Jing Zhang , Kun-Jie Bian , Yi-Jing Wang , Xing-Kai Qian , Jin Geng , Hai-Dong Guo , Li-Wei Zou
{"title":"近红外荧光底物驱动下发现脯氨酸内肽酶天然抑制剂及其抑制α -突触核蛋白聚集和促进自噬","authors":"Jing Zhang , Kun-Jie Bian , Yi-Jing Wang , Xing-Kai Qian , Jin Geng , Hai-Dong Guo , Li-Wei Zou","doi":"10.1016/j.cbi.2025.111733","DOIUrl":null,"url":null,"abstract":"<div><div>Prolyl endopeptidase (PREP) drives neurodegenerative diseases through dual mechanisms involving enzymatic activity and protein-protein interactions (PPIs), yet current inhibitors predominantly target single pathways.</div><div>Prolyl endopeptidase (PREP) fuels neurodegeneration via enzymatic cleavage and pathological PPIs, yet current inhibitors usually target only one facet. In this study, leveraging our developed high-sensitivity and high-specificity near-infrared fluorescent probe Z-GP-ACM, we established and validated a screening platform for PREP inhibitors with mouse brain S9 instead of the human recombinant PREP.</div><div>Screening a library of 110 natural compounds identified a series of flavonoid derivatives (FV64-FV68) as potent PREP inhibitors, with FV67 and FV68 exhibiting particularly strong inhibition (IC<sub>50</sub> values of 0.65 μM and 0.31 μM, respectively). Reversibility assays revealed that all new inhibitors display time-independent potency (IC<sub>50</sub> unchanged after 5 vs 35 min pre-incubation), confirming reversible inhibition.</div><div>Furthermore. Kinetic analyses classified FV66/FV67 as mixed-type and FV64/FV65/FV68 as non-competitive inhibitors. Molecular docking simulations further revealed that FV68 binds the S1 and S2 sub-sites of PREP through hydrogen bonding and π-π stacking, which is the structural basis for its high activity. Further studies showed that both FV67 and FV68 inhibited PREP activity in HT22 and SH-SY5Y cells with a dose-dependent manner. Notably, FV68 enhanced autophagy, reduced α-synuclein aggregation, and mitigated H<sub>2</sub>O<sub>2</sub>-induced oxidative stress. These studies not only provide directions for the development of novel PREP inhibitors derived from natural products, also reveal new mechanisms by which natural compounds may intervene in neurodegenerative diseases by PREP-inhibited modulating PPIs.</div></div>","PeriodicalId":274,"journal":{"name":"Chemico-Biological Interactions","volume":"421 ","pages":"Article 111733"},"PeriodicalIF":5.4000,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"NIR fluorescent substrate-driven discovery of prolyl endopeptidase natural inhibitors and its inhibition of alpha-synuclein aggregation and promotion of autophagy\",\"authors\":\"Jing Zhang , Kun-Jie Bian , Yi-Jing Wang , Xing-Kai Qian , Jin Geng , Hai-Dong Guo , Li-Wei Zou\",\"doi\":\"10.1016/j.cbi.2025.111733\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>Prolyl endopeptidase (PREP) drives neurodegenerative diseases through dual mechanisms involving enzymatic activity and protein-protein interactions (PPIs), yet current inhibitors predominantly target single pathways.</div><div>Prolyl endopeptidase (PREP) fuels neurodegeneration via enzymatic cleavage and pathological PPIs, yet current inhibitors usually target only one facet. In this study, leveraging our developed high-sensitivity and high-specificity near-infrared fluorescent probe Z-GP-ACM, we established and validated a screening platform for PREP inhibitors with mouse brain S9 instead of the human recombinant PREP.</div><div>Screening a library of 110 natural compounds identified a series of flavonoid derivatives (FV64-FV68) as potent PREP inhibitors, with FV67 and FV68 exhibiting particularly strong inhibition (IC<sub>50</sub> values of 0.65 μM and 0.31 μM, respectively). Reversibility assays revealed that all new inhibitors display time-independent potency (IC<sub>50</sub> unchanged after 5 vs 35 min pre-incubation), confirming reversible inhibition.</div><div>Furthermore. Kinetic analyses classified FV66/FV67 as mixed-type and FV64/FV65/FV68 as non-competitive inhibitors. Molecular docking simulations further revealed that FV68 binds the S1 and S2 sub-sites of PREP through hydrogen bonding and π-π stacking, which is the structural basis for its high activity. Further studies showed that both FV67 and FV68 inhibited PREP activity in HT22 and SH-SY5Y cells with a dose-dependent manner. Notably, FV68 enhanced autophagy, reduced α-synuclein aggregation, and mitigated H<sub>2</sub>O<sub>2</sub>-induced oxidative stress. These studies not only provide directions for the development of novel PREP inhibitors derived from natural products, also reveal new mechanisms by which natural compounds may intervene in neurodegenerative diseases by PREP-inhibited modulating PPIs.</div></div>\",\"PeriodicalId\":274,\"journal\":{\"name\":\"Chemico-Biological Interactions\",\"volume\":\"421 \",\"pages\":\"Article 111733\"},\"PeriodicalIF\":5.4000,\"publicationDate\":\"2025-09-05\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Chemico-Biological Interactions\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0009279725003631\",\"RegionNum\":2,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Chemico-Biological Interactions","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0009279725003631","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
NIR fluorescent substrate-driven discovery of prolyl endopeptidase natural inhibitors and its inhibition of alpha-synuclein aggregation and promotion of autophagy
Prolyl endopeptidase (PREP) drives neurodegenerative diseases through dual mechanisms involving enzymatic activity and protein-protein interactions (PPIs), yet current inhibitors predominantly target single pathways.
Prolyl endopeptidase (PREP) fuels neurodegeneration via enzymatic cleavage and pathological PPIs, yet current inhibitors usually target only one facet. In this study, leveraging our developed high-sensitivity and high-specificity near-infrared fluorescent probe Z-GP-ACM, we established and validated a screening platform for PREP inhibitors with mouse brain S9 instead of the human recombinant PREP.
Screening a library of 110 natural compounds identified a series of flavonoid derivatives (FV64-FV68) as potent PREP inhibitors, with FV67 and FV68 exhibiting particularly strong inhibition (IC50 values of 0.65 μM and 0.31 μM, respectively). Reversibility assays revealed that all new inhibitors display time-independent potency (IC50 unchanged after 5 vs 35 min pre-incubation), confirming reversible inhibition.
Furthermore. Kinetic analyses classified FV66/FV67 as mixed-type and FV64/FV65/FV68 as non-competitive inhibitors. Molecular docking simulations further revealed that FV68 binds the S1 and S2 sub-sites of PREP through hydrogen bonding and π-π stacking, which is the structural basis for its high activity. Further studies showed that both FV67 and FV68 inhibited PREP activity in HT22 and SH-SY5Y cells with a dose-dependent manner. Notably, FV68 enhanced autophagy, reduced α-synuclein aggregation, and mitigated H2O2-induced oxidative stress. These studies not only provide directions for the development of novel PREP inhibitors derived from natural products, also reveal new mechanisms by which natural compounds may intervene in neurodegenerative diseases by PREP-inhibited modulating PPIs.
期刊介绍:
Chemico-Biological Interactions publishes research reports and review articles that examine the molecular, cellular, and/or biochemical basis of toxicologically relevant outcomes. Special emphasis is placed on toxicological mechanisms associated with interactions between chemicals and biological systems. Outcomes may include all traditional endpoints caused by synthetic or naturally occurring chemicals, both in vivo and in vitro. Endpoints of interest include, but are not limited to carcinogenesis, mutagenesis, respiratory toxicology, neurotoxicology, reproductive and developmental toxicology, and immunotoxicology.