RESUMO
The stability of bioactive peptides under various food processing conditions is the basis for their use in industrial manufacturing. This study aimed to identify natural ACE inhibitors with excellent stability and investigate their physicochemical properties and putative molecular mechanisms. Five novel ACE inhibitory peptides (QDPLFPL, FPGVSPF, SPAQLLPF, LVPYRP, and WYWPQ) were isolated and identified using RP-HPLC and Nano LC-MS/MS with foxtail millet protein hydrolysates as the raw material. These peptides are non-toxic and exhibit strong ACE inhibitory activity in vitro (IC50 values between 0.13 mg mL-1 and 0.56 mg mL-1). In addition to QDPLFPL, FPGVSPF, SPAQLLPF, LVPYRP, and WYWPQ have excellent human intestinal absorption. Compared to FPGVSPF and SPAQLLPF, the stable helical structure of LVPYRP and WYWPQ allows them to maintain high stability under conditions that mimic gastrointestinal digestion and various food processing (temperatures, pH, sucrose, NaCl, citric acid, sodium benzoate, Cu2+, Zn2+, K+, Mg2+, Ca2+). The results of molecular docking and molecular dynamics simulation suggest that LVPYRP has greater stability and binding capacity to ACE than WYWPQ. LVPYRP might attach to the active pockets (S1, S2, and S1') of ACE via hydrogen bonds and hydrophobic interactions, then compete with Zn2+ in ACE to demonstrate its ACE inhibitory activity. The binding of LVPYRP to ACE enhances the rearrangement of ACE's active structural domains, with electrostatic and polar solvation energy contributing the most energy to the binding. Our findings suggested that LVPYRP derived from foxtail millet protein hydrolysates has the potential to be incorporated into functional foods to provide antihypertensive benefits.
Assuntos
Inibidores da Enzima Conversora de Angiotensina , Simulação de Acoplamento Molecular , Peptídeos , Proteínas de Plantas , Hidrolisados de Proteína , Setaria (Planta) , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Setaria (Planta)/química , Hidrolisados de Proteína/química , Hidrolisados de Proteína/farmacologia , Humanos , Peptídeos/química , Peptídeos/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Espectrometria de Massas em Tandem , Simulação por ComputadorRESUMO
Hypertension is a major controllable risk factor associated with cardiovascular disease (CVD) and overall mortality worldwide. Most people with hypertension must take medications that are effective in blood pressure management but cause many side effects. Thus, it is important to explore safer antihypertensive alternatives to regulate blood pressure. In this study, peanut protein concentrate (PPC) was hydrolyzed with 3-5% Alcalase for 3-10 h. The in vitro angiotensin-converting enzyme (ACE) and renin-inhibitory activities of the resulting peanut protein hydrolysate (PPH) samples and their fractions of different molecular weight ranges were determined as two measures of their antihypertensive potentials. The results show that the crude PPH produced at 4% Alcalase for 6 h of hydrolysis had the highest ACE-inhibitory activity with IC50 being 5.45 mg/mL. The PPH samples produced with 3-5% Alcalase hydrolysis for 6-8 h also displayed substantial renin-inhibitory activities, which is a great advantage over the animal protein-derived bioactive peptides or hydrolysate. Remarkably higher ACE- and renin-inhibitory activities were observed in fractions smaller than 5 kDa with IC50 being 0.85 and 1.78 mg/mL. Hence, the PPH and its small molecular fraction produced under proper Alcalase hydrolysis conditions have great potential to serve as a cost-effective anti-hypertensive ingredient for blood pressure management.
Assuntos
Inibidores da Enzima Conversora de Angiotensina , Arachis , Peptidil Dipeptidase A , Proteínas de Plantas , Hidrolisados de Proteína , Renina , Subtilisinas , Subtilisinas/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Hidrolisados de Proteína/farmacologia , Hidrolisados de Proteína/química , Hidrolisados de Proteína/metabolismo , Arachis/química , Renina/metabolismo , Renina/antagonistas & inibidores , Hidrólise , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Proteínas de Plantas/química , Peptidil Dipeptidase A/metabolismo , Peptidil Dipeptidase A/química , Anti-Hipertensivos/farmacologia , Anti-Hipertensivos/química , HumanosRESUMO
Marine bacterial proteases have rarely been used to produce bioactive peptides, although many have been reported. This study aims to evaluate the potential of the marine bacterial metalloprotease A69 from recombinant Bacillus subtilis in the preparation of peanut peptides (PPs) with antioxidant activity and angiotensin-converting enzyme (ACE)-inhibitory activity. Based on the optimization of the hydrolysis parameters of protease A69, a process for PPs preparation was set up in which the peanut protein was hydrolyzed by A69 at 3000 U g-1 and 60 °C, pH 7.0 for 4 h. The prepared PPs exhibited a high content of peptides with molecular weights lower than 1000 Da (>80%) and 3000 Da (>95%) and contained 17 kinds of amino acids. Moreover, the PPs displayed elevated scavenging of hydroxyl radical and 1,1-diphenyl-2-picryl-hydrazyl radical, with IC50 values of 1.50 mg mL-1 and 1.66 mg mL-1, respectively, indicating the good antioxidant activity of the PPs. The PPs also showed remarkable ACE-inhibitory activity, with an IC50 value of 0.71 mg mL-1. By liquid chromatography mass spectrometry analysis, the sequences of 19 ACE inhibitory peptides and 15 antioxidant peptides were identified from the PPs. These results indicate that the prepared PPs have a good nutritional value, as well as good antioxidant and antihypertensive effects, and that the marine bacterial metalloprotease A69 has promising potential in relation to the preparation of bioactive peptides from peanut protein.
Assuntos
Inibidores da Enzima Conversora de Angiotensina , Antioxidantes , Arachis , Bacillus subtilis , Metaloproteases , Peptídeos , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/química , Antioxidantes/farmacologia , Antioxidantes/química , Metaloproteases/química , Metaloproteases/farmacologia , Arachis/química , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Peptídeos/farmacologia , Peptídeos/química , Hidrólise , Peptidil Dipeptidase A/metabolismo , Peptidil Dipeptidase A/químicaRESUMO
Human somatic angiotensin-converting enzyme (ACE) is a key zinc metallopeptidase that plays a pivotal role in the renin-angiotensin-aldosterone system (RAAS) by regulating blood pressure and electrolyte balance. Inhibition of ACE is a cornerstone in the management of hypertension, cardiovascular diseases, and renal disorders. Recent advances in structural biology techniques have provided invaluable insights into the molecular mechanisms underlying ACE inhibition, facilitating the design and development of more effective therapeutic agents. This review focuses on the latest advancements in elucidating the structural basis for ACE inhibition. High-resolution crystallographic studies of minimally glycosylated individual domains of ACE have revealed intricate molecular details of the ACE catalytic N- and C-domains, and their detailed interactions with clinically relevant and newly designed domain-specific inhibitors. In addition, the recently elucidated structure of the glycosylated form of full-length ACE by cryo-electron microscopy (cryo-EM) has shed light on the mechanism of ACE dimerization and revealed continuous conformational changes which occur prior to ligand binding. In addition to these experimental techniques, computational approaches have also played a pivotal role in elucidating the structural basis for ACE inhibition. Molecular dynamics simulations and computational docking studies have provided atomic details of inhibitor binding kinetics and energetics, facilitating the rational design of novel ACE inhibitors with improved potency and selectivity. Furthermore, computational analysis of the motions observed by cryo-EM allowed the identification of allosteric binding sites on ACE. This affords new opportunities for the development of next-generation allosteric inhibitors with enhanced pharmacological properties. Overall, the insights highlighted in this review could enable the rational design of novel ACE inhibitors with improved efficacy and safety profiles, ultimately leading to better therapeutic outcomes for patients with hypertension and cardiovascular diseases.
Assuntos
Inibidores da Enzima Conversora de Angiotensina , Peptidil Dipeptidase A , Humanos , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Simulação de Dinâmica Molecular , Relação Estrutura-Atividade , Simulação de Acoplamento Molecular , Sistema Renina-Angiotensina/efeitos dos fármacos , Microscopia Crioeletrônica , Ligação Proteica , AnimaisRESUMO
Cardiovascular diseases, including heart failure, stroke, and hypertension, affect 608 million people worldwide and cause 32% of deaths. Combination therapy is required in 60% of patients, involving concurrent Renin-Angiotensin-Aldosterone-System (RAAS) and Neprilysin inhibition. This study introduces a novel multi-target in-silico modeling technique (mt-QSAR) to evaluate the inhibitory potential against Neprilysin and Angiotensin-converting enzymes. Using both linear (GA-LDA) and non-linear (RF) algorithms, mt-QSAR classification models were developed using 983 chemicals to predict inhibitory effects on Neprilysin and Angiotensin-converting enzymes. The Box-Jenkins method, feature selection method, and machine learning algorithms were employed to obtain the most predictive model with ~ 90% overall accuracy. Additionally, the study employed virtual screening of designed scaffolds (Chalcone and its analogues, 1,3-Thiazole, 1,3,4-Thiadiazole) applying developed mt-QSAR models and molecular docking. The identified virtual hits underwent successive filtration steps, incorporating assessments of drug-likeness, ADMET profiles, and synthetic accessibility tools. Finally, Molecular dynamic simulations were then used to identify and rank the most favourable compounds. The data acquired from this study may provide crucial direction for the identification of new multi-targeted cardiovascular inhibitors.
Assuntos
Inibidores da Enzima Conversora de Angiotensina , Simulação por Computador , Simulação de Acoplamento Molecular , Neprilisina , Relação Quantitativa Estrutura-Atividade , Neprilisina/antagonistas & inibidores , Neprilisina/química , Neprilisina/metabolismo , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Humanos , Peptidil Dipeptidase A/metabolismo , Peptidil Dipeptidase A/química , Algoritmos , Simulação de Dinâmica MolecularRESUMO
In this study, we combined AlphaFold-based approaches for atomistic modeling of multiple protein states and microsecond molecular simulations to accurately characterize conformational ensembles evolution and binding mechanisms of convergent evolution for the SARS-CoV-2 spike Omicron variants BA.1, BA.2, BA.2.75, BA.3, BA.4/BA.5 and BQ.1.1. We employed and validated several different adaptations of the AlphaFold methodology for modeling of conformational ensembles including the introduced randomized full sequence scanning for manipulation of sequence variations to systematically explore conformational dynamics of Omicron spike protein complexes with the ACE2 receptor. Microsecond atomistic molecular dynamics (MD) simulations provide a detailed characterization of the conformational landscapes and thermodynamic stability of the Omicron variant complexes. By integrating the predictions of conformational ensembles from different AlphaFold adaptations and applying statistical confidence metrics we can expand characterization of the conformational ensembles and identify functional protein conformations that determine the equilibrium dynamics for the Omicron spike complexes with the ACE2. Conformational ensembles of the Omicron RBD-ACE2 complexes obtained using AlphaFold-based approaches for modeling protein states and MD simulations are employed for accurate comparative prediction of the binding energetics revealing an excellent agreement with the experimental data. In particular, the results demonstrated that AlphaFold-generated extended conformational ensembles can produce accurate binding energies for the Omicron RBD-ACE2 complexes. The results of this study suggested complementarities and potential synergies between AlphaFold predictions of protein conformational ensembles and MD simulations showing that integrating information from both methods can potentially yield a more adequate characterization of the conformational landscapes for the Omicron RBD-ACE2 complexes. This study provides insights in the interplay between conformational dynamics and binding, showing that evolution of Omicron variants through acquisition of convergent mutational sites may leverage conformational adaptability and dynamic couplings between key binding energy hotspots to optimize ACE2 binding affinity and enable immune evasion.
Assuntos
Enzima de Conversão de Angiotensina 2 , Simulação de Dinâmica Molecular , Ligação Proteica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/química , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Humanos , Termodinâmica , Conformação Proteica , Sítios de Ligação , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , COVID-19/virologiaRESUMO
Pelodiscus sinensis meat is a nutritional food and tonic with angiotensin-converting enzyme (ACE) inhibitory activities. To identify the bioactive substances responsible, several bioinformatics methods were integrated to enable a virtual screening for bioactive peptides in proteins identified within a water-soluble protein fraction of Pelodiscus sinensis meat by Shotgun proteomics. The peptides were generated from the identified proteins by in silico proteolysis using six proteases. A comparison of the numbers of proteins suitable for digestion with each enzyme and the iBAQ (intensity-based absolute quantification) values for these proteins revealed that bromelain and papain were the most suitable proteases for this sample. Next, the water solubility, toxicity, and ADMET (absorption/distribution/metabolism/excretion/toxicity) properties of these peptides were evaluated in silico. Finally, a novel ACE inhibitory peptide IEWEF with an IC50 value of 41.33 µM was identified. The activity of the synthesized peptide was verified in vitro, and it was shown to be a non-competitive ACE inhibitor. Molecular docking revealed that IEWEF could tightly bind to C-ACE, and N-ACE with energies less than 0 kJ mol-1, and the peptide IEWEF can form hydrogen bonds with C-ACE and N-ACE respectively. These results provide evidence that bioactive peptides in the water-soluble protein fraction account for (at least) some of the ACE inhibitory activities observed in Pelodiscus sinensis meat. Furthermore, our research provides a workflow for the efficient identification of novel ACE inhibitory peptides from complex protein mixtures.
Assuntos
Inibidores da Enzima Conversora de Angiotensina , Simulação de Acoplamento Molecular , Peptídeos , Hidrolisados de Proteína , Solubilidade , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Hidrolisados de Proteína/química , Hidrolisados de Proteína/metabolismo , Animais , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/metabolismo , Água/química , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Papaína/metabolismo , Papaína/antagonistas & inibidores , Papaína/química , Proteínas de Peixes/química , Proteínas de Peixes/metabolismoRESUMO
The salty oligopeptides from Stropharia rugosoannulata have been proven to be potential ACE inhibitors. To investigate the ACE receptor binding properties and interaction mechanisms of salty oligopeptides, the molecular interaction, dynamics simulation, and antihypertensive evaluation cross-validation strategy were employed to reveal the oligopeptides' binding reactions and modes with the ACE receptor. Single oligopeptide (ESPERPFL, KSWDDFFTR) had exothermic and specific binding reactions with the ACE receptor, driven by hydrogen bonds and van der Waals forces. The coexistence of the multiple oligopeptide molecules did not produce the apparent ACE receptor competition binding reactions. The molecular dynamics simulation verified that the two oligopeptides disturbed the ACE receptor's different residue regions. Both oligopeptides could form stable complexes with the ACE receptor. Based on the classification of 50 oligopeptides' binding modes, ESPERPFL and KSWDDFFTR belonged to different classes, and their receptor binding modes and sites complemented, resulting in a potential synergistic effect on ACE inhibition. The antihypertensive effect of KSWDDFFTR and its distribution in the body were evaluated using SHR rats orally and ICR mice by tail vein injection, and KSWDDFFTR had antihypertensive effects within 8 h. The study provides a theoretical basis for understanding salty oligopeptides' ACE receptor binding mechanism and their antihypertensive effects.
Assuntos
Anti-Hipertensivos , Simulação de Dinâmica Molecular , Oligopeptídeos , Animais , Oligopeptídeos/farmacologia , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Anti-Hipertensivos/farmacologia , Anti-Hipertensivos/química , Ratos , Masculino , Peptidil Dipeptidase A/metabolismo , Peptidil Dipeptidase A/química , Agaricales/química , Agaricales/metabolismo , Camundongos , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Ligação Proteica , Pressão Sanguínea/efeitos dos fármacos , Ratos Endogâmicos SHRRESUMO
Peptides possessing antihypertensive attributes via inhibiting the angiotensin-converting enzyme (ACE) were derived through the enzymatic degradation of Trichiurus lepturus (ribbonfish) using alkaline protease. The resulting mixture underwent filtration using centrifugation, ultrafiltration tubes, and Sephadex G-25 gels. Peptides exhibiting ACE-inhibitory properties and DPPH free-radical-scavenging abilities were isolated and subsequently purified via LC/MS-MS, leading to the identification of over 100 peptide components. In silico screening yielded five ACE inhibitory peptides: FAGDDAPR, QGPIGPR, IFPRNPP, AGFAGDDAPR, and GPTGPAGPR. Among these, IFPRNPP and AGFAGDDAPR were found to be allergenic, while FAGDDAPRR, QGPIGPR, and GPTGPAGP showed good ACE-inhibitory effects. IC50 values for the latter peptides were obtained from HUVEC cells: FAGDDAPRR (IC50 = 262.98 µM), QGPIGPR (IC50 = 81.09 µM), and GPTGPAGP (IC50 = 168.11 µM). Peptide constituents derived from ribbonfish proteins effectively modulated ACE activity, thus underscoring their therapeutic potential. Molecular docking and modeling corroborated these findings, emphasizing the utility of functional foods as a promising avenue for the treatment and prevention of hypertension, with potential ancillary health benefits and applications as substitutes for synthetic drugs.
Assuntos
Inibidores da Enzima Conversora de Angiotensina , Anti-Hipertensivos , Células Endoteliais da Veia Umbilical Humana , Peptídeos , Peptidil Dipeptidase A , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Anti-Hipertensivos/farmacologia , Anti-Hipertensivos/química , Anti-Hipertensivos/isolamento & purificação , Animais , Humanos , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/isolamento & purificação , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Peptidil Dipeptidase A/metabolismo , Peptidil Dipeptidase A/química , Simulação de Acoplamento Molecular , Perciformes/metabolismoRESUMO
Pumpkin (Cucurbita moschata) seed meal (PSM), the major byproduct of pumpkin seed oil industry, was used to prepare angiotensin-converting enzyme (ACE) inhibitory and angiotensin-converting enzyme 2 (ACE2) upregulating peptides. These peptides were isolated and purified from the PSM hydrolysate prepared using Neutrase 5.0 BG by ultrafiltration, Sephadex G-15 column chromatography, and reversed-phase high-performance liquid chromatography. Two peptides with significant ACE inhibition activity were identified as SNHANQLDFHP and PVQVLASAYR with IC50 values of 172.07 and 90.69 µM, respectively. The C-terminal tripeptides of the two peptides contained Pro, Phe, and Tyr, respectively, and PVQVLASAYR also had Val in its N-terminal tripeptide, which was a favorable structure for ACE inhibition. Molecular docking results declared that the two peptides could interact with ACE through hydrogen bonds and hydrophobic interactions. Furthermore, the two peptides performed protective function on EA.hy926 cells by decreasing the secretion of endothelin-1, increasing the release of nitric oxide, and regulating the ACE2 activity. In vitro simulated gastrointestinal digestion showed the two peptides exhibited good stability against gastrointestinal enzyme digestion. In conclusion, PSM is a promising material for preparing antihypertensive peptides.
Assuntos
Enzima de Conversão de Angiotensina 2 , Inibidores da Enzima Conversora de Angiotensina , Cucurbita , Simulação de Acoplamento Molecular , Peptídeos , Peptidil Dipeptidase A , Sementes , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Cucurbita/química , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Sementes/química , Humanos , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Hidrolisados de Proteína/química , Hidrolisados de Proteína/metabolismo , Regulação para Cima/efeitos dos fármacos , Linhagem Celular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismoRESUMO
Angiotensin-converting enzyme (ACE), consisting of N-domain and C-domain, is a key regulator of blood pressure. The use of cACE-specific inhibitors helps minimize side effects in clinical applications. Legumes are a good source of proteins containing ACE inhibitory peptides; however, no studies have reported the identification of cACE-specific inhibitory peptides from Fabaceae. In this study, thermal hydrolysates from seeds, sprouts, pods, seedlings, and flowers of legumes were analyzed. Flowers of legumes exhibited a C-domain-preference ACE inhibition and anti-hypertensive effect in rats. Screening the legume peptide library identified a novel cACE inhibitory peptide, SJ-1. This study reported the first identification of cACE inhibitory peptide from Fabaceae foods. SJ-1, identified from the legume flowers, interacted with active site residues of cACE, leading to the inhibition of ACE activity, downregulation of bradykinin levels, and reduction of blood pressure. These findings also suggested the potential of legume proteins as a source of cACE inhibitory peptides.
Assuntos
Inibidores da Enzima Conversora de Angiotensina , Fabaceae , Biblioteca de Peptídeos , Peptídeos , Peptidil Dipeptidase A , Proteínas de Plantas , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Fabaceae/química , Animais , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Ratos , Proteínas de Plantas/química , Masculino , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Humanos , Pressão Sanguínea/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Hipertensão/metabolismo , Ratos Sprague-DawleyRESUMO
Molecular imprinting techniques have attracted a lot of attention as a potential biomimetic technology, but there are still challenges in protein imprinting. Herein, multifunctional nanosized molecularly imprinted polymers (nanoMIPs) for human angiotensin-converting enzyme 2 (ACE2) were prepared by epitope imprinting of magnetic nanoparticles-anchored peptide (magNP-P) templates, which were further applied to construct a competitive displacement fluorescence assay toward ACE2. A cysteine-flanked dodecapeptide sequence was elaborately selected as an epitope for ACE2, which was immobilized onto the surface of magnetic nanoparticles and served as a magNP-P template for imprinting. During polymerization, fluorescent monomers were introduced to endow fluorescence responsiveness to the prepared self-signaling nanoMIPs. A competitive displacement fluorescence assay based on the nanoMIPs was established and operated in a washing-free manner, yielding a wide range for ACE2 (0.1-6.0 pg/mL) and a low detection limit (0.081 pg/mL). This approach offers a promising avenue in the preparation of nanoMIPs for macromolecule recognition and expands potential application of an MIP in the detection of proteins as well as peptides.
Assuntos
Enzima de Conversão de Angiotensina 2 , Humanos , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Peptidil Dipeptidase A/metabolismo , Peptidil Dipeptidase A/química , Impressão Molecular , Nanopartículas de Magnetita/química , Polímeros Molecularmente Impressos/química , Limite de Detecção , Peptídeos/química , Peptídeos/metabolismoRESUMO
Food-derived peptides with low molecular weight, high bioavailability, and good absorptivity have been exploited as angiotensin-converting enzyme (ACE) inhibitors. In the present study, in-vitro inhibition kinetics of peanut peptides, in silico screening, validation of ACE inhibitory activity, molecular dynamics (MD) simulations, and HUVEC cells were performed to systematically identify the inhibitory mechanism of ACE interacting with peanut peptides. The results indicate that FPHPP, FPHY, and FPHFD peptides have good thermal, pH, and digestive stability. MD trajectories elucidate the dynamic correlation between peptides and ACE and verify the specific binding interaction. Noteworthily, FPHPP is the best inhibitor with a strongest binding affinity and significantly increases NO, SOD production, and AT2R expression, and decreases ROS, MDA, ET-1 levels, ACE, and AT1R accumulation in Ang II-injury HUVEC cells.
Assuntos
Inibidores da Enzima Conversora de Angiotensina , Arachis , Células Endoteliais da Veia Umbilical Humana , Peptídeos , Peptidil Dipeptidase A , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Humanos , Peptidil Dipeptidase A/metabolismo , Peptidil Dipeptidase A/química , Arachis/química , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Simulação de Dinâmica Molecular , Simulação por Computador , Cinética , Ligação ProteicaRESUMO
In the study, papain was used to hydrolyze tilapia (Oreochromis mossambicus) skin to obtain a tilapia skin hydrolysate (TSH) with dual angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities. The resulting TSH was sequentially fractionated by ultrafiltration, size exclusion separation chromatography, and reverse-phase high-performance liquid chromatography. Its inhibitory effects on ACE and DPP-IV were determined by commercial reagent kits. Two peptides purified from TSH were identified as Gly-Pro-Leu-Gly-Ala-Leu (GPLGAL) and Lys-Pro-Ala-Gly-Asn (KPAGN) by the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Inhibitory concentration (IC50) of GPLGAL on ACE and DPP-IV were 117.20 ± 1.69 and 187.10 ± 2.75 µM, respectively. IC50 of KPAGN on ACE and DPP-IV were 137.40 ± 2.33 and 259.20 ± 2.85 µM, respectively. The molecular simulation demonstrated that the binding affinities of GPLGAL to ACE and DPP-IV proteins were -8.5 and -7.4 kcal/mol, respectively, whereas those of KPAGN to ACE and DPP-IV proteins were -7.9 and -6.7 kcal/mol, respectively. GPLGAL interacted with 21 amino acid residues of the ACE active site, whereas KPAGN engaged with 19 amino acid residues. Additionally, GPLGAL interacted with 10 amino acid residues of the DPP-IV active site, whereas KPAGN engaged with 13 amino acid residues. The two peptides predominantly occupied the active sites of ACE (His513, Tyr523, and Ala354) and DPP-IV (Tyr662 and Arg125) through hydrogen bonding. This leads to the deactivation of ACE and DPP-IV. PRACTICAL APPLICATION: Accelerate tilapia skin development and high-value utilization; provide foundation for preparing the peptides with dual ACE and DPP-IV inhibiting activity.
Assuntos
Inibidores da Enzima Conversora de Angiotensina , Dipeptidil Peptidase 4 , Inibidores da Dipeptidil Peptidase IV , Simulação de Acoplamento Molecular , Peptídeos , Peptidil Dipeptidase A , Pele , Tilápia , Animais , Inibidores da Dipeptidil Peptidase IV/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/química , Dipeptidil Peptidase 4/metabolismo , Peptidil Dipeptidase A/metabolismo , Peptidil Dipeptidase A/química , Pele/química , Peptídeos/farmacologia , Peptídeos/química , Proteínas de Peixes/química , Proteínas de Peixes/farmacologia , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Hidrólise , Cromatografia Líquida de Alta Pressão/métodosRESUMO
BACKGROUND: Rice-based distillers' spent cake (RDSC), a by-product of the Chinese liquor (Baijiu) industry, is a potential source of angiotensin-converting enzyme (ACE) inhibitory peptide. Since ACE plays a crucial role in controlling hypertension, inhibition of ACE has been widely emphasized. The ACE inhibitory active peptide derived from by-products of food has been recognized as a safer and cheaper inhibitor. RESULTS: Aimed to discover ACE-inhibiting active peptides in RDSC. Hydrolysis of RDSC by alcalase for 4 h followed by ultrafiltration yielded low-molecular-weight (< 3 kDa) fractions. Subsequently, a comprehensive method using a combination of liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) and LC-Q-Exactive-MS to identify the novel short peptides (3-5 amino acids residues; n = 7) and medium-sized peptides (more than 6 amino acids residues; n = 6). In vitro activity assay showed that the peptides KPFFPGL, GFPRPLL, GPPGVF, and VGK exhibited the highest activity with inhibitory concentration of 50% (IC50) of 11.63, 12.34, 19.55, and 33.54 µmol L-1. Molecular docking reveal that the active and inactive sites (Glu123, Asp121, Arg522, and Lys118) play important roles in enhancing the ACE inhibitory activity of peptides. CONCLUSION: Here we report a comprehensive method that effectively extracted and identified the bioactive peptides from RDSC. Four highly active novel peptides may be the most promising candidates for functional foods against hypertension, provide significant information for enhancing value of rice-based distilled by-products. © 2024 Society of Chemical Industry.
Assuntos
Inibidores da Enzima Conversora de Angiotensina , Simulação de Acoplamento Molecular , Oryza , Peptídeos , Peptidil Dipeptidase A , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Resíduos Industriais/análise , Oryza/química , Peptídeos/química , Peptídeos/farmacologia , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Resíduos/análiseRESUMO
In this study, the effects of Lactiplantibacillus plantarum M11 (Lb. plantarum M11) in conjunction with sodium caseinate on the characteristics and angiotensin converting enzyme (ACE) inhibitory activity of yogurt were investigated. ACE inhibitory peptides (ACEIPs) in yogurt were identified by nano-LC-MS/MS and potential ACEIPs were predicted by in silico and molecular docking methods. The results showed that the ACE-inhibitory activity of yogurt was significantly enhanced (p < 0.05), while maintaining the quality characteristics of the yogurt. Thirteen ACEIPs in the improved yogurt (883 + M11-CS group) were identified, which were more abundant than the other yogurt groups (control 883 group, 883 + M11 group and 883-CS group). Two novel peptides with potential ACE inhibitory activity, YPFPGPIH and NILRFF, were screened. The two peptides showed PeptideRanker scores above 0.8, small molecular weight and strong hydrophobicity, and were non-toxic after prediction. Molecular docking results showed that binding energies with ACE were -9.4 kcal mol-1 and -10.7 kcal mol-1, respectively, and could bind to the active site of ACE. These results indicated that yogurt with Lb. plantarum M11 and sodium caseinate has the potential to be utilized as a functional food with antihypertensive properties. The combination of ACEIP-producing strains and casein fortification could be an effective method to promote the release of ACEIPs from yogurt.
Assuntos
Inibidores da Enzima Conversora de Angiotensina , Lactobacillus plantarum , Inibidores da Enzima Conversora de Angiotensina/química , Caseínas/química , Simulação de Acoplamento Molecular , Espectrometria de Massas em Tandem , Peptidil Dipeptidase A/química , Iogurte , Peptídeos/químicaRESUMO
Faba bean flour, after in vitro gastrointestinal digestion, showed important antioxidant and angiotensin-converting enzyme (ACE) inhibitory activities. In the present study, 11 faba bean- derived peptides were synthesized to confirm their bioactivities and provide a deeper understanding of their mechanisms of action. The results revealed that 7 peptides were potent antioxidants, namely, NYDEGSEPR, TETWNPNHPEL, TETWNPNHPE, VIPTEPPH, VIPTEPPHA, VVIPTEPPHA, and VVIPTEPPH. Among them, TETWNPNHPEL had the highest activity in the ABTS (EC50 = 0.5 ± 0.2 mM) and DPPH (EC50 = 2.1 ± 0.1 mM) assays (p < 0.05), whereas TETWNPNHPE had the highest activity (p < 0.05) in the ORAC assay (2.84 ± 0.08 mM Trolox equivalent/mM). Synergistic and/or additive effects were found when selected peptides (TETWNPNHPEL, NYDEGSEPR, and VVIPTEPPHA) were combined. Four peptides were potent ACE inhibitors, where VVIPTEPPH (IC50 = 43 ± 1 µM) and VVIPTEPPHA (IC50 = 50 ± 5 µM) had the highest activity (p < 0.05), followed by VIPTEPPH (IC50 = 90 ± 10 µM) and then VIPTEPPHA (IC50 = 123 ± 5 µM) (p < 0.05). These peptides were noncompetitive inhibitors, as supported by kinetic studies and a molecular docking investigation. This study demonstrated that peptides derived from faba beans have multifunctional bioactivities, making them a promising food-functional and nutraceutical ingredient.
Assuntos
Antioxidantes , Vicia faba , Antioxidantes/química , Vicia faba/metabolismo , Simulação de Acoplamento Molecular , Cinética , Peptídeos/química , Digestão , Angiotensinas , Peptidil Dipeptidase A/químicaRESUMO
In this study, we used traditional laboratory methods, bioinformatics, and cellular models to screen novel ACE inhibitory (ACEI) peptides with strong ACEI activity, moderate absorption rates, and multiple targets from bovine colostrum immunoglobulin G (IgG). The purified fraction of the compound proteinase hydrolysate of IgG showed good ACEI activity. After nano-UPLC-MS/MS identification and in silico analysis, eight peptides were synthesized and verified. Among them, SFYPDY, TSFYPDY, FSWF, WYQQVPGSGL, and GVHTFP were identified as ACEI peptides, as they exhibited strong ACEI activity (with IC50 values of 104.7, 80.0, 121.2, 39.8, and 86.3 µM, respectively). They displayed good stability in an in vitro simulated gastrointestinal digestion assay. In a Caco-2 monolayer model, SFYPDY, FSWF, and WYQQVPGSGL exhibited better absorption rates and lower IC50 values than the other peptides and were thereby identified as novel ACEI peptides. Subsequently, in a H2O2-induced endothelial dysfunction (ED) model based on HUVECs, SFYPDY, FSWF, and WYQQVPGSGL regulated ED by reducing apoptosis and ROS accumulation while upregulating NOS3 mRNA expression. Network pharmacology analysis and RT-qPCR confirmed that they regulated multiple targets. Overall, our results suggest that SFYPDY, FSWF, and WYQQVPGSGL can serve as novel multitarget ACEI peptides.
Assuntos
Imunoglobulina G , Doenças Vasculares , Humanos , Feminino , Gravidez , Animais , Bovinos , Farmacologia em Rede , Espectrometria de Massas em Tandem , Células CACO-2 , Colostro/metabolismo , Peróxido de Hidrogênio , Peptídeos/química , Peptidil Dipeptidase A/química , Hidrolisados de Proteína/química , Simulação de Acoplamento MolecularRESUMO
Protein hydrolysates from sea cucumber (Apostichopus japonicus) gonads are rich in active materials with remarkable angiotensin-converting enzyme (ACE) inhibitory activity. Alcalase was used to hydrolyze sea cucumber gonads, and the hydrolysate was separated by the ultrafiltration membrane to produce a low-molecular-weight peptide component (less than 3 kDa) with good ACE inhibitory activity. The peptide component (less than 3 kDa) was isolated and purified using a combination method of ACE gel affinity chromatography and reverse high-performance liquid chromatography. The purified fractions were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the resulting products were filtered using structure-based virtual screening (SBVS) to obtain 20 peptides. Of those, three noncompetitive inhibitory peptides (DDQIHIF with an IC50 value of 333.5 µmol·L-1, HDWWKER with an IC50 value of 583.6 µmol·L-1, and THDWWKER with an IC50 value of 1291.8 µmol·L-1) were further investigated based on their favorable pharmacochemical properties and ACE inhibitory activity. Molecular docking studies indicated that the three peptides were entirely enclosed within the ACE protein cavity, improving the overall stability of the complex through interaction forces with the ACE active site. The total free binding energies (ΔGtotal) for DDQIHIF, HDWWKER, and THDWWKER were -21.9 Kcal·mol-1, -71.6 Kcal·mol-1, and -69.1 Kcal·mol-1, respectively. Furthermore, a short-term assay of antihypertensive activity in spontaneously hypertensive rats (SHRs) revealed that HDWWKER could significantly decrease the systolic blood pressure (SBP) of SHRs after intravenous administration. The results showed that based on the better antihypertensive activity of the peptide in SHRs, the feasibility of targeted affinity purification and computer-aided drug discovery (CADD) for the efficient screening and preparation of ACE inhibitory peptide was verified, which provided a new idea of modern drug development method for clinical use.
Assuntos
Anti-Hipertensivos , Pepinos-do-Mar , Ratos , Animais , Anti-Hipertensivos/farmacologia , Inibidores da Enzima Conversora de Angiotensina/química , Cromatografia Líquida , Simulação de Acoplamento Molecular , Pepinos-do-Mar/metabolismo , Espectrometria de Massas em Tandem , Peptídeos/química , Ratos Endogâmicos SHR , Cromatografia de Afinidade , Peptidil Dipeptidase A/química , Hidrolisados de Proteína/química , Gônadas/metabolismo , AngiotensinasRESUMO
Three kinds of coronaviruses are highly pathogenic to humans, and two of them mainly infect humans through Angiotensin-converting enzyme 2 ï¼ACE2ï¼receptors. Therefore, specifically blocking ACE2 binding at the interface with the receptor-binding domain is promising to achieve both preventive and therapeutic effects of coronaviruses. Alternatively, drug-targeted delivery based on ACE2 receptors can further improve the efficacy and safety of inhalation drugs. Here, these two approaches are innovatively combined by designing a nanoemulsion (NE) drug delivery system (termed NE-AYQ) for inhalation that targets binding to ACE2 receptors. This inhalation-delivered remdesivir nanoemulsion (termed RDSV-NE-AYQ) effectively inhibits the infection of target cells by both wild-type and mutant viruses. The RDSV-NE-AYQ strongly inhibits Severe acute respiratory syndrome coronavirus 2 at two dimensions: they not only block the binding of the virus to host cells at the cell surface but also restrict virus replication intracellularly. Furthermore, in the mouse model of acute lung injury, the inhaled drug delivery system loaded with anti-inflammatory drugs (TPCA-1-NE-AYQ) can significantly alleviate the lung tissue injury of mice. This smart combination provides a new choice for dealing with possible emergencies in the future and for the rapid development of inhaled drugs for the treatment of respiratory diseases.