Antioxidant activity of peptide-based angiotensin converting enzyme inhibitors

Angiotensin converting enzyme (ACE) inhibitors are important for the treatment of hypertension as they can decrease the formation of vasopressor hormone angiotensin II (Ang II) and elevate the levels of vasodilating hormone bradykinin. It is observed that bradykinin contains a Ser-Pro-Phe motif near the site of hydrolysis. The selenium analogues of captopril represent a novel class of ACE inhibitors as they also exhibit significant antioxidant activity. In this study, several di- and tripeptides containing selenocysteine and cysteine residues at the N-terminal were synthesized. Hydrolysis of angiotensin I (Ang I) to Ang II by ACE was studied in the presence of these peptides. It is observed that the introduction of L-Phe to Sec-Pro and Cys-Pro peptides significantly increases the ACE inhibitory activity. On the other hand, the introduction of L-Val or L-Ala decreases the inhibitory potency of the parent compounds. The presence of an L-Pro moiety in captopril analogues appears to be important for ACE inhibition as the replacement of L-Pro by L-piperidine 2-carboxylic acid decreases the ACE inhibition. The synthetic peptides were also tested for their ability to scavenge peroxynitrite (PN) and to exhibit glutathione peroxidase (GPx)-like activity. All the selenium-containing peptides exhibited good PN-scavenging and GPx activities.     

Effect of peptide-based captopril analogues on angiotensin converting enzyme activity and peroxynitrite-mediated tyrosine nitration

Angiotensin converting enzyme (ACE) regulates the blood pressure by converting angiotensin I to angiotensin II and bradykinin to bradykinin 1-7. These two reactions elevate the blood pressure as angiotensin II and bradykinin are vasoconstrictory and vasodilatory hormones, respectively. Therefore, inhibition of ACE is an important strategy for the treatment of hypertension. The natural substrates of ACE, i.e., angiotensin II and bradykinin, contain a Pro-Phe motif near the site of hydrolysis. Therefore, there may be a Pro-Phe binding pocket at the active site of ACE, which may facilitate the substrate binding. In view of this, we have synthesized a series of thiol- and selenol-containing dipeptides and captopril analogues and studied their ACE inhibition activities. This study reveals that both the selenol or thiol moiety and proline residues are essential for ACE inhibition. Although the introduction of a Phe residue to captopril and its selenium analogue considerably reduces the inhibitory effect, there appears to be a Phe binding pocket at the active site of ACE.     

Angiotensin II Type I Receptor (AT1R): The Gate towards COVID-19-Associated Diseases

The binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein to its cellular receptor, the angiotensin-converting enzyme 2 (ACE2), causes its downregulation, which subsequently leads to the dysregulation of the renin–angiotensin system (RAS) in favor of the ACE–angiotensin II (Ang II)–angiotensin II type I receptor (AT1R) axis. AT1R has a major role in RAS by being involved in several physiological events including blood pressure control and electrolyte balance. Following SARS-CoV-2 infection, pathogenic episodes generated by the vasoconstriction, proinflammatory, profibrotic, and prooxidative consequences of the Ang II–AT1R axis activation are accompanied by a hyperinflammatory state (cytokine storm) and an acute respiratory distress syndrome (ARDS). AT1R, a member of the G protein-coupled receptor (GPCR) family, modulates Ang II deleterious effects through the activation of multiple downstream signaling pathways, among which are MAP kinases (ERK 1/2, JNK, p38MAPK), receptor tyrosine kinases (PDGF, EGFR, insulin receptor), and nonreceptor tyrosine kinases (Src, JAK/STAT, focal adhesion kinase (FAK)), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. COVID-19 is well known for generating respiratory symptoms, but because ACE2 is expressed in various body tissues, several extrapulmonary pathologies are also manifested, including neurologic disorders, vasculature and myocardial complications, kidney injury, gastrointestinal symptoms, hepatic injury, hyperglycemia, and dermatologic complications. Therefore, the development of drugs based on RAS blockers, such as angiotensin II receptor blockers (ARBs), that inhibit the damaging axis of the RAS cascade may become one of the most promising approaches for the treatment of COVID-19 in the near future. We herein review the general features of AT1R, with a special focus on the receptor-mediated activation of the different downstream signaling pathways leading to specific cellular responses. In addition, we provide the latest insights into the roles of AT1R in COVID-19 outcomes in different systems of the human body, as well as the role of ARBs as tentative pharmacological agents to treat COVID-19.     

Separation and detection of angiotensin peptides by Cu(II) complexation and capillary electrophoresis with UV and electrochemical detection

The use of capillary electrophoresis (CE) with on-capillary Cu(II) complexation for the determination of angiotensin and its metabolites is described. The resulting copper-peptide complexes can be detected using either UV or electrochemical (EC) detection. Optimal reaction and separation conditions for the angiotensin peptides were first determined using CE with UV detection. With UV detection, the limit of detection (signal-to noise ratio S/N = 3) for native angiotensin II was 18 microM, while the limit of detection (LOD) obtained for the copper-angiotensin II complex is 2 microM. CE with EC detection was then evaluated, yielding significantly lower LODs--2 microM for native angiotensin II and 200 nM for the copper-angiotensin II complex. The addition of copper to the run buffer improved the separation and sensitivity for both CE-UV and CE-EC detection. The method was demonstrated by monitoring the conversion of angiotensin I to angiotensin II in plasma via angiotensin-converting enzyme (ACE) and subsequent inhibition of ACE by captopril.     

Inhibitory effects of enalaprilat on rat cardiac fibroblast proliferation via ROS/P38MAPK/TGF-β1 signaling pathway

Enalaprilat (Ena.), an angiotensin II (Ang II) converting enzyme inhibitor (ACEI), can produce some therapeutic effects on hypertension, ventricular hypertrophy and myocardial remodeling in clinic, but its precise mechanism, especially its signaling pathways remain elusive. In this study, cardiac fibroblasts (CFb) was isolated by the trypsin digestion method; a BrdU proliferation assay was adopted to determine cell proliferation; an immunofluorescence assay was used to measure intracellular reactive oxygen species (ROS); immunocytochemistry staining and Western blotting assay were used to detect phosphorylated p38 mitogen activated protein kinase (p-p38MAPK) and transforming growth factor-β(1) (TGF-β(1)) protein expression, respectively. The results showed that Ang II (10(-7) M) stimulated the cardiac fibroblast proliferation which was inhibited by NAC (an antioxidant), SB203580 (a p38MAPK inhibitor) or enalaprilat; Ang II caused an burst of intracellular ROS level within thirty minutes, an increase in p-p38MAPK (3.6-fold of that in the control group), as well as an elevation of TGF-β(1) meantime; NAC, an antioxidant, and enalaprilat treatment attenuated cardiac fibroblast proliferation induced by Ang II and decreased ROS and p-p38MAPK protein levels in rat cardiac fibroblast; SB203580 lowered TGF-β(1) protein expression in rats' CFb in a dose-dependent manner. It could be concluded that enalaprilat can inhibit the cardiac fibroblast proliferation induced by Ang II via blocking ROS/P38MAPK/TGF-β(1) signaling pathways and the study provides a theoretical proof for the application of ACEIs in treating myocardial fibrosis and discovering the primary mechanism through which ACEIs inhibit CFb proliferation.     

Sodium tanshinone IIA sulfonate attenuates angiotensin II-induced collagen type I expression in cardiac fibroblasts in vitro

Cardiac fibrosis occurs after pathological stimuli to the cardiovascular system. One of the most important factors that contribute to cardiac fibrosis is angiotensin II (Ang II). Accumulating studies have suggested that reactive oxygen species (ROS) plays an important role in cardiac fibrosis and sodium tanshinone IIA sulfonate (STS) possesses antioxidant action. We therefore examined whether STS depresses Ang II-induced collagen type I expression in cardiac fibroblasts. In this study, Ang II significantly enhanced collagen type I expression and collagen synthesis. Meanwhile, Ang II depressed matrix metalloproteinase-1 (MMP-1) expression and activity. These responses were attenuated by STS. Furthermore, STS depressed the intracellular generation of ROS, NADPH oxidase activity and subunit p47^phox expression. In addition, N-acetylcysteine the ROS scavenger, depressed effects of Ang II in a manner similar to STS. In conclusion, the current studies demonstrate that anti-fibrotic effects of STS are mediated by interfering with the modulation of ROS.     

Determination of captopril in biological samples by high-performance liquid chromatography with ThioGlo 3 derivatization.

Captopril, a well-known angiotensin converting enzyme (ACE) inhibitor, is widely used for treatment of arterial hypertension. Recent studies suggest that it may also act as a scavenger of free radicals because of its thiol group. Therefore, the present study describes a rapid, sensitive and relatively simple method for the detection of captopril in biological tissues with reverse-phase HPLC. Captopril was first derivatized with ThioGlo 3 [3H-Naphto[2,1-b]pyran,9-acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)phenyl-3-oxo-)]. It was then detected by fluorescence-HPLC using an Astec C(18) column as the stationary phase and a water:acetonitrile:acetic acid:phosphoric acid mixture (50:50; 1 mL/L acids) as the mobile phase (excitation wavelength, 365 nm; emission wavelength, 445 nm). The calibration curve for captopril was linear over a range of 10-2500 nM and the coefficient of variation acquired for the within- and between-run precision for captopril was 0.5 and 3.8%, respectively. The detection limit of captopril with this method was found to be 200 fmol/20 microL injection volume. Its relative recovery from biological samples was determined to the range from 93.3 to 105.3%. Based on these results, we believe that our method is advantageous for captopril determination.     

Development of inhibitors of the aspartyl protease renin for the treatment of hypertension

Renin is the rate-limiting enzyme in the renin-angiotensin-aldosterone system (RAS) which controls blood pressure and volume. The biological function of renin is to cleave the N-terminus of angiotensinogen releasing the decapeptide, angiotensin I (ANGI). Subsequently, angiotensin I is further processed by the angiotensin converting enzyme (ACE) to produce angiotensin II (ANGII). The RAS cascade is a major target for the clinical management of hypertension. Current clinical treatments include angiotensin converting enzyme inhibitors (ACEi) and ANGII receptor blockers (ARBs). As the rate-limiting enzyme in ANGII production, renin inhibitors have been pursued as an additional class of anti-hypertensives. Clinical studies conducted with renin inhibitors have shown them to be as effective as ACE inhibitors in lowering blood pressure. Most importantly, inhibitors of renin may have a number of potential advantages over ACEi and ARBs. Renin is specific for angiotensinogen and will not carry the ancillary pharmacology associated with ACEi or ARBs. To date, no renin inhibitors have made it to market. The development of these inhibitors has been hindered by poor bioavailability and complex synthesis. However, despite the pharmacokinetic challenges of designing renin inhibitors, the enzyme remains a promising target for the development of novel treatments for hypertension. This review will consist of an overview of renin biology, the pharmacology of renin and RAS and focus in on renin as a target for blood pressure regulation. We also cover the evaluation of renin inhibitors in animal models and clinical studies. Presently a number of new generation inhibitors of renin are in development with at least one in the clinic and these will be discussed. Finally we will discuss what might distinguish renin inhibitors from current therapeutic options and discuss other therapeutic indications renin inhibitors might have.     

Effect of myricetin on deoxycorticosterone acetate (DOCA)-salt-hypertensive rats

Chronic administration of myricetin (100 and 300?mg?kg?1, p.o., for 4 weeks) isolated from Vitis vinifera (Vitaceae) ameliorated hypertension and oxidative stress induced by deoxycorticosterone acetate (DOCA)-salt in rats. Myricetin treatment reduced systolic blood pressure, vascular reactivity changes and reversed the DOCA-induced increase in heart rate. Urinary sodium excretion was significantly decreased in animals treated with myricetin compared to the DOCA group when measured by flame photometer. The cumulative concentration response curve of serotonin (5-HT) and angiotensin II (Ang II) were shifted towards the right in rats treated with myricetin using the isolated rat fundus strip and ascending colon, respectively. Increased levels of thiobarbituric acid reactive substances and decreased levels of superoxide dismutase, catalase and reduced glutathione in the heart tissue were observed in animals treated with DOCA, which were reversed by myricetin. Thus, myricetin shows antihypertensive and antioxidant properties in the DOCA model of hypertension.     

Review on the Angiotensin-I-Converting Enzyme (ACE) Inhibitor Peptides from Marine Proteins

Hypertension is now a major problem threatening people health in the world. Angiotensin-I-converting enzyme (ACE) plays an important physiological role in regulation of blood pressure via conversion of angiotensin I to angiotensin II. Inhibition of ACE may have an antihypertensive effect as a consequence of a decrease in blood pressure. A number of terrestrial-derived peptides have been reviewed about their in vitro and in vivo ACE-inhibitory activities. Marine organisms are potentially an untapped source of drugs and value-added food production. The aim of this review is to discuss the marine-derived ACE-inhibitory peptides from sources, production, structure aspects, and their future prospects as functional food or novel therapeutic drug candidates.     

Prediction of COVID-19 manipulation by selective ACE inhibitory compounds of Potentilla reptant root: In silico study and ADMET profile

In the novel SARS-CoV-2 (COVID-19) as a global emergency event, the main reason of the cardiac injury from COVID-19 is angiotensin-converting enzyme 2 (ACE2) targeting in SARS-CoV-2 infection. The inhibition of ACE2 induces an increase in the angiotensin II (Ang II) and the angiotensin II receptor type 1 (AT1R) leading to impaired cardiac function or cardiac inflammatory responses. The ethyl acetate fraction of Potentilla reptans L. root can rescue heart dysfunction, oxidative stress, cardiac arrhythmias and apoptosis. Therefore, isolated components of P. reptans evaluated to identify natural anti-SARS-CoV-2 agents via molecular docking.In silico molecular docking study were carried out using the Auto Dock software on the isolated compounds of Potentilla reptans root. The protein targets of selective ACE and others obtained from Protein Data Bank (PDB). The best binding pose between amino acid residues involved in active site of the targets and compounds was discovered via molecular docking. Furthermore, ADMET properties of the compounds were evaluated.The triterpenoids of P. reptans showed more ACE inhibitory potential than catechin in both domains. They were selective on the nACE domain, especially compound 5. Also, the compound 5 & 6 had the highest binding affinity toward active site of nACE, cACE, AT1R, ACE2, and TNF-α receptors. Meanwhile, compound 3 showed more activity to inhibit TXA2. Drug likeness and ADMET analysis showed that the compounds passed the criteria of drug likeness and Lipinski rules. The current study depicted that P. reptans root showed cardioprotective effect in COVID-19 infection and manipulation of angiotensin II-induced side effects.     

The Two-Way Switch Role of ACE2 in the Treatment of Novel Coronavirus Pneumonia and Underlying Comorbidities

December 2019 saw the emergence of the coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which has spread across the globe. The high infectivity and ongoing mortality of SARS-CoV-2 emphasize the demand of drug discovery. Angiotensin-converting enzyme II (ACE2) is the functional receptor for SARS-CoV-2 entry into host cells. ACE2 exists as a membrane-bound protein on major viral target pulmonary epithelial cells, and its peptidase domain (PD) interacts SARS-CoV-2 spike protein with higher affinity. Therefore, targeting ACE2 is an important pharmacological intervention for a SARS-CoV-2 infection. In this review, we described the two-way switch role of ACE2 in the treatment of novel coronavirus pneumonia and underlying comorbidities, and discussed the potential effect of the ACE inhibitor and angiotensin receptor blocker on a hypertension patient with the SARS-CoV-2 infection. In addition, we analyzed the S-protein-binding site on ACE2 and suggested that blocking hot spot-31 and hot spot-353 on ACE2 could be a therapeutic strategy for preventing the spread of SARS-CoV-2. Besides, the recombinant ACE2 protein could be another potential treatment option for SARS-CoV-2 induced acute severe lung failure. This review could provide beneficial information for the development of anti-SARS-CoV-2 agents via targeting ACE2 and the clinical usage of renin-angiotensin system (RAS) drugs for novel coronavirus pneumonia treatment.     

Heme peroxidase-catalyzed iodination of human angiotensins and the effect of iodination on angiotensin converting enzyme activity

The heme peroxidase-catalyzed iodination of human angiotensins I and II is described. It is observed that lactoperoxidase (LPO) can effectively and selectively iodinate the tyrosyl residues in angiotensin peptides. The thiourea/thiouracil-based peroxidase inhibitors effectively inhibit the iodination reactions, indicating that iodination is an enzymatic reaction and the mechanism of iodination is similar to that of peroxidase-catalyzed iodination of thyroglobulin. This study also shows that the monoiodo Ang I is a better substrate for the angiotensin converting enzyme than the native peptide.     

The Renin-Angiotensin System: A Key Role in SARS-CoV-2-Induced COVID-19

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), was first identified in Eastern Asia (Wuhan, China) in December 2019. The virus then spread to Europe and across all continents where it has led to higher mortality and morbidity, and was declared as a pandemic by the World Health Organization (WHO) in March 2020. Recently, different vaccines have been produced and seem to be more or less effective in protecting from COVID-19. The renin–angiotensin system (RAS), an essential enzymatic cascade involved in maintaining blood pressure and electrolyte balance, is involved in the pathogenicity of COVID-19, since the angiotensin-converting enzyme II (ACE2) acts as the cellular receptor for SARS-CoV-2 in many human tissues and organs. In fact, the viral entrance promotes a downregulation of ACE2 followed by RAS balance dysregulation and an overactivation of the angiotensin II (Ang II)–angiotensin II type I receptor (AT1R) axis, which is characterized by a strong vasoconstriction and the induction of the profibrotic, proapoptotic and proinflammatory signalizations in the lungs and other organs. This mechanism features a massive cytokine storm, hypercoagulation, an acute respiratory distress syndrome (ARDS) and subsequent multiple organ damage. While all individuals are vulnerable to SARS-CoV-2, the disease outcome and severity differ among people and countries and depend on a dual interaction between the virus and the affected host. Many studies have already pointed out the importance of host genetic polymorphisms (especially in the RAS) as well as other related factors such age, gender, lifestyle and habits and underlying pathologies or comorbidities (diabetes and cardiovascular diseases) that could render individuals at higher risk of infection and pathogenicity. In this review, we explore the correlation between all these risk factors as well as how and why they could account for severe post-COVID-19 complications.     

Molecular dynamics simulations of angiotensin II in aqueous and dimethyl sulfoxide environments

Angiotensin II (Ang II) is an octapeptidic hormone, which plays an important role in the mechanisms of blood pressure control. In this work, extensive molecular dynamics (MD) simulations have been carried out on this peptide, both in aqueous and in dimethyl sulfoxide (DMSO) environments. Experimentally proposed models for the structure of angiotensin II in both environments are not consensual and the results obtained have provided some further insight about the structural properties of this hormone. In these simulations, the N-terminus of Ang II in the aqueous environment has been associated with a considerable larger flexibility than the correspondent C-terminus, but this was not found in the case of the DMSO environment. This is consistent with the assumption that the biological activity of Ang II is associated with its C-terminal residues embedded in a hydrophobic environment of its AT1 receptor. Other features detected in DMSO environment were an H(His6 imidazole)-O(Phe8 carboxylate) hydrogen bond and a salt-bridge structure involving the Asp1 and Arg2 side chains. An additional important conformational feature is the spatial proximity between Tyr4 and His6 in both water and DMSO environments. This molecular feature may trigger the interest for the synthetic chemists to apply rational design for the synthesis of novel AT1 antagonists.     

Angiotensin converting enzyme I/D, angiotensinogen T174M-M235T and angiotensin II type 1 receptor A1166C gene polymorphisms in Turkish hypertensive patients

Essential hypertension is a multifactorial disease in which genetic and enviromental factors play an important role. These factors differ in each population. As there are no existing data for the Turkish population, we investigated four Renin Angiotensin System (RAS) gene polymorphisms, the angiotensin converting enzyme (ACE), angiotensinogen (AGN) M235T/T174M and angiotensin II type 1 receptor A1166C polymorphism in 109 hypertensive and 86 normotensive Turkish subjects. Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP), and agarose gel electrophoresis tecniques were used to determine these polymorphism. The frequencies of person that carry ACE D allel (DD+ID) was significantly higher in hypertensive group (99.1%) than controls (80%) (P 0.000). M235T TT genotype was also found significantly higher in hypertensives than control group (20% vs 2.7%; P 0.001). The frequency of AGN 174M allele was higher in the hypertensive group than control subjects (8.76% vs 4.81%). Frequency of ATR1 C allele (AC+CC genotypes) was found higher hypertensives than controls (39.4% vs 25.9%; P = 0.054). Our results suggest that an interaction exists between the RAS genes and hypertension in Turkish population.     

Sensitive assay for serum angiotensin-converting enzyme and separation of angiotensin analogues by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the assay of angiotensin-converting enzyme in human serum and for the separation of angiotensins and their analogues after pre-column fluorescence derivatization with benzoin. Angiotensin II, formed enzymatically from angiotensin I, is converted into a fluorescent derivative which is then separated isocratically from the substrate and biological substances in the enzyme reaction mixture on a reversed-phase column (TSK gel ODS-120T). The lower limit of detection for angiotensin II is 0.66 pmol per enzyme assay tube. The method is simple and sensitive, and requires as little as 5 microliter of human serum. Angiotensin analogues can also be separated and quantified by the chromatographic technique, and thus this method permits the use of the analogues of angiotensin I as substrates.     

Synthesis and characterization of captopril derivatives

To develop more potential angiotensin converting enzyme (ACE) inhibitors, a series of captopril (Cap) derivatives were synthesized, including Cap-glycine methyl ester, Cap-l-alanine methyl ester, Cap-l-aspartic acid dimethyl ester, Cap-l-lysine methyl ester, Cap-O-acylisourea, acetyl captopril, and benzoyl captopril. The resulting products were characterized by IR and UV–visible spectroscopy and MS, which showed the desired products were successfully synthesized. This could serve as a guide for rational design of highly potent ACE inhibitors.     

The Synthetic Strategy toward of ACE-Inhibitors

Angiotensin II is an important octapeptide which is responsible for the increase in blood pressure in three major mechanisms. It acts as a hormone to attack the receptor on the blood vessels, which cause strong vasoconstriction. It is also the major stimulus for release another hormone, aldolsterone, which promote the excretion of potassium ion and retention of sodium and waster. Both of the above effects increase the blood pressure. On the other hand, ACE (Angiotensin Converting Enzyme) catalyzes the hydrolysis of bradykinin that is a potent vasodilator. Therefore, the inhibitor of ACE can act as an efficient anti-hypertensive agent through multiple routes.     

Quantitation of plasma angiotensin II in healthy Chinese subjects by a validated liquid chromatography tandem mass spectrometry method

Quantitation of plasma angiotensin (Ang) II, the active mediator of the renin–angiotensin system, is challenging owing to its low physiological concentration. We report a validated liquid chromatography–mass spectrometry (LCMS) method to overcome this challenge. Ang II was extracted from EDTA plasma by an offline solid-phase extraction procedure with a Waters MAX μElution plate. LCMS quantitation was performed on the Waters TQS system, monitoring the 3+ ions of the peptide. The analytical performance of the LCMS method was validated. The stability of Ang II was studied with or without the presence of a protease inhibitor. Local reference intervals were established from 143 healthy normotensive subjects (57% female, 21–60 years old). The Ang II LCMS method had a measurable range of 3.3–700 pmol/L. The between-batch precision coefficient of variation was <7% over Ang II concentrations of 8.6–110 pmol/L. No significant matrix interference and carryover were observed. There was no significant difference in Ang II concentration in EDTA blood and plasma for at least 2h and 1 h at room temperature, respectively. Ang II was stable for at least 1 year when stored at −80°C, with or without the protease inhibitor. Age-dependent Ang II reference intervals were established: 4.4–17.7 pmol/L (21–30 years) and 3.9–12.8 pmol/L (31–60 years). The present LCMS method is suitable for quantitation of plasma Ang II to study the renin-angiotensin system.