Molecular Mechanisms of Drug Elimination

肾脏消除阴离子药物、外源药物和毒素对哺乳动物物种的生存是必要的。这一过程是通过近端小管上皮基底外侧和根尖膜特定转运蛋白的协同作用，从血液到尿液的载体运输介导的。这个过程的第一步是从小管周围血浆中提取有机阴离子，部分通过有机阴离子转运体（OAT）途径进入近端小管细胞。因此，OAT途径是机体药物清除/解毒的主要位点之一，但也是药物-药物相互作用和药物致肾毒性的位点。为了最大限度地提高治疗效果和减少毒性，必须确定转运蛋白的结构-功能关系，包括药物/抑制剂结合位点，以及转运机制的调节。本应用程序的总体目标是通过OAT途径确定药物消除的分子机制。我们最近从小鼠肾脏中分离出一个编码OAT家族第一个成员的cDNA。我们已经证明OAT功能通过两种不同的机制下调：a) PKC的激活（它不会使OAT磷酸化），b)一种未知的激酶使OAT的丝氨酸磷酸化。因此提出了两个具体目标（SA）。在SA-1中，我们将通过结合生化、生物物理和基因工程方法来确定OAT的功能决定因素。在SA-2中，我们将确定OAT功能的短期调节机制。我们将首先通过质谱和定点诱变的结合确定丝氨酸磷酸化位点和磷酸化OAT的激酶。然后，我们将探讨激酶/磷酸化影响OAT功能的机制。从这些研究中获得的知识对于合理设计新药和抑制剂以优化药物治疗同时避免不必要的药物相互作用将是无价的。这些研究也应该提供适用于整个oat样转运蛋白家族的重要的基础机制信息。最终，这项工作将允许深入了解正常肾脏和各种获得性和遗传性疾病中有机阴离子运输的机制和调节。

Renal elimination of anionic drugs, xenobiotics and toxins are necessary for the survival of mammalian species. This process is mediated by vectorial transport from blood into the urine through the cooperative function of specific transporters in the basolateral and apical membranes of the proximal tubule epithelium. The first step of this process is the extraction of organic anions from the peritubular blood plasma into proximal tubule cells through, in part, the organic anion transporter (OAT) pathway. As a result, OAT pathway is one of the major sites for body drug clearance/detoxification but it is also the site for drug-drug interaction and drug-induced nephrotoxicity. To maximize therapeutic efficacy and minimize toxicity, the transporter structure-function relationships, including drug/inhibitor binding sites, and the regulation of transport mechanisms must be defined. The overall objective of this application is to define the molecular mechanisms underlying drug elimination through the OAT pathway. We have recently isolated a cDNA from mouse kidney which encodes the first member of the OAT family. We have shown that OAT function is down-regulated by two distinct mechanisms: a) activation of PKC (which does not phosphorylate OAT), and b) serine-phosphorylation of OAT by an unknown kinase. Two specific aims (SA) are therefore proposed. In SA-1, we will identify the functional determinants of OAT through a combination of biochemical, biophysical and genetic engineering approaches. In SA-2, we will identify the short-term regulatory mechanisms of OAT function. We will first identify the site(s) of serine phosphorylation and the kinases which phosphorylate OAT through a combination of mass spectra and site-directed mutagenesis. We will then explore the mechanisms by which kinases/phosphorylationj influence OAT function. The knowledge gained from these studies will be invaluable toward the rational design of novel drugs and inhibitors to optimize drug therapy while avoiding unwanted drug interactions. These studies should also provide fundamental important mechanistic information applicable to the whole family of OAT-like transporters. Ultimately, this work should permit insight into the mechanism and regulation of organic anion transport in normal kidney and in various acquired and inheritable disorders.