LC-MS-MS

液质联用-质谱

基本信息

批准号：
7614516
负责人：
RICHARD B VAN BREEMEN
金额：
$ 23.81万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7614516
关键词：
Affinity Androgen Receptor Animals Binding Biological Biological Assay Biological Availability Biological Factors Blood Caco-2 Cells Cell model Chemoprevention Clinic Collaborations Complement Complex Mixtures Cytochrome P450 Data Databases Development Dose Drug Interactions Drug Kinetics Enzymes Epithelial Epoxy Compounds Estrogen Receptor alpha Evaluation Exhibits Fractionation Glutathione Hepatic Hepatocyte Human In Vitro Incubated Intestinal Absorption Intestines Intravenous Investigational Drugs Isoenzymes Laboratories Lead Libraries Ligands Liver Microsomes Mass Spectrum Analysis Measurement Measures Metabolic Metabolic Activation Metabolism Methods Mining Modeling Molecular Weight Monoclonal Antibodies Mus NADP Oral Pattern Permeability Pharmaceutical Preparations Pharmacologic Substance Phase Physiologic pulse Plant Extracts Preclinical Drug Development Preparation Process Productivity Proteins Protons Pulse taking Quinones RXR Rate Rattus Reaction Recombinants Research Research Personnel Resolution Safety Sampling Science Screening procedure Serum Standards of Weights and Measures Structure-Activity Relationship System Testing Tissue Sample Toxic effect Transferase Translations Ultrafiltration Urine Xenobiotics adduct analog analytical method antibody inhibitor base benzoquinone cancer chemoprevention cofactor combinatorial cyclooxygenase 2 drug discovery drug metabolism high throughput screening in vivo marine organism monolayer pre-clinical preclinical study programs quinone methide receptor research study sulfotransferase

项目摘要

Based on the method of pulsed ultrafiltration mass spectrometry, which was invented in the laboratory of Dr. van Breemen, natural product extracts will be screened rapidly for the discovery of specific compounds that bind to important target molecules. These high throughput screening assays will enhance the productivity of the entire program. Existing ultrafiltration screening assays for ligands to cyclooxygenase-2 and the estrogen receptors-alpha and -beta will be utilized as well as new assays that will be developed to screen for ligands to receptors such as the androgen receptor and RXR. Hits obtained during ultrafiltration mass spectrometric screening will be identified based on molecular weight, elemental composition (determined using exact mass measurement), tandem mass spectrometric (MS-MS) fragmentation patterns, comparison with standards available at the UIC Program for Collaborative Research in the Pharmaceutical Sciences, dereplication using the UIC NAPRALERT database of natural products and proton NMR and 13C-NMR analysis of HPLC-purified material. Since this screening approach will identify lead compounds based on binding to a specific target protein, these hits will be assayed in Core B to confirm their biological activity. Next, the Phase I and Phase II metabolism of the lead compounds from all parts of the program will be assessed in vitro by incubation with human hepatocytes and human liver microsomes followed by analysis using LC-MS and LC-MS-MS. The in vivo metabolism of cancer chemoprevention agents will be evaluated by LC-MS and LC-MS-MS analysis of blood and urine samples from animal studies. In addition, pharmacokinetic studies of these compounds will be carried out in animals. In another set of preclinical drug development studies, the human intestinal permeability of each lead compound will be evaluated using the Caco-2 cell monolayer model. Finally, LC-MS and LC-MS-MS will be used to confirm the molecular weights, elemental compositions, fragmentation patterns, and purity of compounds. In summary, this project will interact with all of the other Projects within this program by screening natural product extracts for the rapid identification of ligands for specific target proteins, by providing preclinical evaluation of hepatic metabolism, pharmacokinetics and intestinal permeability of lead compounds, and by providing mass spectrometric characterization of isolated and synthetic compounds.

基于在Van Breemen博士实验室发明的脉冲超滤质谱法的方法，将迅速筛选自然产物提取物，以发现与重要靶分子结合的特定化合物。这些高吞吐量筛选分析将提高整个计划的生产率。现有的超滤筛选测定法对环氧酶-2和雌激素受体-Alpha和-beta的配体以及将开发的新测定方法将用于筛选出雄激素受体和RXR等受体的配体。将根据分子量，元素组成（使用精确质量测量），串联质谱（MS-MS）片段化模式来鉴定在超滤质谱筛选期间获得的点击率，并与在UIC计划中可用的标准差进行比较药物科学，使用天然产物的UIC Napralert数据库和Proton NMR的消除以及HPLC纯化材料的13C-NMR分析。由于这种筛选方法将基于与特定靶蛋白的结合确定铅化合物，因此核心B将测定点击以确认其生物学活性。接下来，通过与人肝细胞和人肝微粒体孵育，将在体外评估该铅化合物的I期和II期代谢，然后使用LC-MS和LC-MS-MS进行分析。癌症化学预防剂的体内代谢将通过LC-MS和LC-MS-MS分析动物研究中的血液和尿液样本分析。此外，这些化合物的药代动力学研究将在动物中进行。在另一组临床前药物开发研究中，将使用CACO-2细胞单层模型评估每种铅化合物的人类肠道通透性。最后，LC-MS和LC-MS-MS将用于确认化合物的分子量，元素组成，碎片模式和纯度。总而言之，该项目将通过筛选自然产品提取物来与该计划中的所有其他项目进行互动，以快速鉴定特定靶蛋白的配体，并通过对肝代谢，药代动力学和铅化合物的肠道渗透率进行临床前评估，并通过提供相互隔离的混合物的质量分析表征。