Targeted covalent inhibitors,primarily targeting cysteine residues,have attracted great attention as potential drug candidates due to good potency and prolonged duration of action.However,their discovery is challengin...Targeted covalent inhibitors,primarily targeting cysteine residues,have attracted great attention as potential drug candidates due to good potency and prolonged duration of action.However,their discovery is challenging.In this research,a database-assisted liquid chromatography-tandem mass spectrometry(LC-MS/MS)strategy was developed to quickly discover potential cysteine-targeting compounds.First,compounds with potential reactive groups were selected and incubated with N-acetyl-cysteine in microsomes.And the precursor ions of possible cysteine-adducts were predicted based on covalent binding mechanisms to establish in-house database.Second,substrate-independent product ions produced from N-acetyl-cysteine moiety were selected.Third,multiple reaction monitoring scan was conducted to achieve sensitive screening for cysteine-targeting compounds.This strategy showed broad applicability,and covalent compounds with diverse structures were screened out,offering structural resources for covalent inhibitors development.Moreover,the screened compounds,norketamine and hydroxynorketamine,could modify synaptic transmission-related proteins in vivo,indicating their potential as covalent inhibitors.This experimental-based screening strategy provides a quick and reliable guidance for the design and discovery of covalent inhibitors.展开更多
Objective:To screen potential serum differential proteins in insulin resistance(IR)complicated with polycystic ovary syndrome(PCOS)using isobaric tags for relative and absolute quantitation(i TRAQ)combined with ultra-...Objective:To screen potential serum differential proteins in insulin resistance(IR)complicated with polycystic ovary syndrome(PCOS)using isobaric tags for relative and absolute quantitation(i TRAQ)combined with ultra-high performance liquid chromatography-tandem mass spectrometry(LC-MS/MS).Methods:A total of 20 patients diagnosed with PCOS in our hospital were selected,including 10 cases of simple PCOS and 10 cases of PCOS+IR.i TRAQ combined with LC-MS/MS was used for proteomic analysis to identify serum differential proteins.Bioinformatics analyses,including gene ontology(GO),Kyoto encyclopedia of genes and genomes(KEGG),and protein-protein interaction(PPI)analysis,were conducted to understand the biological processes,cellular components,and molecular functions of the differentially expressed proteins detected by the two methods.Results:A total of 454,675 secondary spectra were detected by iTRAQ,with 14,376 matched spectra.Combined with LC-MS/MS,74,386peptides,47,542 unique peptide sequences,and 54,675 proteins were identified.A total of 249 differentially expressed proteins with a fold change≥1.30 or≤0.83 were found,among which 9 had a P-value<0.05.There were 5 up-regulated and 4 down-regulated proteins,namely RPAP3,ALDH6A1,COX20,RASSF3,ALPK2,NANOS1,FAM210A,CHGA,and CGA.These differential proteins were imported into the STRING database for PPI network analysis,which revealed 21 nodes and 69 edges with a PPI enrichment P-value<0.001.KEGG enrichment results included hsa04913(Ovarian steroidogenesis),hsa04024(cAMP signaling pathway),and hsa04080(Neuroactive ligand-receptor interaction).Conclusion:The combination of i TRAQ and LC-MS/MS technologies identified nine differential proteins including CHGA and CGA,which are mainly enriched in pathways related to ovarian steroidogenesis,c AMP signaling,and neuroactive ligand-receptor interaction.This combination provides a powerful platform for exploring the pathogenesis of IR complicated with PCOS and discovering new biomarkers.展开更多
3-(4-Chlorophenyl)-7-[2-(piperazin-1-yl)ethoxy]-4H-chromen-4-one(CPEO-43)is a derivative of soybean isoflavone(SI),synthesized by introducing a chlorine atom and a piperazine group into the structure of natural SI.In ...3-(4-Chlorophenyl)-7-[2-(piperazin-1-yl)ethoxy]-4H-chromen-4-one(CPEO-43)is a derivative of soybean isoflavone(SI),synthesized by introducing a chlorine atom and a piperazine group into the structure of natural SI.In vitro experiments have demonstrated that CPEO-43 exhibits a notable inhibitory effect on both A549 cells and HCT116 cells.For the further development and utilization of CPEO-43,this study aims to establish and validate a liquid chromatography-tandem mass spectrometry(LC-MS/MS)quantitative analysis method for the pharmacokinetic study of CPEO-43.Normal rats were intragastrically administered different doses(2,6,and 20 mg/kg)of CPEO-43,and blood was taken from the ocular venous plexus at different time points.The blood concentration of CPEO-43 at different time points was determined using LC-MS/MS technology,and the pharmacokinetic parameters of the compound were calculated using the pharmacokinetic software DAS.The results indicated that the established LC-MS/MS method complies with the standards for bioanalytical method validation in the Chinese Pharmacopoeia(CHP)and can be applied to the pharmacokinetic study of CPEO-43.The pharmacokinetic software DAS non-compartmental model was successfully used to calculate the C_(max),T_(max),t_(1/2),AUC_(0-∞),MRTs,CL,and Vd pharmacokinetic parameters of the compound at different doses.The results were as follows:62.0±10.5,222.0±28.7,and 1384.5±376.4 ng/mL;8.5±1.2,6.0±0.0,and 11.0±6.2 h;15.6,15.0,and 18.5 h;1517.8±317.0,5328.7±864.4,and 45556.3±22735.6 ng·h/mL;17.8±1.2,17.7±0.8,and 20.0±3.2 h;1370.3±305.9,1153.5±205.6,and 505.3±179.8 mL/kg/h;30843.0±7458.0,24344.0±5237.0,and 13950.3±5996.9 mL/kg.These characteristics are of great significance for understanding the in vivo process of the drug,formulating dosing regimens,and evaluating the safety and efficacy of the drug.展开更多
基金supported by the Science and Technology Development Fund,Macao SAR,China(Grant Nos.:FDCT 0001/2020/AKP and 006/2023/SKL)Guangxi Science and Technology Major Program,China(Program No.:Guike AA22096022).
文摘Targeted covalent inhibitors,primarily targeting cysteine residues,have attracted great attention as potential drug candidates due to good potency and prolonged duration of action.However,their discovery is challenging.In this research,a database-assisted liquid chromatography-tandem mass spectrometry(LC-MS/MS)strategy was developed to quickly discover potential cysteine-targeting compounds.First,compounds with potential reactive groups were selected and incubated with N-acetyl-cysteine in microsomes.And the precursor ions of possible cysteine-adducts were predicted based on covalent binding mechanisms to establish in-house database.Second,substrate-independent product ions produced from N-acetyl-cysteine moiety were selected.Third,multiple reaction monitoring scan was conducted to achieve sensitive screening for cysteine-targeting compounds.This strategy showed broad applicability,and covalent compounds with diverse structures were screened out,offering structural resources for covalent inhibitors development.Moreover,the screened compounds,norketamine and hydroxynorketamine,could modify synaptic transmission-related proteins in vivo,indicating their potential as covalent inhibitors.This experimental-based screening strategy provides a quick and reliable guidance for the design and discovery of covalent inhibitors.
文摘Objective:To screen potential serum differential proteins in insulin resistance(IR)complicated with polycystic ovary syndrome(PCOS)using isobaric tags for relative and absolute quantitation(i TRAQ)combined with ultra-high performance liquid chromatography-tandem mass spectrometry(LC-MS/MS).Methods:A total of 20 patients diagnosed with PCOS in our hospital were selected,including 10 cases of simple PCOS and 10 cases of PCOS+IR.i TRAQ combined with LC-MS/MS was used for proteomic analysis to identify serum differential proteins.Bioinformatics analyses,including gene ontology(GO),Kyoto encyclopedia of genes and genomes(KEGG),and protein-protein interaction(PPI)analysis,were conducted to understand the biological processes,cellular components,and molecular functions of the differentially expressed proteins detected by the two methods.Results:A total of 454,675 secondary spectra were detected by iTRAQ,with 14,376 matched spectra.Combined with LC-MS/MS,74,386peptides,47,542 unique peptide sequences,and 54,675 proteins were identified.A total of 249 differentially expressed proteins with a fold change≥1.30 or≤0.83 were found,among which 9 had a P-value<0.05.There were 5 up-regulated and 4 down-regulated proteins,namely RPAP3,ALDH6A1,COX20,RASSF3,ALPK2,NANOS1,FAM210A,CHGA,and CGA.These differential proteins were imported into the STRING database for PPI network analysis,which revealed 21 nodes and 69 edges with a PPI enrichment P-value<0.001.KEGG enrichment results included hsa04913(Ovarian steroidogenesis),hsa04024(cAMP signaling pathway),and hsa04080(Neuroactive ligand-receptor interaction).Conclusion:The combination of i TRAQ and LC-MS/MS technologies identified nine differential proteins including CHGA and CGA,which are mainly enriched in pathways related to ovarian steroidogenesis,c AMP signaling,and neuroactive ligand-receptor interaction.This combination provides a powerful platform for exploring the pathogenesis of IR complicated with PCOS and discovering new biomarkers.
基金Scientific Research Projects of Hebei North University(Grant No.XJ2024022).
文摘3-(4-Chlorophenyl)-7-[2-(piperazin-1-yl)ethoxy]-4H-chromen-4-one(CPEO-43)is a derivative of soybean isoflavone(SI),synthesized by introducing a chlorine atom and a piperazine group into the structure of natural SI.In vitro experiments have demonstrated that CPEO-43 exhibits a notable inhibitory effect on both A549 cells and HCT116 cells.For the further development and utilization of CPEO-43,this study aims to establish and validate a liquid chromatography-tandem mass spectrometry(LC-MS/MS)quantitative analysis method for the pharmacokinetic study of CPEO-43.Normal rats were intragastrically administered different doses(2,6,and 20 mg/kg)of CPEO-43,and blood was taken from the ocular venous plexus at different time points.The blood concentration of CPEO-43 at different time points was determined using LC-MS/MS technology,and the pharmacokinetic parameters of the compound were calculated using the pharmacokinetic software DAS.The results indicated that the established LC-MS/MS method complies with the standards for bioanalytical method validation in the Chinese Pharmacopoeia(CHP)and can be applied to the pharmacokinetic study of CPEO-43.The pharmacokinetic software DAS non-compartmental model was successfully used to calculate the C_(max),T_(max),t_(1/2),AUC_(0-∞),MRTs,CL,and Vd pharmacokinetic parameters of the compound at different doses.The results were as follows:62.0±10.5,222.0±28.7,and 1384.5±376.4 ng/mL;8.5±1.2,6.0±0.0,and 11.0±6.2 h;15.6,15.0,and 18.5 h;1517.8±317.0,5328.7±864.4,and 45556.3±22735.6 ng·h/mL;17.8±1.2,17.7±0.8,and 20.0±3.2 h;1370.3±305.9,1153.5±205.6,and 505.3±179.8 mL/kg/h;30843.0±7458.0,24344.0±5237.0,and 13950.3±5996.9 mL/kg.These characteristics are of great significance for understanding the in vivo process of the drug,formulating dosing regimens,and evaluating the safety and efficacy of the drug.
