Angiotensin I-converting Enzyme Inhibitor Research Articles

Peptide inhibitors of angiotensin-I converting enzyme based on angiotensin (1–7) with selectivity for the C-terminal domain

The renin-angiotensin system (RAS) is a key regulator of human arterial pressure. Several of its effects are modulated by angiotensin II, an octapeptide originating from the action of angiotensin-I converting enzyme (ACE) on the decapeptide angiotensin-I. ACE possess two active sites (nACE and cACE) that have their own kinetic and substrate specificities. ACE inhibitors are widely used as the first-line treatment for hypertension and other heart-related diseases, but because they inactivate both ACE domains, their use is associated with serious side effects. Thus, the search for domain-specific ACE inhibitors has been the focus of intense research. Angiotensin (1–7), a peptide that also belongs to the RAS, acts as a substrate of nACE and an inhibitor of cACE. We have synthetized 15 derivatives of Ang (1–7), sequentially removing the N-terminal amino acids and modifying peptides extremities, to find molecules with improved selectivity and inhibition properties. Ac-Ang (2–7)-NH2 is a good ACE inhibitor, resistant to cleavage and with improved cACE selectivity. Molecular dynamics simulations provided a model for this peptide’s selectivity, due to Val3 and Tyr4 interactions with ACE subsites. Val3 has an important interaction with the S3 subsite, since its removal greatly reduced peptide-enzyme interactions. Taken together, our findings support ongoing studies using insights from the binding of Ac-Ang (2–7)-NH2 to develop effective cACE inhibitors.

Effects of in vitro digestion on protein degradation, phenolic compound release, and bioactivity of black bean tempeh.

The nutritional value and bioactivity of black beans are enhanced when fermented as tempeh, but their bioaccessibility and bioactivity after ingestion remain unclear. In this study, black bean tempeh and unfermented black beans were digested in vitro and changes in protein degradation, phenolic compound release, angiotensin I-converting enzyme (ACE)-inhibitory activity, and antioxidant activity between the two groups were compared. We observed that the soluble protein content of digested black bean tempeh was generally significantly higher than that of digested unfermented black beans at the same digestion stage (P < 0.05). The degree of protein hydrolysis and the content of <10 kDa peptides were also significantly higher in the digested black bean tempeh than in digested unfermented black beans (P < 0.05). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and reversed-phase high-performance liquid chromatography (RP-HPLC) analysis showed that most macromolecular proteins in tempeh had been degraded during fermentation and more of the small peptides were released from black bean tempeh during digestion, respectively. Compared to that of the unfermented black beans, the level of ACE inhibition of black bean tempeh was lower, but this significantly increased to 82.51% following digestion, closing the gap with unfermented black beans. In addition, the total respective levels of phenolics, flavonoids, and proanthocyanidins released from black bean tempeh were 1.21, 1.40, and 1.55 times those of unfermented black beans following in vitro digestion, respectively. Antioxidant activity was also significantly higher in digested black bean tempeh than in digested unfermented black beans and showed a positive correlation with phenolic compound contents (P < 0.05). The results of this study proved that, compared to unfermented black beans, black bean tempeh retained protein and phenolic compound bioaccessibility and antioxidant activity and showed an improved ACE-inhibitory activity even after consumption.

Angiotensin-converting Enzyme Inhibitors and Angiotensin Receptor Blockers as Potential Therapeutic Options for Pancreatic Cancer.

The renin-angiotensin system (RAS) has been reported to have a role in carcinogenesis, and therefore it may be of value as a potential therapeutic target in inhibiting tumor growth. It has been shown that inhibition of RAS via angiotensin I-converting enzyme inhibitors (ACEIs) and angiotensin II type-1 receptor (ARBs) inhibitors may have a protective effect against several malignancies. Here, we provide an overview of the potential value of the RAS pathway and targeting via ACE/ARB inhibitors in pancreatic cancer. Whilst the potential role of RAS as a target for the treatment of pancreatic cancer has been reported, the use of candesartan with gemcitabine failed to improve outcomes in pancreatic cancer. Another study of 1-3 years using ARB was found to reduce the risk of pancreatic cancer. In line with these trials, others have demonstrated that the ARBs in combination with gemcitabine might improve clinical outcomes in patients with advanced pancreatic cancer. Prospective trials are warranted to investigate this hypothesis.

The addition of probiotic promotes the release of ACE-I peptide of Cheddar cheese: Peptide profile and molecular docking

The use of probiotics to promote the release of bioactive peptides during Cheddar cheese ripening was investigated. The proteolytic and angiotensin-converting enzyme inhibitory (ACE-I) activities and ACE-I peptide profile of Cheddar cheese with different probiotics were analysed and compared. Molecular docking was used to analyse the binding site of peptide to ACE molecule. Compared with other cheeses, ACE-I activity of Lactobacillus helveticus cheese was the highest (79.71%), and the degree of proteolysis was higher and the molecular mass was lower. Most of the peptides were less than 1.6 kDa. In addition, 43.31–51.59% of the peptides in cheese originated from β-casein, mainly from the degradation of starter and probiotics. SKVLPQ and YQEPVLGP, which were uniquely identified in L.helveticus cheese, could interact with the S1 and S2 active pocket of ACE, respectively. Especially, YQEPVLGP could bind to ACE at multiple sites and was an efficient ACE inhibitory peptide.

Identification, in silico screening, and molecular docking of novel ACE inhibitory peptides isolated from the edible symbiot Boletus griseus-Hypomyces chrysospermus

The aim of this study is to find angiotensin-Ⅰ-converting enzyme inhibitory (ACEI) peptides from the edible symbiot Boletus griseus-Hypomyces chrysospermus (BGHC). The peptides from the methanol extract of BGHC were identified through ultraperformance liquid chromatography quadrupole Orbitrap tandem mass spectrometry analysis coupled with de novo sequencing by using Peak Studio software. Subsequently, two ACEI peptides, being KYPHVF and LFRPE, were screened on the basis of PeptideRanker and the AHTpin online server. The in silico absorption, distribution, metabolism, excretion and toxicities (ADMET) of the two peptides were predicted with AdmetSAR software, and the two peptides showed good absorption and nontoxicity. Meanwhile, the ACEI mechanism of KYPHVF and LFRPE was analyzed through molecular docking with SYBYL-X 2.0 software. The molecular docking study revealed that the total scores of the interactions of KYPHVF and LFRPE with ACE were 12.53 and 11.34, respectively. KYPHVF and LFRPE could establish hydrogen bonds and hydrophobic interactions with the active site of ACE. Furthermore, the ACEI activities of KYPHVF and LFRPE were determined in vitro with the IC50 values of 5.99 and 58.15 μmol/L, respectively.

Novel angiotensin I-converting enzyme (ACE) inhibitory peptides from walnut protein isolate: Separation, identification and molecular docking study.

Walnut protein isolate was hydrolyzed using alcalase® to obtain angiotensin-I-converting enzyme (ACE) inhibitory (ACEI) peptides. The components with high ACEI activity were successfully purified from walnut protein isolate hydrolysates (WPIH) by ultrafiltration and G-25 gel chromatography. The 1520 peptides were identified by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Then the screening model of ACEI peptides was established by in silico approach. It was found that four ACEI active peptides (PPKP, YPQY, YLPP, and PKPP) were obtained with IC50 values ranging from 506 to 89 μmol/L, among which PPKP had the highest ACEI activity (IC50 =89 ± 1μmol/L). The four peptides mentioned above were novel, non-toxic, and resistant to gastrointestinal digestion. The molecular docking studies showed that the ACEI effect of ACEI peptide was mainly due to the interaction with residues of Gln281 and His353 in the ACE active pockets. In vivo availability of ACEI peptides showed that the probability of PPKP binding to ACE was 37.9% in the human body. Our studies suggest that the ACEI peptides derived from the WPIH can be considered functional foods that can prevent hypertension. PRACTICAL APPLICATIONS: Hypertension is a significant risk factor for cardiovascular and cerebrovascular disease, the leading cause of death worldwide. This study used a cost-effective method to isolate and identify potential ACEI peptides from the walnut meal. Since the walnut meal is often discarded in the processing of walnut products and thus pollutes the environment, the preparation of walnut meal into ACEI peptides can reduce the impact of hypertension on people and reduce environmental pollution. The experimental results show that walnut ACEI peptides are a safe and healthy nutritional product.

Synthesis and evaluation of new phosphonated δ‐lactam and glutarimide derivatives as angiotensin converting enzyme inhibitors

AbstractAn efficient synthesis of new γ,δ‐insaturated δ‐lactam and glutarimide derivatives bearing a phosphonomethyl group from a common allylphosphonate precursor is described. Our approach is based on a two‐step procedure involving the preparation of phosphonated‐1,5‐ketoester and −1,5‐diester followed by an amidation–heterocyclization sequence. The first step proceeds via Michael's addition of ethyl acetoacetate and diethyl malonate on an allylphosphonate starting material. The second step consists of a base‐promoted intramolecular amidation‐cyclization sequence with primary amines, which accounts for the construction of δ‐lactams and glutarimide skeletons. We performed the evaluation of angiotensin I‐converting enzyme (ACE) inhibition using an in vitro enzyme assay on six new compounds. Five compounds showed potent ACE inhibitory activity, with IC50 values ranging from 0.02 to 0.27 mg/ml. Compared with Captopril, used as a reference drug, two new glutarimide derivatives exhibited higher efficiency ACE inhibition activity.

Mechanistic Insights into Angiotensin I-Converting Enzyme Inhibitory Tripeptides to Decipher the Chemical Basis of Their Activity.

Food proteins are an important source of bioactive peptides, and the angiotensin I-converting enzyme (ACE) inhibitors are worthy of attention for their possible beneficial effects in subjects with mild hypertension. However, the chemical basis underpinning their activity is not well-understood, hampering the discovery of novel inhibitory sequences from the plethora of peptides encrypted in food proteins. This work combined computational and in vitro investigations to describe precisely the chemical basis of potent inhibitory tripeptides. A substantial set of previously uncharacterized tripeptides have been investigated in silico and in vitro, and LCP was described for the first time as a potent ACE inhibitory peptide with IC50 values of 8.25 and 6.95 μM in cell-free and cell-based assays, respectively. The outcomes presented could serve to better understand the chemical basis of already characterized potent inhibitory tripeptides or as a blueprint to design novel and potent inhibitory peptides and peptide-like molecules.

Corn distillers solubles by two-step proteolytic hydrolysis as a new source of plant-based protein hydrolysates with ACE and DPP4 inhibition activities

Proteins of low-value and underexplored corn distillers solubles (CDS) have not been considerably valorized. Hence, the influence of one-step enzymatic hydrolysis of proteins with alcalase (A), trypsin (T) or flavourzyme (F) and two steps with AT, TA, AF, FA, TF, or FT was assessed to release peptides with angiotensin-I converting enzyme inhibition (ACEi) and dipeptidyl peptidase4 inhibition (DPP4i). The AF hydrolysate was the best sample in terms of yield, protein content, degree of hydrolysis, ACEi (97.68 ± 1.09 %), and DPP4i (51.51 ± 0.28 %). Mass spectrometry of the most active AF hydrolysate (<3 kDa) identified new major peptides like APLA, PLFP, LFLP, LPPYL, PLYPLP, NDWHTGPL, LPPYLPS, GSPFLGQ, SWQQPIVGG. Bioinformatic analysis showed these can inhibit both ACE and DPP4. This is because peptides contain functional groups and adopt conformations significantly binding with other functional groups at enzyme active sites (p < 0.05). This establishes dual bioactivity of peptides, which may have applications in food, feed, and pharmaceutical industries.

Enzymatic hydrolysis of marine fish gelatin for producing ACE inhibitor peptides: meta-analysis

Bioactive peptides showing Angiotensin-I-Converting Enzyme inhibitory (ACEi) activity can control blood pressure. They can be produced from various protein sources, including fish gelatin. This article provides a systematic review and meta-analysis on gelatin extraction, conditions of enzymatic hydrolysis, characteristics of ACEi peptides, and effect of hydrolysis procedure on ACEi peptides. Fish gelatin preparation uses various solutions such as acids, bases and distilled water. Enzymatic hydrolysis is carried out with an E/S ratio of 0.2 – 7% and temperature of 28.9 oC (using bromelain) and 60.6 oC (using Alcalase). Level of pH in hydrolysis also varied greatly, from 2 (using Pepsin) to 10 (using Purafect enzyme). The molecular weight of fish gelatin ACEi peptides ranged from 186-829 Da and the sequence of peptides was dominated by hydrophobic and aliphatic amino acids. Based on a meta-analysis, hydrolysis using enzyme combination resulted in more satisfying product than a single enzyme, represented with a combined effect size value of 0.593.

A novel angiotensin-I-converting enzyme inhibitory peptide from oyster: Simulated gastro-intestinal digestion, molecular docking, inhibition kinetics and antihypertensive effects in rats.

In this study, a novel peptide, AEYLCEAC with high angiotensin-I-converting enzyme inhibitory (ACEI) activity was screened from oyster (Crassostrea gigas) hydrolysates, which was obtained from simulated gastro-intestinal digestion. Candidate peptides were confirmed to have a higher binding to angiotensin-I-converting enzyme (ACE) than the positive drug phosphoinic tripeptide calculated by Discovery Studio, and AEYLCEAC showed the highest ACE inhibition rate in vitro with a IC50 of 4.287 mM. Lineweaver-Burk plots confirmed that the peptidic inhibitory type of ACE is competitive. The molecular docking showed that ACEI activity of the AEYLCEAC was mainly due to the hydrogen bonding interactions with the active pockets (S1 and S2) of ACE. In vivo, AEYLCEAC effectively reduced diastolic blood pressure (DBP) and Systolic blood pressure (SBP) in hypertensive rats. These results indicate that AEYLCEAC might act as a helpful ingredient in functional foods or pharmaceuticals for the prevention and treatment of hypertension.

Peptides inhibiting angiotensin-I-converting enzyme: Isolation from flavourzyme hydrolysate of Protaetia brevitarsis larva protein and identification

Many angiotensin-I-converting enzyme (ACE) inhibitory peptides are used to prevent and manage hypertension. In this study, ACE inhibitory peptides were isolated from an insect protein that is attracting attention for it potential antihypertensive activity. Protaetia brevitarsis larva protein was enzymatically hydrolyzed by Flavourzyme®, and the hydrolysate was shown to inhibit ACE. Subsequent fractionation, using ultrafiltration and gel permeation chromatography followed by liquid chromatography-tandem mass spectrometry analysis, identified four previously unknown peptides with significant ACE inhibition characteristics (Ser-Tyr, Pro-Phe, Tyr-Pro-Tyr, and Trp-Ile). The highest inhibition activity observed for Trp-Ile. These peptides stimulated production of NO in human umbilical vein endothelial cells and, based on molecular docking analysis, exerted their inhibitory effects via hydrogen bonding with the ACE receptor active site. Thus, the identified peptides can be considered as promising candidates for ACE inhibition and have potential to be used as functional food ingredients.

Isolation and identification of novel angiotensin I-converting enzyme (ACE) inhibitory peptides from Pony Seed and evaluation of the inhibitory mechanisms

In this work, four novel angiotensin I-converting enzyme inhibitory (ACEI) peptides were isolated and identified from peony (Paeonia suffruticosa Andr.) seed hydrolysate (PSH) by consecutive chromatography and UPLC-ESI-QTOF-MS/MS. The IC50 values of HWS (0.640 ±0.016 mg/mL) and VLSGF (0.328 ± 0.040 mg/mL) were significantly (p < 0.05) lower than those of the other two peptides. The Lineweaver-Burk plots showed that VLSGF is competitive inhibition against ACE and HWS acted as a mixed-type inhibitor. Meanwhile, the results of molecular docking showed hydrogen interactions between VLSGF/HWS and ACE. In human endothelial cells, the pretreatments of VLSGF and HWS could increase the production of nitric oxide (NO) and the expression of endothelial nitric oxide synthase (eNOS). In addition, they also decreased the reactive oxygen species (ROS) production. These results indicated that it may be possible to use peony seed protein hydrolysate to create ingredients for functional foods.

Isolation, identification, molecular docking analysis, and cytoprotection of seven novel angiotensin I-converting enzyme inhibitory peptides from miiuy croaker byproducts-swim bladders

For efficiently utilizing the processing byproducts of miiuy croaker to prepare novel angiotensin I-converting enzyme (ACE) inhibitory (ACEi) peptides, in vitro gastrointestinal (GI) digestion method was screened and employed to prepare swim bladder hydrolysate with the highest ACEi activity. Subsequently, seven novel ACEi peptides were isolated from the hydrolysate and identified as DEGPE, EVGIQ, SHGEY, GPWGPA, GPFGTD, SPYGF, and VIGPF with molecular weights of 545.49, 544.58, 591.55, 583.63, 592.59, 569.60, and 531.63 Da, respectively. SHGEY and SPYGF exhibited remarkable ACEi activity with IC50 values of 0.86 ± 0.12 and 0.37 ± 0.06 mg/mL. Molecular docking experiment illustrated that the significant ACEi activity of SHGEY and SPYGF with the affinity of -8.7 and -9.7 kcal/mol mainly attributed to effectively combining with the ACEi active sites by hydrophobic interaction, electrostatic force and hydrogen bonding. Moreover, SHGEY and SPYGF could significantly up-regulate the nitric oxide (NO) production and decrease the endothelin-1 (ET-1) secretion in human umbilical vein endothelial cells (HUVECs), but also abolished the negative impacting of norepinephrine to the levels of NO and ET-1. Furthermore, SHGEY and SPYGF showed significant protection to HUVECs against H2O2 damage by increasing superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity to lower the contents of reactive oxide species and malondialdehyde. Consequently, ACEi peptides derived from miiuy croaker swim bladders, especially SHGEY and SPYGF, are health-promoting ingredients for functional products as a supplementary treatment to hypertension and cardiovascular diseases.

Novel angiotensin-converting enzyme inhibitory peptides from tuna byproducts-milts: Preparation, characterization, molecular docking study, and antioxidant function on H2O2-damaged human umbilical vein endothelial cells.

To prepare peptides with high angiotensin-converting enzyme (ACE) inhibitory (ACEi) activity, Alcalase was screened from five proteases and employed to prepare protein hydrolysate (TMH) of skipjack tuna (Katsuwonus pelamis) milts. Subsequently, 10 novel ACEi peptides were isolated from the high-ACEi activity TMH and identified as Tyr-Asp-Asp (YDD), Thr-Arg-Glu (TRE), Arg-Asp-Tyr (RDY), Thr-Glu-Arg-Met (TERM), Asp-Arg-Arg-Tyr-Gly (DRRYG), Ile-Cys-Tyr (ICY), Leu-Ser-Phe-Arg (LSFR), Gly-Val-Arg-Phe (GVRF), Lys-Leu-Tyr-Ala-Leu-Phe (KLYALF), and Ile-Tyr-Ser-Pro (IYSP) with molecular weights of 411.35, 404.41, 452.45, 535.60, 665.69, 397.48, 521.61, 477.55, 753.91, and 478.53 Da, respectively. Among them, the IC50 values of ICY, LSFR, and IYSP on ACE were 0.48, 0.59, and 0.76 mg/mL, respectively. The significant ACEi activity of ICY, LSFR, and IYSP with affinities of −7.0, −8.5, and −8.3 kcal/mol mainly attributed to effectively combining with the ACEi active sites through hydrogen bonding, electrostatic force, and hydrophobic interaction. Moreover, ICY, LSFR, and IYSP could positively influence the production of nitric oxide (NO) and endothelin-1 (ET-1) secretion in human umbilical vein endothelial cells (HUVECs) and weaken the adverse impact of norepinephrine (NE) on the production of NO and ET-1. In addition, ICY, LSFR, and IYSP could provide significant protection to HUVECs against H2O2 damage by increasing antioxidase levels to decrease the contents of reactive oxide species and malondialdehyde. Therefore, the ACEi peptides of ICY, LSFR, and IYSP are beneficial functional molecules for healthy foods against hypertension and cardiovascular diseases.

Formation and inhibition mechanism of novel angiotensin I converting enzyme inhibitory peptides from Chouguiyu

Angiotensin I converting enzyme (ACE) inhibitory peptides from fermented foods exhibit great potential to alleviate hypertension. In this study, the peptide extract from Chouguiyu exhibited a good inhibition effect on ACE, and the inhibition rate was significantly enhanced after fermentation for 8 days. The ACE inhibitory peptides were further identified, followed by their inhibition and formation mechanisms using microbiome technology and molecular docking. A total of 356 ACE inhibitory peptides were predicted using in silico, and most ACE inhibitory peptides increased after fermentation. These peptides could be hydrolyzed from 94 kinds of precursor proteins, mainly including muscle-type creatine kinase, nebulin, and troponin I. P1 (VEIINARA), P2 (FAVMVKG), P4 (EITWSDDKK), P7 (DFDDIQK), P8 (IGDDPKF), P9 (INDDPKIL), and P10 (GVDNPGHPFI) were selected as the core ACE inhibitory peptides according to their abundance and docking energy. The salt bridge and conventional hydrogen bond connecting unsaturated oxygen atoms in the peptides contributed most to the ACE inhibition. The cleavage proteases from the microbial genera in Chouguiyu for preparing these 7 core ACE inhibitory peptides were further analyzed by hydrolysis prediction and Pearson's correlation. The correlation network showed that P7, P8, and P9 were mainly produced by the proteases from LAB including Lactococcus, Enterococcus, Vagococcus, Peptostreptococcus, and Streptococcus, while P1, P2, P4, and P10 were mainly Produced by Aeromonas, Bacillus, Escherichia, and Psychrobacter. This study is helpful in isolating the proteases and microbial strains to directionally produce the responding ACE inhibitory peptides.

Effects of Inherent Lactic Acid Bacteria on Inhibition of Angiotensin I-Converting Enzyme and Antioxidant Activities in Dry-Cured Meat Products.

The aim of this study was to investigate the inherent bacteria that contribute to expressing the angiotensin I-converting enzyme (ACE) inhibitory activity and the antioxidant activity of dry-cured meat products without a bacterial starter. Among the ten dry-cured meat product samples, Coppa and Milano salami exhibited high ACE inhibitory activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability, and oxygen radical absorbance capacity (ORAC). No consistent trend was observed in the pH values or the total peptide and imidazole dipeptide concentration of the products that exhibited high ACE inhibitory and antioxidant activities in the tested samples. To investigate the bacteria contributing to the ACE inhibitory and antioxidant activities of the product, 16S rRNA sequencing analysis, isolation, and identification of bacteria were performed using not only Coppa and Milano salami but also the Jamon Serrano and Parma prosciutto products that had low functional activities. Results suggest the Lactobacillales order, particularly the species Latilactobacillus sakei and Pediococcus pentosaceus, were the main inherent bacteria in Coppa and Milano salami, respectively, compared with the Jamon Serrano and Parma prosciutto products. Therefore, the inherent lactic acid bacteria in dry-cured meat products without bacterial starter is important for ACE inhibitory and antioxidant activities of the products.

Functional properties and consumer acceptance of whey‐oat beverages under different ultrasonication times and inulin concentration

This study aimed to determine the influence of ultrasound (3 vs. 15 min) and inulin (1% vs. 2% w/v) on the functional and sensory properties of whey-oat beverages. The treatments were identified as I1-U3, I2-U3, I1-U15, and I2-U15 referring to the inulin (I) percentage (1 or 2%) and the ultrasound (U) time (min), respectively. The acidity of whey and oat solutions decreased (p < .05) during storage while the antioxidant activity increased without effect on angiotensin-converting enzyme inhibitory (ACEI) activity. The whey-oat beverage with the highest antioxidant and ACEI activities was I1-U15. The beverages with inulin and ultrasound application were more accepted than the control beverage. Overall, the greatest outcomes were observed in the beverage containing 1% w/v inulin and 15 min ultrasound. Whey, oat, and inulin are potential ingredients that confer functional properties for the formulation of value-added beverages. Ultrasound technology can be a strategy to improve the functional properties of whey-oat beverages. Practical applications Ultrasound is a green, versatile, and non-thermal technology with an ample scope for development and commercialization in beverage processing industries. The use of ultrasound and inulin addition yielded in a whey-oat beverage with enhanced bioactive properties on cold storage. The synergistic effect of a whey-oat matrix added with inulin and treated with ultrasound has clearly evidenced its benefits in the design of new functional whey-oat-based beverages to be marketed as healthy, fast, and convenient supplementary foods.

Structural basis for the inhibition of human angiotensin-1 converting enzyme by fosinoprilat.

Human angiotensin I-converting enzyme (ACE) has two isoforms, somatic ACE (sACE) and testis ACE (tACE). The functions of sACE are widespread, with its involvement in blood pressure regulation most extensively studied. sACE is composed of an N-domain (nACE) and a C-domain (cACE), both catalytically active but have significant structural differences, resulting in different substrate specificities. Even though ACE inhibitors are used clinically, they need much improvement because of serious side effects seen in patients (~ 25-30%) with long-term treatment due to nonselective inhibition of nACE and cACE. Investigation into the distinguishing structural features of each domain is therefore of vital importance for the development of domain-specific inhibitors with minimal side effects. Here, we report kinetic data and high-resolution crystal structures of both nACE (1.75 Å) and cACE (1.85 Å) in complex with fosinoprilat, a clinically used inhibitor. These structures allowed detailed analysis of the molecular features conferring domain selectivity by fosinoprilat. Particularly, altered hydrophobic interactions were observed to be a contributing factor. These experimental data contribute to improved understanding of the structural features that dictate ACE inhibitor domain selectivity, allowing further progress towards designing novel 2nd-generation domain-specific potent ACE inhibitors suitable for clinical administration, with a variety of potential future therapeutic benefits. DATABASE: The atomic coordinates and structure factors for nACE-fosinoprilat and cACE-fosinoprilat structures have been deposited with codes 7Z6Z and 7Z70, respectively, in the RCSB Protein Data Bank, www.pdb.org.

QGC606: A Best-in-Class Orally Active Centrally Acting Aminopeptidase A Inhibitor Prodrug for Treating Heart Failure Following Myocardial Infarction.

Blockade of brain renin-angiotensin system (RAS) overactivity by firibastat, the first centrally acting aminopeptidase A (APA) inhibitor prodrug, has already demonstrated its effectiveness in improving cardiac function after myocardial infarction (MI). We developed QGC606, a more potent and more selective APA inhibitor prodrug and studied its effects after long-term oral administration in mice post-MI. Two days after MI induced by the left anterior descending artery ligation, adult male mice were randomized into 4 groups to receive oral treatment during 4 weeks with vehicle; QGC606; firibastat; or the angiotensin-I converting enzyme inhibitor ramipril, used as positive control. Four weeks post-MI, brain APA was overactivated in vehicle-treated MI mice. QGC606 treatment normalized brain APA hyperactivity to control values measured in sham-operated mice. Four weeks post-MI, QGC606-treated mice had higher left ventricular (LV) ejection fractions, significantly smaller LV end-systolic diameter and volume, significantly lower HF biomarkers mRNA expression (Myh7 and Anf) and plasma N-terminal pro B-type natriuretic peptide (NT-pro-BNP) and noradrenaline levels than saline-treated mice. QGC606 treatment significantly improved the dP/dt max and min, LV end-diastolic pressure without affecting blood pressure (BP), whereas we observed a decrease in BP in ramipril-treated mice. We observed also a reduction of cardiac fibrosis, highlighted by lower connective tissue growth factor mRNA levels and a reduction of both the fibrotic area and MI size in QGC606-treated mice. Chronic oral QGC606 administration in post-MI mice showed beneficial effects in improving cardiac function and reducing cardiac remodeling and fibrosis but, unlike ramipril, without lowering BP.