ABSTRACT
Over 300 Bi-binding peptides from 166 proteins in H. pylori were identified by Bi-IMAC. Bi(3+) exhibits high selectivity towards peptide enriched by cysteines and histidines with dominated motif patterns of CXnC, CXnH and HXnH. Structural rationalization and functional categorization on the identified Bi-binding peptides and proteins provide an insight into the inhibitory action of bismuth drugs.
Subject(s)
Bismuth/metabolism , Chromatography, Affinity , Helicobacter pylori/metabolism , Bismuth/chemistry , Coordination Complexes/chemistry , Coordination Complexes/metabolism , Drug Delivery Systems , Helicobacter pylori/chemistry , Peptides/metabolism , Protein Binding , Protein Structure, TertiaryABSTRACT
Protein disulfide isomerase (PDI) family members including PDI and ERp57 emerge as novel targets for anti-thrombotic treatments, but chemical agents with selectivity remain to be explored. We previously reported a novel derivative of danshensu (DSS), known as ADTM, displayed strong cardioprotective effects against oxidative stress-induced cellular injury in vitro and acute myocardial infarct in vivo. Herein, using chemical proteomics approach, we identified ERp57 as a major target of ADTM. ADTM displayed potent inhibitory effects on the redox activity of ERp57, inhibited the adenosine diphosphate (ADP)-induced expressions of P-selectin and αIIbß3 integrin, and disrupted the interaction between ERp57 and αIIbß3. In addition, ADTM inhibited both arachidonic acid (AA)-induced and ADP-induced platelet aggregation in vitro. Furthermore, ADTM significantly inhibited rat platelet aggregation and thrombus formation in vivo. Taken together, ADTM represents a promising candidate for anti-thrombotic therapy targeting ERp57.
Subject(s)
Fibrinolytic Agents/pharmacology , Protein Disulfide-Isomerases/metabolism , Adenosine Diphosphate/metabolism , Animals , Blood Platelets/drug effects , Blood Platelets/metabolism , Cell Adhesion Molecules/metabolism , Chlorides/adverse effects , Disease Models, Animal , Enzyme Activation , Ferric Compounds/adverse effects , Fibrinolytic Agents/chemistry , Gene Expression Regulation/drug effects , Heme Oxygenase-1/metabolism , Humans , Lactates/chemistry , Lactates/pharmacology , Microfilament Proteins/metabolism , Models, Biological , P-Selectin/genetics , P-Selectin/metabolism , Phosphoproteins/metabolism , Phosphorylation , Platelet Activation , Platelet Aggregation/drug effects , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Protein Binding , Proteomics , Rats , Thrombosis/drug therapy , Thrombosis/etiology , Venous Thrombosis/etiologyABSTRACT
Although zebrafish has become a popular animal model for drug discovery and screening, drug metabolism in zebrafish remains largely unknown. In this study, we probed the metabolic capability of zebrafish larvae with calycosin, one of the major isoflavone constituents of Radix Astragali that was previously demonstrated to be angiogenic in the zebrafish model. The metabolism of calycosin and accumulation of its metabolites in zebrafish larvae were determined using an LC-MS/MS method. Calycosin showed a slow but steady decrease from the culture medium as well as a steady accumulation in zebrafish larvae. Calycosin underwent major conjugation and minor oxidation in zebrafish larvae. A total of ten calycosin metabolites formed from glucuronidation, glucosylation, sulfation, oxidation or a combination of two of these metabolisms were identified, most of which were reported for the first time. Most metabolites increased steadily in the larvae over 24-h experimental period. The dominant phase II conjugation of calycosin in zebrafish larvae matched well with existing knowledge of isoflavone metabolism in mammalians. The findings shed a light in certain degree of similarity of phase II drug metabolism between zebrafish larvae and mammals and warrant further investigation on feasibility of adopting the zebrafish larvae as a whole-organism model for examining drug metabolism.
Subject(s)
Drugs, Chinese Herbal/chemistry , Isoflavones/metabolism , Zebrafish/metabolism , Animals , Astragalus Plant/chemistry , Astragalus propinquus , Calibration , Chromatography, High Pressure Liquid , Culture Media/chemistry , Isoflavones/chemistry , Isoflavones/pharmacology , Larva/drug effects , Larva/metabolism , Mass Spectrometry , Metabolic Networks and Pathways/drug effects , Time FactorsABSTRACT
Flavonoids isolated from Herba Epimedii such as icaritin, icariin and epimedin C have been suggested as potential bone anabolic compounds. However, the "specific localized effects" of these flavonoids in bone, in vivo, and the metabolism of these flavonoids in zebrafish larvae have never been demonstrated. In this study, we used multiple methods including in vivo imaging, drug metabolites profiling, transcriptomic and proteomic approaches to determine the mechanisms involved in the distribution and metabolism of the flavonoids in zebrafish larvae by measuring the fluorescence emission, in vivo, of icaritin and its glycoside derivatives. The fluorescence emission mechanism of icaritin in vitro was identified by spectrophotometric analysis, and the fluorescent property of icaritin was used as a probe to visualize the metabolism and distribution of icaritin and its glycoside derivatives in zebrafish larvae. Phase I and phase II metabolism of icaritin and its derivatives were identified in zebrafish by mass spectrometry. The combined transcriptomics and proteomics demonstrate a high degree of conservation of phase I and phase II drug metabolic enzymes between zebrafish larvae and mammals. Icaritin and its glycoside derivatives were demonstrated using combined approaches of in vivo imaging, drug metabolites identification, and transcriptomic and proteomic profiling to illustrate phase I and phase II metabolism of the flavonoids and their distribution in bone of zebrafish larvae. This study provides a new methodological model for use of the zebrafish larvae to examine drug metabolism.
