Molecular Mechanisms of Drug Elimination

肾脏清除阴离子药物、异物和毒素对于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膜、侧膜和顶膜上特定转运体的协同作用，由血液向尿液的载体转运所介导的。这一过程的第一步是部分通过有机阴离子转运体(OAT)途径将有机阴离子从肾小管周围血浆中提取到近端小管细胞中。因此，OAT途径是体内药物清除/解毒的主要部位之一，但也是药物相互作用和药物所致肾毒性的部位。为了使治疗效果最大化和毒性最小化，必须确定转运体的结构-功能关系，包括药物/抑制剂结合部位，以及对转运体机制的调节。这项应用的总体目标是确定通过燕麦酶途径消除药物的分子机制。我们最近从小鼠肾脏中分离到一个编码燕麦家族第一个成员的基因。我们已经证明OAT功能被两种不同的机制下调：a)激活PKC(不使OAT磷酸化)，b)未知的激酶使OAT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的方法来确定燕麦的功能决定因素。在SA-2中，我们将确定燕麦功能的短期调节机制。我们将首先通过质谱学和定点突变相结合的方法确定丝氨酸磷酸化的位点(S)和使燕麦蛋白磷酸化的激酶。然后，我们将探索激酶/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非常宝贵的。这些研究还应提供适用于燕麦类运输器整个家族的基本、重要的机械学信息。最终，这项工作应该能够深入了解正常肾脏和各种后天和遗传性疾病中有机阴离子转运的机制和调节。

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.