Studies of P-glycoprotein drug interactions

项目摘要/摘要 项目名称：P-糖蛋白药物相互作用研究 P-糖蛋白(Pgp)是一种分子泵，通过转运数百种结构上的 无关的毒素排出细胞。PGP限制了肠道的摄取，并促进了药物的排泄 肝、肾和血脑屏障，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。它是七个最重要的交通工具之一 负责管理现在需要药物文件的药物吸收和处置 美国食品和药物管理局(FDA)批准任何新药的互动。PGP是一种 具有两个跨膜区和两个核苷酸结合的ATP结合盒转运体 域(NBD)。它利用三磷酸腺苷的水解作用将底物泵过细胞膜。我们最近的X- PGP的射线结构确定了有助于结合PGP的疏水和芳香氨基酸 不同的抑制剂对药物结合部位的影响。在这个提案中，我们将检验这样一种假设，即治疗 药物与蛋白质TMD中不同亚组的残基结合。我们的 一般的方法是在战略位置引入色氨酸(Trp)，以监测药物 有约束力的。TRP将在新的全功能无TRP PGP或低TRP PGP的背景下推出。 Trp PGP在细胞质结构域中保留三个天然的构象敏感的TRPs。vbl.使用 荧光的变化，如猝灭，以及共振能量转移(FRET)，我们将绘制出来 纯化蛋白与生物化学定义的原型底物相互作用的位置 和不同的结合部位，以及常见治疗药物和新发现的抑制剂的结合部位。 我们将进一步使用琥珀终止密码子将荧光Trp类似物(L-ANAP)插入到野生型PGP中 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这个多结构域的协作性和构象动力学 我们将对药物结合的机制及其对Pgp的影响有独特的见解。 功能。通过这些新的方法，我们将解决分子机制和动力学 药物/抑制剂结合，确定协同效应，细化药物-药物作用机制 PGP中的相互作用。这些信息将为精制PGP药物的新分析方法铺平道路 新旧药物的相互作用研究，对临床重新设计药物具有重要的价值 良好的药代动力学，加速药物治疗的发展。

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.