radicals generated in free solution at approximately diffusion-controled rates (10¹⁰ M^-1s^-1) as established by the deoxyribose assay in the presence of EDTA. The compounds also inhibited deoxyribose degradation in reaction mixtures which did not contain EDTA but not so effectively. This later finding also suggests that they have some degree of metal-binding capability. Chemiluminescence assays of oxidation of hypoxanthine by xanthine oxidase in the presence of luminol, confirm that the three ACE inhibitors are oxygen free radical scavengers. Our results indicate that the presence of a sulphydryl group in the chemical structure of ACE inhibitors is not relevant for their oxygen free radical scavenging ability.     

Free radical scavenging: a potentially beneficial action of thiol-containing angiotensin converting enzyme inhibitors

Free radical (FR) scavenging may be a therapeutically useful adjunctive property of angiotensin converting enzyme (ACE) inhibitors. In this study we have shown that SH-containing ACE inhibitors (captopril, epicaptopril, zofenopril) are potent FR scavengers at a concentration of 4 x 10(-5) M whereas non-SH ACE inhibitors (enalaprilat, quinaprilat and perindoprilat) have no FR-scavenging activity at this concentration. Furthermore, the SH-containing agents preferentially scavenged general radicals rather than superoxide radicals, i.e. suggesting that these drugs would be effective in quenching the culprit FR in ischaemia/reperfusion injury.     

The structure of testis angiotensin-converting enzyme in complex with the C domain-specific inhibitor RXPA380

Angiotensin I-converting enzyme (ACE) is central to the regulation of the renin-angiotensin system and is a key therapeutic target for combating hypertension and related cardiovascular diseases. Currently available drugs bind both active sites of its two homologous domains, although it is now understood that these domains function differently in vivo. The recently solved crystal structures of both domains (N and C) open the door to new domain-specific inhibitor design, taking advantage of the differences between these two large active sites. Here we present the first crystal structure at a resolution of 2.25 A of testis ACE (identical to the C domain of somatic ACE) with the highly C-domain-specific phosphinic inhibitor, RXPA380. Testis ACE retains the same conformation as seen in previously determined inhibitor complexes, but the RXPA380 central backbone conformation is more similar to that seen for the inhibitor captopril than enalaprilat. The RXPA380 molecule occupies more subsites of the testis ACE active site than the previously determined inhibitors and possesses bulky moieties that extend into the S2' and S2 subsites. Thus the high affinity of RXPA380 for the testis ACE/somatic ACE C domain is explained by the interaction of these bulky moieties with residues unique to these domains, specifically Phe 391, Val 379, and Val 380, that are not found in the N domain. The characterization of the extended active site and the binding of a potent C-domain-selective inhibitor provide the first structural data for the design of truly domain-specific pharmacophores.     

Thermodynamic determination of the binding constants of angiotensin-converting enzyme inhibitors by a displacement method

Somatic angiotensin I-converting enzyme (s-ACE) plays a central role in blood pressure regulation and has been the target of most antihypertensive drugs. A displacement isothermal titration calorimetry method has been used to accurately determine the binding constant of three strong s-ACE inhibitors. Under the experimental conditions studied in this work, the relative potency of the inhibitors was determined to be enalaprilat>lisinopril>captopril. We analyze the thermodynamic behaviour of the binding process using the new structural information provided by the ACE structures, as well as the conformational changes that occur upon binding.     

The two homologous domains of human angiotensin I-converting enzyme interact differently with competitive inhibitors.

The endothelial angiotensin I-converting enzyme (ACE; EC 3.4.15.1) has recently been shown to contain two large homologous domains (called here the N and C domains), each being a zinc-dependent dipeptidyl carboxypeptidase. To further characterize the two active sites of ACE, we have investigated their interaction with four competitive ACE inhibitors, which are all potent antihypertensive drugs. The binding of [3H] trandolaprilat to the two active sites was examined using the wild-type ACE and four ACE mutants each containing only one intact domain, the other domain being either deleted or inactivated by point mutation of the zinc-coordinating histidines. In contrast with all the previous studies, which suggested the presence of a single high affinity inhibitor binding site in ACE, the present study shows that both the N and C domains of ACE contain a high affinity inhibitor binding site (KD = 3 and 1 X 10(-10) M, respectively, at pH 7.5, 4 degrees C, and 100 mM NaCl). Chloride stabilizes the enzyme-inhibitor complex for each domain primarily by slowing its dissociation rate, as the k-1 values of the N and C domains are markedly decreased (about 30- and 1100-fold, respectively) by 300 mM NaCl. At high chloride concentrations, the chloride effect is much greater for the C domain than for the N domain resulting in a higher affinity of this inhibitor for the C domain. In addition, the inhibitory potency of captopril (C), enalaprilat (E), and lisinopril (L) for each domain was assayed by hydrolysis of Hip-His-Leu. Their Ki values for the two domains are all within the nanomolar range, indicating that they are all highly potent inhibitors for both domains. However, their relative potencies are different for the C domain (L greater than E greater than C) and the N domain (C greater than E greater than L). The different inhibitor binding properties of the two domains observed in the present study provide strong evidence for the presence of structural differences between the two active sites of ACE.     

Different inhibition of enalaprilat and imidaprilat on bradykinin metabolizing enzymes

Effects of angiotensin-converting enzyme (ACE) inhibitors, enalaprilat and imidaprilat, on bradykinin (BK) metabolizing enzymes, aminopeptidase P (APP), neutral endopeptidase (NEP) and carboxypeptidase N (CPN), were examined. APP activity in the mouse lung was inhibited by enalaprilat in a concentration-dependent manner while imidaprilat did not influence the enzyme activity. The inhibitory effects of these ACE inhibitors on the NEP activity in the mouse lung and the CPN activity in the mouse serum were negligible. These data suggested that the influence of enalaprilat on the APP activity and subsequent BK metabolism are different from those of imidaprilat.     

Lack of direct interaction between enalaprilat and the kinin B1 receptors

It has been recently proposed that the second extracellular loop of the human bradykinin (BK) B1 receptor (B1R) contains a conserved HExxH motif also present in peptidases possessing a Zn2+ prosthetic group, such as angiotensin converting enzyme (ACE), and that ACE inhibitors directly activate B1R signaling in endothelial cells. However, the binding of ACE inhibitors to the B1Rs has never been directly evaluated. Information about binding of a radiolabeled inhibitor to natural or recombinant ACE in intact cells (physiologic ionic composition) was also collected. We used the tritiated form of an ACE inhibitor previously proposed to activate the B1R, enalaprilat, to address these questions using recombinant human B1Rs and naturally expressed or recombinant ACE. [3H]Lys-des-Arg9-BK bound to the human recombinant B1Rs with high affinity (KD 0.35 nM) in HEK 293a cells. [3H]Enalaprilat (0.25-10 nM) did not bind to cells expressing recombinant human B1R, but bound with a subnanomolar affinity to recombinant ACE or to naturally expressed ACE in human umbilical vein endothelial cells. The radioligand was further validated using a binding competition assay that involved unlabeled ACE inhibitors or their prodrug forms in endothelial cells. Membranes of HEK 293a cells that expressed B1Rs did not hydrolyze hippuryl-glycylglycine (an ACE substrate). Enalaprilat did not stimulate calcium signaling in HEK 293a cells that expressed B1Rs. A typical ACE inhibitor did not bind to nor stimulate the human B1Rs; nevertheless, several other indirect mechanisms could connect ACE inhibition to B1R stimulation in vivo.     

Sialosylglobotetraosylceramide: a marker for amyotropic lateral sclerosis

Modification of alanyl proline by introduction of both zinc coordinating and S1 subsite binding interactions affords potent new carboxy- and mercapto-acyl dipeptide angiotensin-converting enzyme (ACE) inhibitors. Design of these inhibitors was guided by an extension of the hypothetical ACE active site model originally used to derive captopril. Significant increases in ACE inhibitory activity were observed by introduction of conformation constraint into acyclic acyl dipeptides, thus further defining the three dimensional structure of the ACE active site.     

Contribution of captopril thiol group to the prevention of spontaneous hypertension

We aimed to compare the effect of angiotensin converting enzyme (ACE) inhibitors captopril (containing thiol group) and enalapril (without thiol group) on the development of spontaneous hypertension and to analyze mechanisms of their actions, particularly effects on oxidative stress and NO production. Six-week-old SHR were divided into three groups: control, group receiving captopril (50 mg/kg/day) or enalapril (50 mg/kg/day) for 6 weeks. At the end of experiment, systolic blood pressure (SBP) increased by 41 % in controls. Both captopril and enalapril prevented blood pressure increase, however, SBP in the captopril group (121+/-5 mmHg) was significantly lower than that in the enalapril group (140+/-5 mmHg). Concentration of conjugated dienes in the aorta was significantly lower in the captopril group compared to the enalapril group. Captopril and enalapril increased NO synthase activity in the heart and aorta to the similar level. Neither captopril nor enalapril was, however, able to increase the expression of eNOS. Both ACE inhibitors increased the level of cGMP. However, cGMP level was significantly higher in the aorta of captopril group. We conclude that captopril, beside inhibition of ACE, prevented hypertension by increasing NO synthase activity and by simultaneous decrease of oxidative stress which resulted in increase of cGMP concentration.     

Conformational Changes of Blood ACE in Chronic Uremia

Background

The pattern of binding of monoclonal antibodies (mAbs) to 16 epitopes on human angiotensin I-converting enzyme (ACE) comprise a conformational ACE fingerprint and is a sensitive marker of subtle protein conformational changes.

Hypothesis

Toxic substances in the blood of patients with uremia due to End Stage Renal Disease (ESRD) can induce local conformational changes in the ACE protein globule and alter the efficacy of ACE inhibitors.

Methodology/Principal Findings

The recognition of ACE by 16 mAbs to the epitopes on the N and C domains of ACE was estimated using an immune-capture enzymatic plate precipitation assay. The precipitation pattern of blood ACE by a set of mAbs was substantially influenced by the presence of ACE inhibitors with the most dramatic local conformational change noted in the N-domain region recognized by mAb 1G12. The “short” ACE inhibitor enalaprilat (tripeptide analog) and “long” inhibitor teprotide (nonapeptide) produced strikingly different mAb 1G12 binding with enalaprilat strongly increasing mAb 1G12 binding and teprotide decreasing binding. Reduction in S-S bonds via glutathione and dithiothreitol treatment increased 1G12 binding to blood ACE in a manner comparable to enalaprilat. Some patients with uremia due to ESRD exhibited significantly increased mAb 1G12 binding to blood ACE and increased ACE activity towards angiotensin I accompanied by reduced ACE inhibition by inhibitory mAbs and ACE inhibitors.

Conclusions/Significance

The estimation of relative mAb 1G12 binding to blood ACE detects a subpopulation of ESRD patients with conformationally changed ACE, which activity is less suppressible by ACE inhibitors. This parameter may potentially serve as a biomarker for those patients who may need higher concentrations of ACE inhibitors upon anti-hypertensive therapy.     

Accurate aqueous proton dissociation constants calculations for selected angiotensin-converting enzyme inhibitors

Studies that allow computing values of aqueous proton dissociation constants (pKa), gas phase proton affinities, and the free energy of solvation have been performed for six members of angiotensin-I-converting enzyme (ACE) inhibitor family (captopril, enalaprilat, imidaprilat, ramiprilat, perindoprilat, and spiraprilat). Density functional theory (DFT) calculations using PBE1PBE functional on optimized molecular geometries have been carried out to investigate the thermodynamics of gas-phase protonation. The conductor-like polarizable continuum model (CPCM) solvation method at various levels of theory was applied to calculate the free energy of solvation for the ACE inhibitors and their respective anions. The CPCM solvation calculations were performed on both gas-phase and solvent-phase optimized structures. The combination of gas-phase and solvation energies according to the thermodynamic cycle enabled us to compute accurate pKa values for the all studied molecules.     

High-Resolution Crystal Structures of Drosophila melanogaster Angiotensin-Converting Enzyme in Complex with Novel Inhibitors and Antihypertensive Drugs

Angiotensin I-converting enzyme (ACE), one of the central components of the renin-angiotensin system, is a key therapeutic target for the treatment of hypertension and cardiovascular disorders. Human somatic ACE (sACE) has two homologous domains (N and C). The N- and C-domain catalytic sites have different activities toward various substrates. Moreover, some of the undesirable side effects of the currently available and widely used ACE inhibitors may arise from their targeting both domains leading to defects in other pathways. In addition, structural studies have shown that although both these domains have much in common at the inhibitor binding site, there are significant differences and these are greater at the peptide binding sites than regions distal to the active site. As a model system, we have used an ACE homologue from Drosophila melanogaster (AnCE, a single domain protein with ACE activity) to study ACE inhibitor binding. In an extensive study, we present high-resolution structures for native AnCE and in complex with six known antihypertensive drugs, a novel C-domain sACE specific inhibitor, lisW-S, and two sACE domain-specific phosphinic peptidyl inhibitors, RXPA380 and RXP407 (i.e., nine structures). These structures show detailed binding features of the inhibitors and highlight subtle changes in the orientation of side chains at different binding pockets in the active site in comparison with the active site of N- and C-domains of sACE. This study provides information about the structure-activity relationships that could be utilized for designing new inhibitors with improved domain selectivity for sACE.     

Novel mechanism of inhibition of human angiotensin-I-converting enzyme (ACE) by a highly specific phosphinic tripeptide

Human ACE (angiotensin-I-converting enzyme) has long been regarded as an excellent target for the treatment of hypertension and related cardiovascular diseases. Highly potent inhibitors have been developed and are extensively used in the clinic. To develop inhibitors with higher therapeutic efficacy and reduced side effects, recent efforts have been directed towards the discovery of compounds able to simultaneously block more than one zinc metallopeptidase (apart from ACE) involved in blood pressure regulation in humans, such as neprilysin and ECE-1 (endothelin-converting enzyme-1). In the present paper, we show the first structures of testis ACE [C-ACE, which is identical with the C-domain of somatic ACE and the dominant domain responsible for blood pressure regulation, at 1.97? (1 ?=0.1 nm)] and the N-domain of somatic ACE (N-ACE, at 2.15?) in complex with a highly potent and selective dual ACE/ECE-1 inhibitor. The structural determinants revealed unique features of the binding of two molecules of the dual inhibitor in the active site of C-ACE. In both structures, the first molecule is positioned in the obligatory binding site and has a bulky bicyclic P(1)' residue with the unusual R configuration which, surprisingly, is accommodated by the large S(2)' pocket. In the C-ACE complex, the isoxazole phenyl group of the second molecule makes strong pi-pi stacking interactions with the amino benzoyl group of the first molecule locking them in a 'hand-shake' conformation. These features, for the first time, highlight the unusual architecture and flexibility of the active site of C-ACE, which could be further utilized for structure-based design of new C-ACE or vasopeptidase inhibitors.     

Accurate Aqueous Proton Dissociation Constants Calculations for Selected Angiotensin-Converting Enzyme Inhibitors

Abstract

Studies that allow computing values of aqueous proton dissociation constants (pK_a), gas phase proton affinities, and the free energy of solvation have been performed for six members of angiotensin-I-converting enzyme (ACE) inhibitor family (captopril, enalaprilat, imidaprilat, ramiprilat, perindoprilat, and spiraprilat). Density functional theory (DFT) calculations using PBE1PBE functional on optimized molecular geometries have been carried out to investigate the thermodynamics of gas-phase protonation. The conductor-like polarizable continuum model (CPCM) solvation method at various levels of theory was applied to calculate the free energy of solvation for the ACE inhibitors and their respective anions. The CPCM solvation calculations were performed on both gas-phase and solvent-phase optimized structures. The combination of gas-phase and solvation energies according to the thermodynamic cycle enabled us to compute accurate pK_a values for the all studied molecules.     

Effects of enalaprilat on neointimal growth of cultured rabbit aorta following balloon injury

Our objective was to determine if the ability of an angiotensin-converting enzyme (ACE) inhibitor to attenuate neointima formation in balloon-damaged vessel is expressed in an isolated organ culture model of neointimal growth. In vivo balloon angioplasty in combination with in vitro organ culture was used to produce a unique model of vascular neointima formation. Aortic segments were cultured in medium containing a broad concentration range of the ACE inhibitor enalaprilat (0-100 microM). Cell proliferative indices and neointima:media thickness ratios were determined from vessel segments after 1, 4, and 7 days in culture. We observed no significant effect on either parameter at any dose of enalaprilat. Linear regression analysis on the rate of increase in intima to media thickness ratios during the 7 days of culture also showed no effect of enalaprilat at any concentration. We conclude that enalaprilat has no effect on neointimal growth or cell proliferation in this vascular organ culture model, and it is suggested that ACE inhibitors may act by mechanisms other than local converting enzyme inhibition to attenuate neointimal growth in rabbits following vascular ballooning in vivo.