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Studies of P-glycoprotein drug interactions

P-糖蛋白药物相互作用的研究

基本信息

批准号：
10366914
负责人：
INA L URBATSCH
金额：
$ 31.92万
依托单位：
TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10366914
关键词：
ABCB1 gene AIDS/HIV problem ATP Hydrolysis ATP-Binding Cassette Transporters Address Affinity Amber Amino Acids Antineoplastic Agents Area Aromatic Amino Acids Binding Binding Sites Biochemical Biological Biological Assay Biological Availability Biomedical Engineering Blood - brain barrier anatomy Cardiovascular Agents Cardiovascular system Cell membrane Cells Chemicals Clinical Codon Nucleotides Cryoelectron Microscopy Crystallization Cytoplasmic Tail Development Dimerization Disease Documentation Drug Binding Site Drug Combinations Drug Interactions Drug Kinetics Drug Monitoring Drug Transport Drug resistance Drug usage Energy Transfer Engineering Environment Excretory function Fluorescence Fluorescence Spectroscopy Fluorescent Probes Goals Human Hydrophobicity Intestines Kidney Kinetics Knowledge Laboratories Learning Ligand Binding Lipid Bilayers Liver Malignant Neoplasms Maps Measures Membrane Mental disorders Molecular Molecular Conformation Monitor Multi-Drug Resistance Nifedipine Nucleotides Paclitaxel Pharmaceutical Preparations Positioning Attribute Prazosin Property Proteins Pump Recombinant Proteins Rhodamine 123 Roentgen Rays Scientist Screening procedure Site Structure Surface System Technology Terminator Codon Tertiary Protein Structure Testing Therapeutic Toxin Transmembrane Domain Tryptophan United States Food and Drug Administration Variant Vinblastine X-Ray Crystallography absorption analog L biophysical techniques cancer therapy clinically relevant combat efflux pump experience experimental study functional group inhibitor insight molecular pump nanodisk novel strategies novel therapeutics prevent rational design reconstitution single molecule success tool tryptophan analog uptake

项目摘要

PROJECT SUMMARY/ABSTRACT PROJECT TITLE: Studies of P-glycoprotein drug interactions P-glycoprotein (Pgp) is a molecular pump that detoxifies cells by transporting hundreds of structurally unrelated toxins out of the cell. Pgp limits uptake in the intestines, and enhances excretion of drugs in the liver, kidney and blood-brain barrier, of many drugs that are used for treatment of cancers, HIV/AIDS, psychiatric illnesses, and cardiovascular conditions. It is among the seven most important transporters responsible for regulating drug absorption and disposition that now require documentation of drug interactions for approval of any new drugs by the US Food and Drug Administration (FDA). Pgp is an ATP-binding cassette transporter with two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). It uses ATP hydrolysis to pump substrates across the cell membranes. Our recent X- ray structures of Pgp identified hydrophobic and aromatic amino acids that contribute to binding of different inhibitors to the drug-binding site. In this proposal, we will test the hypothesis that therapeutic drugs bind to different subsets of residues within defined subpockets in the TMDs of the protein. Our general approach is to introduce tryptophans (Trps) at strategic positions in order to monitor drug binding. The Trps will be introduced on the background of a new fully functional Trp-less Pgp, or a low- Trp Pgp that retains three native conformationally sensitive Trps in the cytoplasmic domains. Using fluorescence changes, such as quenching, and resonance energy transfer (FRET), we will map out sites of interaction of the purified protein with prototypical substrates that occupy biochemically defined and distinct binding sites, as well as those of common therapeutic drugs and newly identified inhibitors. We will further insert a fluorescent Trp analog (L-Anap) into wild-type Pgp using the amber stop codon suppression strategy to explore monitoring drug binding in biological cell membranes. By determining how drugs and inhibitors modulate the cooperativity and conformational dynamics of this multidomain transporter, we will gain unique insight into the mechanisms of drug binding and their effects on Pgp function. With these new approaches, we will address the molecular mechanism and kinetics of drug/inhibitor binding, determine synergistic effects, and refine the mechanisms of drug-drug interactions in Pgp. The information will pave the way to new analytical approaches to refine Pgp drug interaction studies of old and new drugs, and will be invaluable to redesign drugs with clinically favorable pharmacokinetics and accelerate pharmacotherapeutic developments.

项目总结/摘要项目名称：P-糖蛋白药物相互作用研究 P-糖蛋白（Pgp）是一种分子泵，通过转运数百种结构上的把不相关的毒素排出细胞Pgp限制了肠道的吸收，并增强了药物在肠道中的排泄。肝、肾和血脑屏障，许多用于治疗癌症、艾滋病毒/艾滋病的药物，精神疾病和心血管疾病。它是七种最重要的运输工具之一负责管理药物吸收和处置，现在需要记录药物美国食品药品监督管理局（FDA）批准任何新药的相互作用。Pgp是一个具有两个跨膜结构域（TMD）和两个核苷酸结合的ATP结合盒转运蛋白域（NBD）。它利用ATP水解将底物泵送穿过细胞膜。我们最近的X- Pgp的X射线结构鉴定了有助于结合药物结合位点的不同抑制剂。在这个建议中，我们将测试的假设，治疗药物结合于蛋白质的TMD中限定的亚口袋内的不同残基子集。我们一般方法是在战略位置引入色氨酸（Trps）以监测药物约束力Trps将在新的全功能Trp-少Pgp或低- 在胞质结构域中保留三个天然构象敏感Trp的Trp Pgp。使用荧光变化，如淬灭，共振能量转移（FRET），我们将绘制出纯化的蛋白质与原型底物的相互作用位点，和不同的结合位点，以及常见的治疗药物和新鉴定的抑制剂的结合位点。我们将进一步插入荧光色氨酸类似物（L-Anap）到野生型Pgp使用琥珀终止密码子抑制策略，以探索监测生物细胞膜中的药物结合。通过确定药物和抑制剂如何调节这种多结构域的协同性和构象动力学转运蛋白，我们将获得独特的见解药物结合的机制和它们对Pgp的影响功能通过这些新的方法，我们将解决的分子机制和动力学，药物/抑制剂结合，确定协同效应，并完善药物-药物的机制 Pgp的相互作用这些信息将为改进Pgp药物的新分析方法铺平道路新老药物的相互作用研究，并将是非常宝贵的重新设计药物与临床有利的药代动力学和加速药理学发展。