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Understanding polyspecific drug binding in P-glycoprotein

了解 P-糖蛋白中的多特异性药物结合

基本信息

批准号：
8365444
负责人：
INA L URBATSCH
金额：
$ 34.75万
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：
2012
资助国家：
美国
起止时间：
2012-09-21 至 2015-09-20
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8365444
关键词：
ATP Hydrolysis ATP-Binding Cassette Transporters Address Affinity Antineoplastic Agents Antineoplastic Combined Chemotherapy Protocols Aromatic Amino Acids Aromatic Compounds Binding Binding Sites Biological Assay Cells Chemicals Chemotherapy-Oncologic Procedure Development Doxorubicin Drug Binding Site Drug Kinetics Drug Monitoring Drug resistance Drug usage Engineering Environment Fluorescence Fluorescence Resonance Energy Transfer Fluorescence Spectroscopy Goals HIV In Vitro Kinetics Knowledge Laboratories Learning Length Ligand Binding Location Malignant Neoplasms Maps Measures Mediating Membrane Membrane Proteins Molecular Molecular Conformation Multi-Drug Resistance Mutagenesis P-Glycoprotein Paclitaxel Pharmaceutical Preparations Positioning Attribute Prazosin Proteins Pump Rhodamine Rhodamine 123 Roentgen Rays Scientist Series Site Structural Protein Structure Substrate Specificity Surface System Technology Testing Toxin Transmembrane Domain Tryptophan United States National Institutes of Health Vinblastine Work Yeasts base chemosensitizing agent chemotherapy clinically relevant design directed evolution functional group high throughput screening inhibitor/antagonist killings mutant neoplastic cell novel strategies prevent repository research study small molecule success tool

项目摘要

DESCRIPTION (provided by applicant): Multidrug resistance (MDR) mediated by P-glycoprotein (Pgp) is a significant problem in the treatment of many cancers, HIV, and psychiatric illnesses. Pgp is an ATP-binding cassette transporter that pumps many structurally unrelated drugs out of the cell through an ATP-dependent mechanism. Our recent X- ray structure of Pgp identified hydrophobic and aromatic amino acids that contribute to binding of two different inhibitors to the drug-binding site. In this proposal, we will test the hypothesis tht anticancer drugs bind to different subsets of residues within defined subpockets in the transmembrane regions of the protein. Using tryptophan (Trp) fluorescence quenching, we will map out sites of interaction of the purified protein with three prototypical substrates that occupy biochemically defined and distinct binding sites, as well as those of common anticancer drugs and newly identified inhibitors. The novelty of this proposal is our development of a functional Trp-free Pgp, and the introduction into this Trp-free background of one or more Trps at strategic positions to monitor drug binding. With this new approach, we will address the molecular mechanism and kinetics of drug/inhibitor binding and determine the mechanisms of action of the recently-identified blockers. We plan to obtain direct information on how different surfaces of the protein subpockets interact with anticancer drugs, and how different blockers work. The latter will be invaluable to develop mechanistically- and structurally-based panels of potential blockers for high-throughput screening. PUBLIC HEALTH RELEVANCE: P-glycoprotein (Pgp) is the cell's cleaning machine, pumping harmful substances to the outside of the cell. In cancer chemotherapy, Pgp can cause problems by removing chemotherapy drugs from the tumor cells that they were intended to kill. By learning more about how Pgp recognizes the chemicals that it carries out of the cell, scientists may devise new drugs to prevent Pgp from interfering with the valuable effects of anticancer drugs.

描述（由申请人提供）：p -糖蛋白（Pgp）介导的多药耐药（MDR）是许多癌症、HIV和精神疾病治疗中的一个重要问题。Pgp是一种atp结合盒转运体，通过atp依赖机制将许多结构无关的药物泵出细胞。我们最近的Pgp X射线结构鉴定了疏水性和芳香氨基酸，它们有助于两种不同抑制剂与药物结合位点的结合。在这个提议中，我们将测试一个假设，即抗癌药物结合到蛋白质跨膜区域中定义的亚袋内的不同残基亚群。利用色氨酸（Trp）荧光猝灭，我们将绘制出纯化蛋白与占据的三个原型底物相互作用的位点

项目成果

期刊论文数量（20）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Subcellular Localization of Signal Peptide Fusion Proteins Expressed in E. coli.

大肠杆菌中表达的信号肽融合蛋白的亚细胞定位。

DOI：
10.1101/pdb.prot102145
发表时间：
2021
期刊：
Cold Spring Harbor protocols
影响因子：
0
作者：
Kielkopf,ClaraL;Bauer,William;Urbatsch,InaL
通讯作者：
Urbatsch,InaL

Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions.

通过定向进化取代十一种天然色氨酸，产生具有位点特异性、非保守取代的活性 P-糖蛋白。

DOI：
10.1038/s41598-020-59802-w
发表时间：
2020
期刊：
Scientific reports
影响因子：
4.6
作者：
Swartz,DouglasJ;Singh,Anukriti;Sok,Narong;Thomas,JoshuaN;Weber,Joachim;Urbatsch,InaL
通讯作者：
Urbatsch,InaL

Bradford Assay for Determining Protein Concentration.