Studies of P-glycoprotein Drug Interactions - Administrative Supplement for Equipment Purchase

P-糖蛋白药物相互作用研究-设备采购行政补充

基本信息

批准号：
10795338
负责人：
INA L URBATSCH
金额：
$ 6.75万
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/10795338
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 equipment acquisition 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 (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（原始资助的项目） P-糖蛋白（PGP）是一种分子泵，通过运输数百种结构无关的细胞排毒细胞。毒素从细胞中出来。 PGP限制了肠中的摄取，并增强了肝脏，肾脏和血脑屏障，用于治疗癌症，艾滋病毒，精神病和治疗的许多药物心血管疾病。它是负责调节药物的七个最重要的转运蛋白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是一个ATP结合盒式转运蛋白，有两个跨膜结构域（TMD）和两个核苷酸结合结构域（NBD）。它使用ATP水解泵送整个细胞膜的底物。我们最近的PGP X射线结构鉴定了疏水和芳香族有助于不同抑制剂与药物结合部位结合的氨基酸。在此建议中，我们将测试治疗药物与TMDS中定义的子口腔中的不同子集结合的假设蛋白质。我们的一般方法是在战略位置引入色氨酸（TRP），以监视药物结合。 TRP将在新的全功能无效PGP或低trp的背景下引入 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），我们将绘制出相互作用的位点纯化的蛋白质具有典型的底物，占据生化定义和不同结合位点的蛋白质作为常见治疗药物和新鉴定的抑制剂的药物。我们将进一步插入荧光TRP 使用Amber终止密码子抑制策略来探索监测药物在生物细胞膜中结合。通过确定药物和抑制剂如何调节合作性和该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/抑制剂结合的动力学，确定协同作用并完善药物 - 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药物相互作用对新旧药物的研究，对于用临床上有利的药代动力学的重新设计药物将是无价的并加速了药物治疗发展。