Developmental Pharmacology of Mitochondrial and Cytosolic GSTZ1

项目摘要。 这项研究的广泛，长期的目标是了解谷胱甘肽转移酶Z1的性质 （GSTZ 1）及其在人体内消除研究药物二氯乙酸（DCA）的作用 寿命这一点很重要，因为虽然DCA是线粒体丙酮酸脱氢酶激酶的抑制剂， 作为代谢调节剂，在治疗乳酸性酸中毒、某些实体瘤和肺肿瘤中具有治疗益处， 由于动脉高血压，它的药代动力学表现出明显的年龄和遗传相关的个体差异 与DCA的副作用有关DCA代谢的第一步是脱氯生成乙醛酸， 由GSTZ 1催化。GSTZ 1也被称为马来酰乙酰乙酸异构酶，其在哺乳动物中起着重要的生理作用。 在酪氨酸催化途径的内源性活性代谢物异构化中的作用，马来酰乙酰乙酸 和马来酰丙酮。GSTZ 1主要在肝脏中表达，但也在肾脏、肠、脑和心脏中表达。是 已知催化DCA脱卤成其主要代谢物乙醛酸的唯一酶。虽然 众所周知，DCA抑制其自身代谢以及其内源性底物的代谢， GSTZ 1的失活，DCA的药代动力学的显著个体差异的原因， 重复剂量只是部分理解。大多数成年人清除重复剂量的DCA比 在儿童和成人中，清除率依赖于GSTZ 1单倍型。初步证据 DCA代谢的肝外位点相对于DCA后的肝脏变得更重要 治疗，这是年龄依赖性的。最后，氯离子（Cl-）浓度影响灭活率， GSTZ 1通过DCA以单倍型依赖的方式;与细胞质相比，线粒体中的[Cl-]低得多 被认为是线粒体基质酶更快失活的一个因素， 在观察到的年龄依赖性差异的因素，细胞内[Cl-]随年龄而变化。的机理 Cl-保护GSTZ 1免于DCA依赖性失活是未知的。 本申请试图研究与年龄和遗传相关的表达和活性变化的原因， GSTZ 1在人体内提出了三个具体目标。第一个具体目标是研究microRNA的作用， (miR)调节肝脏GSTZ 1表达的年龄相关变化。这一目标将使用 不同年龄供体的人类肝脏库以识别miR，以及基于细胞的系统以验证参与。 第二个具体的目的是研究GSTZ 1在细胞中保护免于DCA失活的机制。 Cl-和某些其他阴离子的存在，以及单倍型的影响。表达的重组GSTZ 1A和 1B的结晶和DCA和阴离子的结合位点进行了研究。第三个具体目标将使用青少年 和成年大鼠作为儿童和成人的模型，以检查肝外组织相对于肝脏的作用， 多次DCA给药后GSTZ 1的表达和活性，以模拟临床应用。

Project Summary. The broad, long-term objectives of this research are to understand the properties of glutathione transferase Z1 (GSTZ1) and its role in the elimination of the investigational drug dichloroacetic acid (DCA) across the human lifespan. This is important because while DCA, an inhibitor of mitochondrial pyruvate dehydrogenase kinase, has therapeutic benefits as a metabolic regulator in treating lactic acidosis, certain solid tumors and pulmonary arterial hypertension, it exhibits marked age- and genetics- related individual variability in pharmacokinetics that are linked to DCA's side effects. The first step in DCA metabolism is dechlorination to glyoxylate, catalyzed by GSTZ1. Also known as maleylacetoacetate isomerase, GSTZ1 plays an important physiological role in isomerizing endogenous reactive metabolites of the tyrosine catabolism pathway, maleylacetoacetate and maleylacetone. GSTZ1 is expressed chiefly in the liver, but also in kidney, intestine, brain and heart. It is the only enzyme known to catalyze the dehalogenation of DCA to its primary metabolite, glyoxylate. Although it is well recognized that DCA inhibits its own metabolism as well as that of its endogenous substrates, through inactivation of GSTZ1, the reasons for the marked individual variability in pharmacokinetics of DCA after repeated doses are only partially understood. Most adults clear repeated doses of DCA more slowly than children, and clearance is GSTZ1 haplotype dependent in children and adults. There is preliminary evidence that extrahepatic sites of DCA metabolism become of greater importance relative to liver following DCA treatment, and that this is age-dependent. Finally, chloride (Cl-) concentration affects the rate of inactivation of GSTZ1 by DCA in a haplotype-dependent manner; the much lower [Cl-] in mitochondria compared with cytosol is thought to be a factor in the more rapid inactivation of the mitochondrial matrix enzyme, and could be a factor in the observed age-dependent differences, as intracellular [Cl-] varies with age. The mechanism of how Cl- protects GSTZ1 from DCA-dependent inactivation is not known. This application seeks to examine reasons for age- and genetics- related changes in expression and activity of GSTZ1 in people. Three specific aims are proposed. The first specific aim will study the role of microRNA (miR) in regulating the documented age-related changes in hepatic GSTZ1 expression. This aim will use banked human liver of different aged donors to identify miRs, and cell-based systems to verify involvement. The second specific aim will investigate the mechanism of protection of GSTZ1 from inactivation by DCA in the presence of Cl- and certain other anions, and the influence of haplotype. Expressed recombinant GSTZ1A and 1B will be crystallized and the binding sites of DCA and anions studied. The third specific aim will use juvenile and adult rats as models of children and adults to examine the roles of extrahepatic tissues relative to liver in the expression and activity of GSTZ1 following multiple DCA doses, to mimic the clinical use.