Studies of P-glycoprotein Drug Interactions - Administrative Supplement for Undergraduate Summer Research

P-糖蛋白药物相互作用的研究 - 本科生暑期研究行政补充

基本信息

批准号：
10810072
负责人：
INA L URBATSCH
金额：
$ 0.75万
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/10810072
关键词：
ABCB1 gene ATP Hydrolysis ATP-Binding Cassette Transporters Acceleration Administrative Supplement 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 Cytoplasm Cytoplasmic Tail Development Dimerization Disease Documentation Drug Binding Site Drug Combinations Drug Design Drug Efflux Drug Interactions Drug Kinetics Drug Metabolic Detoxication Drug Monitoring Drug Transport Drug resistance Drug usage Energy Transfer Engineering Environment Excretory function Fluorescence Fluorescence Spectroscopy Fluorescent Probes Funding Goals HIV/AIDS Hydrophobicity Intestines Kidney Kinetics Knowledge Laboratories Learning Ligand Binding Lipid Bilayers Liver Maps Measures Membrane Mental disorders Molecular Molecular Conformation Monitor Multi-Drug Resistance Nifedipine Nucleotides Paclitaxel Pharmaceutical Preparations Positioning Attribute Prazosin Property 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 biophysical techniques cancer drug resistance cancer therapy clinically relevant combat efflux pump experience experimental study functional group inhibitor insight molecular pump nanodisk novel strategies novel therapeutics prevent protein purification protein reconstitution rational design reconstitution single molecule success summer research tool tryptophan analog undergraduate student uptake

项目摘要

PROJECT SUMMARY/ABSTRACT (of the original funded project) 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.

项目摘要/摘要（原始资助项目） P-糖蛋白 (Pgp) 是一种分子泵，通过运输数百个结构上不相关的分子来为细胞解毒。毒素排出细胞。 Pgp限制肠道的摄取，并增强药物在肝脏、肾脏和肾脏的排泄。血脑屏障，许多用于治疗癌症、艾滋病毒/艾滋病、精神疾病和心血管疾病。它是负责调节药物的七个最重要的转运蛋白之一现在需要记录药物相互作用以批准任何新药的吸收和处置美国食品和药物管理局（FDA）。 Pgp 是一种 ATP 结合盒式转运蛋白，具有两个跨膜结构域（TMD）和两个核苷酸结合结构域（NBD）。它利用 ATP 水解来泵送穿过细胞膜的底物。我们最近的 Pgp X 射线结构鉴定出疏水性和芳香性有助于不同抑制剂与药物结合位点结合的氨基酸。在这个提案中，我们将测试假设治疗药物与 TMD 中定义的子口袋内的不同残基子集结合的蛋白质。我们的一般方法是在战略位置引入色氨酸（Trps）以监测药物结合。 Trp 将在新的全功能 Trp-less Pgp（或低 Trp）的背景下引入 Pgp 在细胞质结构域中保留了三个天然构象敏感的色氨酸。使用荧光变化，例如猝灭和共振能量转移（FRET），我们将绘制出相互作用的位点纯化的蛋白质具有占据生化定义的独特结合位点的原型底物，以及如常见的治疗药物和新发现的抑制剂。我们将进一步插入荧光色氨酸使用琥珀终止密码子抑制策略将类似物（L-Anap）转化为野生型Pgp以探索监测药物结合在生物细胞膜上。通过确定药物和抑制剂如何调节协同性和这种多域转运蛋白的构象动力学，我们将获得对药物机制的独特见解结合及其对 Pgp 功能的影响。通过这些新方法，我们将解决分子机制和药物/抑制剂结合动力学，确定协同效应，并完善药物-药物机制 Pgp 中的交互。这些信息将为改进 Pgp 药物相互作用的新分析方法铺平道路新旧药物的研究，对于重新设计具有临床有利药代动力学的药物具有不可估量的价值并加速药物治疗的发展。