Preparation And Structure-activity Relationship Of ACE Inhibitory Peptide From Takifugu Flavidus

Angiotensin-I-converting enzyme inhibitors（ACEI）have been extensively utilized in clinics as efficacious antihypertensive agents.However,the majority of ACE inhibitors are chemically synthesized and often cause significant adverse effects.Consequently,there has been a growing interest in exploring ACE-inhibitory peptides as a more economically viable and less side-effect prone alternative.Nevertheless,only a limited number of these peptides have been successfully developed into clinically effective drugs.Furthermore,the available mechanistic studies on ACE-inhibitory peptides are insufficient.The proteins of Takifugu flavidus are rich in hydrophobic and aromatic amino acids,make them a viable candidate for the production of ACE-inhibitory peptides.In this study,T.flavidus peptides（TFP）were prepared from the skins via alcalase hydrolysis and ultrafiltration.Additionally,the antihypertensive mechanism and molecular simulation of the novel identified ACE-inhibitory peptides were investigated.Moreover,structural modifications were executed to enhance the ACE-inhibitory activity of peptides.The main findings are as follows:（1）Preparation and antihypertensive activity of TFPBased on ACE inhibition rate and the degree of hydrolysis,the enzymatic hydrolysis effects of different proteases on the skins of T.flavidus were analyzed.T.flavidus hydrolysates（TFH）with various molecular weights（<1、1～3、3～10、10～50 and>50 k Da）were seperated using ultrafiltration.T.flavidus peptides（TFP）with MWs<1 k Da showed the strongest ACE-inhibitory activity with a half-maximal inhibitory concentration(IC₅₀)of 0.58 mg/m L.TFP was orally administered to spontaneously hypertensive rats（SHR）and exerted significant blood pressure-lowering effects,reducing systolic blood pressure to 168 mm Hg within 6 hours.Meanwhile,the levels of renin（REN）was decreased in the kidneys of SHR.The findings of the gut flora analysis indicate that the oral administration of TFP resulted in alterations to the composition of the gut microbiome.Specifically,TFP was found to increase the relative abundance of Firmicutes,while decreasing the relative abundance of Bacteroidetes,Proteobacteria,and Verrucomicrobia at the phylum level.Additionally,TFP was observed to increase the relative abundance of Bacteroides and Blautia,while decreasing the relative abundance of Ruminococcus and Prevotella at the genus level.（2）Screening and mechanism-analysis of novel ACE-inhibitory peptides from TFPTFP was further purified using semi-preparative liquid chromatography,gel filtration chromatography and RP-HPLC.Among the fractions obtained,fraction A7-c-2 exhibited the most potent ACE-inhibitory activity(IC₅₀=0.24 mg/m L),and was subsequently subjected to LC-MS/MS analysis.A novel potential ACE-inhibitory peptide,PPLLFAAL(non-competitive suppression mode;IC₅₀of 28μmmol/L)was identified.Furthermore,TFP was subjected to purification through affinity adsorption using an ACE-sepharose 4B column,resulting in the identification of 24 peptides.Among these peptides,TLFGL(IC₅₀=57.5μmol/L),TLRFALHGME(IC₅₀=93.5μmol/L),YVLL(IC₅₀=153.8μmol/L)with the highest ACE-inhibitory activity were selected.The results of molecular docking analysis indicated that four peptides were capable of interacting with the active site of ACE via hydrogen bonds and hydrophobic interactions.Subsequent molecular dynamics simulations demonstrated that the total binding energy（ΔGbinding）of the four peptides was calculated as-111.2265,-82.7382,-66.8471 and-47.5478 k J/mol,suggesting a favorable binding affinity between the peptides and ACE.Furthermore,the binding affinity of the ACE-peptide complexes was confirmed by surface plasmon resonance（SPR）assay.（3）In vivo antihypertensive effects and mechanism analysis of ACE-inhibitory peptides in SHRThe in vivo antihypertensive efficacies of PPLLFAAL,TLRFALHGME,TLFGL,and YVLL were investigated in terms of changes in blood pressure after intravenous administration to SHR.The findings demonstrated that PPLLFAAL,TLRFALHGME,and TLFGL significantly decreased the systolic blood pressure（SBP）of SHR,with PPLLFAAL exhibiting the most pronounced antihypertensive effect.The peptides exhibited a predominant enrichment in the colon and were subsequently excreted through the feces.Notably,the Firmicutes to Bacteroidetes（F/B）ratios of PPLLFAAL,TLRFALHGME,and TLFGL groups were significantly decreased by 47.8%,57.4%and 57.5%,respectively,compared to the SHR group.The abundance of some fecal microorganisms changed significantly before and after administration,including Porphyromonadaceae,Pasteurellaceae,and Saccharibacteria.Multi-omics analysis identified 104 differentially expressed genes,29 differentially expressed proteins,and 48 differentially expressed metabolites.The enriched pathways for the differential genes and proteins were primarily related to bile secretion,glycan degradation,and lysosome function.The main pathways affecting metabolism involved neuroactive ligand-receptor interaction and lysine biosynthesis.（4）Modification and structure-activity relationship analysis of ACE-inhibitory peptidesThe effects of different chemical structure modifications on the pharmacological properties of ACE-inhibitory peptides were investigated.The acylation derivative of PPLLFAAL showed an increased ACE inhibitory activity,but a decreased antihypertensive effect.The acetylation and myristoylation derivatives of TLRFALHGME exhibited a 26.2%and 11.1%increase in ACE inhibitory activity,respectively,along with a corresponding increase in blood pressure-lowering effect by160.7%and 125.7%,respectively.Acetylation modification of TLFGL resulted in a28.3%improvement in ACE inhibitory activity,but a decreased antihypertensive effect.Acetylation modification of YVLL had led to a significant increase of 394.7%in ACE inhibitory activity and a 145.2%enhancement in antihypertensive effect.Molecular simulation results demonstrate that acetylation and myristoylation derivatives of TLRFALHGME,as well as acetylation derivatives of YVLL,exhibit weakened hydrogen bond interactions with ACE.However,their hydrophobic interactions and van der Waals potential energy interactions at the binding sites had significantly increased.The calculated binding free energy were increased by 10.1%,11.1%,and 40.5%for the respective modifications compared to the unmodified structures.In summary,four novel ACE-inhibitory peptides had been successfully identified from the skins of T.flavidus.The structure-activity relationship and mechanism of anti-hypertensive effects had been elucidated.Additionally,the impact of structural modifications on the activity of ACE-inhibitory peptides had been preliminarily investigated,laying the foundation for the development and utilization of ACE-inhibitory peptides.