Developmental Pharmacology of cytosolic and mitochondrial GSTZ1-1/MAAI

描述（由申请人提供）：本研究的广泛，长期目标是了解谷胱甘肽转移酶Z1-1 （GSTZ1-1）的性质及其在人类生命周期中消除卤代乙酸，特别是二氯乙酸（DCA）中的作用。这一点很重要，因为尽管DCA在治疗某些实体瘤、乳酸性酸中毒和肺动脉高压方面具有治疗效果，但它在药代动力学方面表现出明显的个体差异，并且在高剂量时可能引起副作用，特别是在成人中。DCA代谢的第一步是由GSTZ1-1催化脱氯生成乙醛酸盐。GSTZ1-1也被称为马来酰乙酰乙酸异构酶，在酪氨酸的分解代谢中起重要作用，主要在肝脏中表达，是唯一已知的催化包括DCA在内的卤化乙酸脱卤的酶。虽然DCA抑制自身代谢以及马来酰乙酸酯及其脱羧产物马来酰丙酮的代谢，几乎可以肯定是因为DCA灭活GSTZ1-1，但DCA重复给药后药代动力学个体差异显著的原因才刚刚开始被理解。大多数成年人反复服用DCA的清除速度比大多数儿童慢。具有不同GSTZ1-1单倍型的人对重复剂量的DCA的反应似乎不同。最近的体外研究表明，氯浓度以单倍型依赖的方式影响DCA对GSTZ1-1的失活速率。除了众所周知的存在于肝细胞质中，GSTZ1-1最近被发现存在于线粒体基质中。这与DCA的药效学作用位点丙酮酸脱氢酶激酶相同。DCA代谢物glyoxylate的大部分下游代谢发生在线粒体中，这表明该部位在DCA的整体处置中很重要。该应用程序旨在检查GSTZ1-1在整个人类寿命中的表达和活性的年龄相关变化，并确定新发现的线粒体酶的特性。提出了三个具体目标。第一个具体目标是研究肝脏GSTZ1-1在线粒体和细胞质中表达和活性的年龄相关变化。第二个特定目标将检查线粒体酶的特性，在体内暴露于DCA后，线粒体相对于细胞质GSTZ1-1的相对损失，以及线粒体GSTZ1-1/MAAI将GSTZ1-1生理上重要的底物，马来酰丙酮转化为马来酰丙酮的能力。第三个具体目标是利用体外研究来研究线粒体GSTZ1-1对DCA失活的敏感性，以及氯离子和单倍型作为胞质、线粒体和表达GSTZ1-1被DCA失活的速率和机制的决定因素。

DESCRIPTION (provided by applicant): The broad, long-term objectives of this research are to understand the properties of glutathione transferase Z1- 1 (GSTZ1-1) and its role in the elimination of haloacetic acids, particularly dichloroacetic acid (DCA) across the human lifespan. This is important because while DCA has therapeutic benefits in treating certain solid tumors, lactic acidosis and pulmonary arterial hypertension, it exhibits marked individual variability in pharmacokinetics, and can cause side effects at high doses, particularly in adults. The first step in DCA metabolism is dechlorination to glyoxylate, catalyzed by GSTZ1-1. Also known as maleylacetoacetate isomerase, and playing an important role in catabolism of tyrosine, GSTZ1-1 is expressed mainly in the liver and is the only enzyme known to catalyze the dehalogenation of haloacetic acids, including DCA. Although it is well recognized that DCA inhibits its own metabolism and the metabolism of maleylacetoacetate and its decarboxylation product, maleylacetone, almost certainly because DCA inactivates GSTZ1-1, the reasons for the marked individual variability in pharmacokinetics of DCA after repeated doses are only beginning to be understood. Most adults clear repeated doses of DCA more slowly than most children. Persons with different GSTZ1-1 haplotypes appear to differ in their response to repeated doses of DCA. Recent in vitro work showed that chloride concentration affected the rate of inactivation of GSTZ1-1 by DCA in a haplotype-dependent manner. In addition to its well-known presence in liver cytosol, GSTZ1-1 has recently been found to exist in the mitochondrial matrix. This is the same location as pyruvate dehydrogenase kinase, DCA's pharmacodynamics site of action. Most of the downstream metabolism of the DCA metabolite, glyoxylate, occurs in the mitochondria suggesting the importance of this site in the overall disposition of DCA. This application seeks to examine age-related changes in expression and activity of GSTZ1-1 across the human lifespan and ascertain the properties of the newly-discovered mitochondrial enzyme. Three specific aims are proposed. The first specific aim will investigate age-related changes in hepatic GSTZ1-1 expression and activity in mitochondria and cytosol. The second specific aim will examine the properties of the mitochondrial enzyme, with respect to the relative loss of mitochondrial versus cytosolic GSTZ1-1 following in vivo exposure to DCA, and the ability of the mitochondrial GSTZ1-1/MAAI to convert the physiologically important substrate of GSTZ1-1, maleylacetone, to fumarylacetone. The third specific aim will use in vitro studies to investigate sensitivity of mitochondrial GSTZ1-1 to inactivation by DCA and the role of chloride and haplotype as determinants of the rate and mechanism of inactivation of cytosolic, mitochondrial and expressed GSTZ1-1 by DCA.