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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）：由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结合盒转运蛋白，通过ATP依赖性机制将许多结构无关的药物泵出细胞。我们最近的X-射线结构的Pgp确定的疏水性和芳香族氨基酸，有助于结合两种不同的抑制剂的药物结合位点。在这个提议中，我们将测试抗癌药物结合到蛋白质跨膜区域中定义的亚口袋内的不同残基子集的假设。使用色氨酸（Trp）荧光猝灭，我们将绘制出纯化蛋白与三种原型底物相互作用的位点，生物化学定义和独特的结合位点，以及常见的抗癌药物和新发现的抑制剂。该提案的新奇在于我们开发了功能性无Trp Pgp，并在战略位置引入一个或多个Trp以监测药物结合。通过这种新的方法，我们将解决药物/抑制剂结合的分子机制和动力学，并确定最近发现的阻滞剂的作用机制。我们计划获得直接的信息，蛋白质亚口袋与抗癌药物相互作用，以及不同的阻断剂如何起作用。后者将是非常宝贵的开发机制和结构为基础的面板的潜在阻滞剂的高通量筛选。公共卫生相关性：P-糖蛋白（Pgp）是细胞的清洁机器，将有害物质泵送到细胞外。在癌症化疗中，Pgp可能会通过将化疗药物从它们打算杀死的肿瘤细胞中移除而引起问题。通过更多地了解Pgp如何识别它携带出细胞的化学物质，科学家们可能会设计出新的药物来防止Pgp干扰抗癌药物的有效作用。

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.