Captopril Binding Research Articles

Abstract P117: MECHANISMS OF SIGNALING PATHWAYS TRIGGERED BY ANGIOTENSIN I CONVERTING ENZYME IN CHO ECA CELLS TREATED WITH CAPTOPRIL

Renin-Angiotensin System (RAS) is a hormonal system associated with hydroelectrolytic homeostasis and blood pressure control. Angiotensin II (Ang II) is the main and most potent biologically active product in the system, which is produced by the action of Angiotensin Converting Enzyme (ACE) on Ang I. Dysregulation of RAS is an important factor in the development and progression of cardiovascular/renal diseases and diabetes. Experiments and clinical practice have shown that the use of an ACE inhibitor (ACEi) is an effective treatment to attenuate hypertension and diabetic renal damage. In addition, recently the use of ACEi has also been associated with the activation of signaling pathways thus suggesting that ACE can act as a signal transductional molecule. The purpose of this study is to demonstrate the proteins that are differentially regulated when CHO-ECA cells are treated with the ACE inhibitor, captopril. The CHO-ACE cells were cultured in DMEM-SILAC medium in different media: Medium with light chain lysine (LV), medium with heavy chain lysine (P), and control (CT), with DMEM-High Glucose medium. At the 75% confluency, the cells were deprived of serum and then stimulated with captopril (1μM) or vehicle for 2 and 5 minutes (min). CHO-ACE cells were also cultured for the assay analysis of the Kinase Array. ACE activity was determined spectrofluorimetrically using ZPhe-His-Leu as substrate. With the SILAC medium, it was not possible to detect the differently modulated proteins by captopril. The kinase array demonstrated that some signaling pathways were not modulated after Captopril (eg, MSK1/2 and CREB). Other kinases were positively modulated after 2 min stimulation (eg, AMPKa1, HSP27, Akt 1/2/3 T308) and 5 min stimulation (STATa/b, HSP60, b-Catenin) with Captopril. Our screening also has shown that some kinases were negatively modulated following Captopril (eg, p53, PLC-γ1, Pyk2, eNOS). ACE activity in cell lysate group was inexpressive when treated with captopril. Our results expanded the repertoire of kinases modulated by the binding of Captopril to ACE, as well as, the role of ACE as a receptor and the modulation of signaling pathways triggered by this binding, which may imply in the treatment of hypertension.

Study of L-captopril binding to VIM-20 by X-ray crystallography method

Study of <scp>L</scp>-captopril binding to VIM-20 by X-ray crystallography method

Investigation of the Captopril-Insulin Interaction by Mass Spectrometry and Computational Approaches Reveals that Captopril Induces Structural Changes in Insulin.

Post-translational modifications remarkably regulate proteins’ biological function. Small molecules such as reactive thiols, metabolites, and drugs may covalently modify the proteins and cause structural changes. This study reports the covalent modification and noncovalent interaction of insulin and captopril, an FDA-approved antihypertensive drug, through mass spectrometric and computation-based approaches. Mass spectrometric analysis shows that captopril modifies intact insulin, reduces it into its “A” and “B” chains, and covalently modifies them by forming adducts. Since captopril has a reactive thiol group, it might reduce the insulin dimer or modify it by reacting with cysteine residues. This was proven with dithiothreitol treatment, which reduced the abundance of captopril adducts of insulin A and B chains and intact Insulin. Liquid chromatography tandem mass spectrometric analysis identified the modification of a total of four cysteine residues, two in each of the A and B chains of insulin. These modifications were identified to be Cys6 and Cys7 of the A chain and Cys7 and Cys19 of the B chain. Mass spectrometric analysis indicated that captopril may simultaneously modify the cysteine residues of intact insulin or its subunits A and B chains. Biophysical studies involving light scattering and thioflavin T assay suggested that the binding of captopril to the protein leads to the formation of aggregates. Docking and molecular dynamics studies provided insights into the noncovalent interactions and associated structural changes in insulin. This work is a maiden attempt to understand the detailed molecular interactions between captopril and insulin. These findings suggest that further investigations are required to understand the long-term effect of drugs like captopril.

The binding of captopril to angiotensin I-converting enzyme triggers activation of signaling pathways.

Hypertension is a global health problem, and angiotensin I (ANG I)-converting enzyme (ACE) inhibitors are largely used to control this pathology. Recently, it has been shown that ACE can also act as a transducer signal molecule when its inhibitors or substrates bind to it. This new role of ACE could contribute to understanding some of the effects not explained by its catalytic activity only. In this study, we investigated signaling pathway activation in Chinese hamster ovary (CHO) cells stably expressing ACE (CHO-ACE) under different conditions. We also investigated gene modulation after 4 h and 24 h of captopril treatment. Our results demonstrated that CHO-ACE cells when stimulated with ANG I, ramipril, or captopril led to JNK and ERK1/2 phosphorylation. To verify any physiological role at the endogenous level, we made use of primary cultures of mesangial cells from spontaneously hypertensive rats (SHR) and Wistar rats. Our results showed that ERK1/2 activation occurred mainly in primary cultures of mesangial cells from SHR rats upon captopril stimulation, suggesting that this signaling pathway could be differentially regulated during hypertension. Our results also showed that captopril treatment leads to a decrease of cyclooxygenase 2, interleukin-1β, and β-arrestin2 and a significant increase of AP2 gene expression levels. Our findings strengthen the fact that, in addition to the blockage of enzymatic activity, ACE inhibitors also trigger signaling pathway activation, and this may contribute to their beneficial effects in the treatment of hypertension and other pathologies.

Analysis of Binding Interaction between Captopril and Human Serum Albumin

The interaction between captopril, an inhibitor of angiotensin converting enzyme and human serum albumin, a principal plasma protein in the liver has been investigated in vitro under a simulated physiological condition by UV-vis spectrophotometry and fluorescence spectrometry. The intrinsic fluorescence intensity of human serum albumin was strongly quenched by captopril. The binding constants and the number of binding sites can be calculated from the data obtained from fluorescence quenching experiments. The negative value of ΔG0 reveals that the binding process is a spontaneous process. According to the van’t Hoff equation, the standard enthalpy change (ΔH0) and standard entropy change (ΔS0) for the reaction were calculated to be 35.98 KJ●mol-1 and 221.25 J●mol-1 K. It indicated that the hydrophobic interactions play a main role in the binding of captopril to human serum albumin. In addition, the distance between captopril (acceptor) and tryptophan residues of human serum albumin (donor) was estimated to be 1.05 nm according to the Förster’s resonance energy transfer theory. The results obtained herein will be of biological significance in pharmacology and clinical medicine.

Captopril directly inhibits matrix metalloproteinase-2 activity in continuous ambulatory peritoneal dialysis therapy

Background Matrix metalloproteinase (MMP)-2 plays an important role in tissue remodeling related to inflammation during continuous ambulatory peritoneal dialysis (CAPD) therapy. But its inhibitors were not applied clinically. We determined whether an angiotensin-converting enzyme (ACE) inhibitor, captopril, inhibits MMP-2 activity in peritoneal effluents from patients on CAPD, and simulated molecular models of the MMP-2–captopril complex. Methods The inhibitory effect of captopril on MMP-2 activity was measured in peritoneal effluents from 17 patients on CAPD. Molecular models of the MMP-2–captopril complex were simulated by 1000 iterations of random docking and energy minimization. Results Captopril directly inhibited MMP-2 activity in peritoneal effluents from patients on CAPD (IC 50; 48 µmol/l), and that captopril binding to the MMP-2 active site could be formed in each complex model without molecular distortion. Conclusion ACE inhibitors, such as captopril, may be applied as important compounds for MMP-2 inhibition in inflammation caused by CAPD.

Protective ability and binding affinity of captopril towards serum albumin in an in vitro glycation model of diabetes mellitus

One-quarter of adult population in different world regions are reported to live with hypertension, of whom, a high percentage had a diabetes mellitus (DM). This co-morbid state is believed to act synergistically on accelerating the long-term diabetic complications. Therefore, adequate treatment of high blood pressure is essential for diabetic patients, and should be always directed to their benefits. Albumin glycation is still the most important explanation for the pathogenesis of chronic diabetic complications. Our in vitro experiments induce non-enzymatic glycation of bovine serum albumin (BSA) under physiological conditions. The levels of advanced glycation end products (AGEs) were measured by their characteristic intrinsic fluorescence. Additions of captopril at concentrations from 5 to 50 μM caused 10–47% reduction in the formation of AGEs. Captopril binding properties of native and glycosylated BSA were characterized, and its affinity towards the native albumin was unaltered by the in vitro glycation. Therefore, we concluded that captopril could protect against albumin glycation, and it has a similar binding affinities towards native and glcosylated protein. Anti-glycation effect may help to attenuate the serious long-term diabetes related complications. Besides, the unchanged pharmacokinetic parameters provide an essential extra beneficial effect for diabetic hypertensive patients.

Binding of D- and L-captopril inhibitors to metallo-beta-lactamase studied by polarizable molecular mechanics and quantum mechanics.

The bacterial Zn2+ metallo-beta-lactamase from B. fragilis is a zinc-enzyme with two potential metal ion binding sites. It cleaves the lactam ring of antibiotics, thus contributing to the acquired resistance of bacteria against antibiotics. The present study bears on the binuclear form of the enzyme. We compare several possible binding modes of captopril, a mercaptocarboxamide inhibitor of several zinc-metalloenzymes. Two diastereoisomers of captopril were considered, with either a D- or an L-proline residue. We have used the polarizable molecular mechanics procedure SIBFA (Sum of Interactions Between Fragments ab initio computed). Two beta-lactamase models were considered, encompassing 104 and 188 residues, respectively. The energy balances included the inter and intramolecular interaction energies as well as the contribution from solvation computed using a continuum reaction field procedure. The thiolate ion of the inhibitor is binding to both metal ions, expelling the bridging solvent molecule from the uncomplexed enzyme. Different competing binding modes of captopril were considered, either where the inhibitor binds in a monodentate mode to the zinc cations only with its thiolate ion, or in bidentate modes involving additional zinc binding by its carboxylate or ketone carbonyl groups. The additional coordination by the inhibitor's carboxylate or carbonyl group always occurs at the zinc ion, which is bound by a histidine, a cysteine, and an aspartate side chain. For both diastereomers, the energy balances favor monodentate binding of captopril via S-. The preference over bidentate binding is small. The interaction energies were recomputed in model sites restricted to captopril, the Zn2+ cations, and their coordinating end side chains from beta-lactamase (98 atoms). The interaction energies and their ranking among competing arrangements were consistent with those computed by ab initio HF and DFT procedures.

Enthalpy of captopril-angiotensin I-converting enzyme binding

High-sensitivity titration calorimetry is used to measure changes in enthalpy, heat capacity and protonation for the binding of captopril to the angiotensin I-converting enzyme (ACE; EC 3.4.15.1). The affinity of ACE to captopril is high and changes slightly with the pH, because the number of protons linked to binding is low. The determination of the enthalpy change at different pH values suggests that the protonated group in the captopril-ACE complex exhibits a heat protonation of approximately −30 kJ/mol. This value agrees with the protonation of an imidazole group. The residues which may become protonated in the complex could be two histidines existing in two active sites, which are joined to the amino acids coordinated to Zn 2+. Calorimetric measurements indicate that captopril binds to two sites in the monomer of ACE, this binding being enthalpically unfavorable and being dominated by a large positive entropy change. Thus, binding is favored by both electrostatic and hydrophobic interactions. The temperature dependence of the free energy of binding Δ G° is weak because of the enthalpy-entropy compensation caused by a large heat capacity change, Δ C p=−4.3±0.1 kJ/K/mol of monomeric ACE. The strong favorable binding entropy and the negative Δ C p indicate both a large contribution to binding due to hydrophobic effects, which seem to originate from dehydration of the ligand-protein interface, and slight conformational changes in the vicinity of the active sites.

Nuclear magnetic resonance studies of the binding of captopril and penicillamine by serum albumin

The metabolism of the thiol-containing drugs penicillamine (β,β-dimethylcysteine) and captopril ( d-3-mercapto-2-methylpropanoyl- l-proline) involves the formation of mixed disulfides, including mixed disulfides with serum albumin. The reactions of penicillamine and captopril with serum albumin in aqueous solution and in intact human blood plasma have been studied by 500 MHz 1H NMR spectroscopy. Penicillamine was found to react rapidly at the albumin-cysteine mixed disulfide bond to form penicillamine-cysteine mixed disulfide and to react more slowly at other albumin disulfide bonds. The amino acid cysteine was found to react with albumin by the same two pathways. In contrast, captopril rapidly associates with albumin to form noncovalent albumin-captopril complexes. Exchange of captopril between its free and noncovalently bound forms takes place on the NMR time scale. On a longer time scale, captopril reacts with albumin by thiol/disulfide interchange reactions. Noncovalently bound captopril displaced lactate from its albumin binding sites, both in aqueous solution and in human plasma. The results demonstrate that 1H NMR is a useful method for characterizing the state of drug molecules in human plasma and for detecting and monitoring perturbations by drugs of delicately balanced binding equilibria involving endogenous small molecules and macromolecules in plasma.

Angiotensin-converting enzyme localized in the rat pituitary and adrenal glands by [3H]captopril autoradiography.

We have localized angiotensin-converting enzyme (EC 3.14.5.1) in the rat pituitary and adrenal glands by [3H]captopril autoradiography. The maximal numbers of [3H] captopril-binding sites are: posterior pituitary, 4500 fmol/mg protein; anterior pituitary, 1950 fmol/mg; intermediate pituitary, less than 100 fmol/mg; adrenal medulla, 480 fmol/mg; and adrenal cortex, less than 25 fmol/mg. The distribution within the posterior pituitary and adrenal medulla is homogeneous, whereas that in the anterior pituitary is patchy. Subcellular fractionation of the bovine adrenal medulla reveals enrichment of angiotension-converting enzyme in plasma membrane fractions, but not in chromaffin granules. [3H]Captopril autoradiography in the rat pituitary gland is unaltered by dehydration, adrenalectomy, or reserpine treatment and in Brattleboro rats. [3H]Captopril binding in the adrenal medulla is increased by 75% 3 weeks after hypophysectomy and is elevated by 80% after reserpine treatment. The change after hypophysectomy is not reversed by dexamethasone treatment.

Inhibition of [ 3H]captopril binding by peptide analog angiotensin converting enzyme inhibitors

Ketomethylene containing peptide analogs, modeled after a snake venom pentapeptide, have been shown to be potent angiotensin converting enzyme inhibitors. Although the most potent compounds are up to five times more potent than captopril in inhibiting angiotensin converting enzyme activity, they are relatively weak inhibitors of [ 3H]captopril binding to membrane bound angiotensin converting enzyme. This indicates that inhibition of [ 3H]captopril binding and enzymatic activity is due to binding to distinct sites. These results suggest that the inhibitors bind to an additional site on the enzyme distinct from the captopril binding site.

Endocrinological control and cellular localization of rat testicular angiotensin-converting enzyme (EC 3.4.15.1).

Hypophysectomy of prepubescent (3-week-old) rats prevented the pubertal development of testicular, but not pulmonary, angiotensin-converting enzyme (EC 3.4.15.1). Additionally, hypophysectomy resulted in a loss of testicular converting enzyme activity in 10-week-old rats that had achieved puberty and had developed enzyme activity. Hormone regimens consisting of FSH/LH (7.5 U/rat X day), hCG (10 U/rat X day), or testosterone (1 mg/rat X day) were employed to ascertain their ability to maintain activity in hypophysectomized rats. All three of the above hormone regimens, if initiated on the first day after hypophysectomy of 10-week-old rats, were capable of maintaining testicular converting enzyme activity. Centrifugal elutriation of dispersed testicular cells indicated that the majority of enzyme activity in mature rats was associated with the germinal cells, a result consistent with the data accumulated from the hormonal studies. Lastly, [3H]captopril bound specifically to cellular fractions enriched in germinal cells. The above studies suggest that the pituitary gland is required for the development and maintenance of testicular angiotensin-converting enzyme in the rat by stimulating steroidogenesis in the testes. Furthermore, the sensitivity of converting enzyme activity to androgen coupled with the centrifugal elutriation and [3H] captopril binding studies strongly support the notion that testicular converting enzyme is associated with germinal cells.

Autoradiographic visualization of angiotensin-converting enzyme in rat brain with [3H]captopril: localization to a striatonigral pathway.

We have visualized angiotensin-converting enzyme (ACE; dipeptidyl carboxypeptidase, peptidylpeptide hydrolase, EC 3.4.15.1) in rat brain by in vitro [3H]captopril autoradiography. [3H]Captopril binding to brain slices displays a high affinity (Kd = 1.8 X 10(-9) M) and a pharmacological profile similar to that of ACE activity. Very high densities of [3H]captopril binding were found in the choroid plexus and the subfornical organ. High densities were present in the caudate putamen and substantia nigra, zona reticulata. Moderate levels were found in the entopeduncular nucleus, globus pallidus, and median eminence of the hypothalamus. Lower levels were detectable in the supraoptic and paraventricular nuclei of the hypothalamus, the medial habenula, the median preoptic area, and the locus coeruleus. Injection of ibotenic acid or colchicine into the caudate putamen decreased [3H]captopril-associated autoradiographic grains by 85% in the ipsilateral caudate putamen and by greater than 50% in the ipsilateral substantia nigra. Thus, ACE in the substantia nigra is located on presynaptic terminals of axons originating from the caudate putamen, and ACE in the caudate putamen is situated in neuronal perikarya or at the terminals of striatal interneurons. The lack of effect of similar injections into the substantia nigra confirmed that the caudate putamen injections did not cause trans-synaptic changes. The presence of [3H]captopril binding is consistent with an ACE-mediated production of angiotensin II in some brain regions. Although [3H]captopril autoradiography reveals ACE in a striatonigral pathway, there is no evidence for angiotensin II involvement in such a neuronal pathway.

3H]Captopril binding to membrane associated angiotensin converting enzyme

[ 3H]Captopril binding to membrane fractions of rat tissues is saturable and reversible with a K D of 2.4nM. [ 3H]Captopril binding and angiotensin converting enzyme measured with hippuryl-L-histidine-L-leucine are distributed in parallel between different tissues and brain regions, with highest levels in the choroid plexus, lung and corpus striatum. Captopril, N-(1(S)-carboxy-3-phenyl-propyl)-L-alanyl-L-proline, N-(1(S)-carboxy-3-phenyl-propyl)-L-lysyl-L-proline, teprotide, thiorphan and S-acetylcaptopril each have similar potencies for inhibition of [ 3H]captopril binding and of angiotensin converting enzyme. These data strongly indicate that [ 3H]Captopril binding evaluation should help clarify the localization and function of angiotensin converting enzyme and assist in defining pharmacologic actions of captopril.