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：
10661486
负责人：
INA L URBATSCH
金额：
$ 30.6万
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/10661486
关键词：
ABCB1 gene ATP Hydrolysis ATP-Binding Cassette Transporters Acceleration 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 Goals HIV/AIDS Human 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 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.

项目摘要/摘要项目名称：P-糖蛋白药物相互作用的研究 p-糖蛋白（PGP）是一种分子泵，通过在结构上运输数百种对细胞的排毒无关的毒素从细胞中出来。 PGP限制了肠中的摄取，并增强了药物排泄许多药物的肝，肾脏和血脑屏障，用于治疗癌症，艾滋病毒/艾滋病，精神病和心血管疾病。它是最重要的七个运输车之一负责调节目前需要药物记录的药物吸收和处置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）。它使用ATP水解将底物泵送到整个细胞膜上。我们最近的X- PGP的射线结构确定了有助于结合的疏水性和芳香氨基酸药物结合部位的不同抑制剂。在此提案中，我们将测试治疗性的假设药物与蛋白质TMDS定义的子腔内中的不同子集结合。我们的一般方法是在战略位置引入色氨酸（TRP）以监测药物结合。 TRP将在新的全功能无TRP PGP或低 - 的背景下引入 TRP PGP保留了细胞质结构域中三个天然构象敏感的TRP。使用荧光变化，例如淬火和共振能量传递（FRET），我们将绘制纯化蛋白质与原型底物相互作用的位点，这些底物占据了生化定义的以及独特的结合部位，以及常见的治疗药物和新鉴定的抑制剂。我们将使用Amber终止密码子进一步将荧光TRP模拟（L-ANAP）插入野生型PGP 探索监测生物细胞膜中药物结合的抑制策略。通过确定药物和抑制剂如何调节该多域的合作性和构象动力学转运蛋白，我们将获得对药物结合机制及其对PGP的影响的独特见解功能。通过这些新方法，我们将解决分子机制和动力学药物/抑制剂结合，确定协同作用并完善药物的机制 PGP中的相互作用。该信息将为新的分析方法铺平道路，以完善PGP药物新旧药物的相互作用研究将是对临床上的重新设计药物的宝贵有利的药代动力学和加速药物治疗发展。