Pharmacogenetics of Human Carbonyl Reductases

描述（由申请人提供）：人羰基还原酶（CBRs）催化几种广泛应用于临床实践的药物的还原，包括抗癌蒽环类药物阿霉素和柔红霉素。这两种药物的药效学是不可预测的。我们假设CBR活性的个体间差异导致了阿霉素和柔红霉素不可预测的药效学特征。因此，我们的主要目标是表征可变CBR活性的分子基础，作为设计更有效的抗癌疗法的先决条件。到目前为止，我们已经(1)鉴定了羰基还原酶1 （CBR1）和羰基还原酶3 （CBR3）的功能等位变异；(2)记录了CBR1和CBR3在肝组织中的表达变异性；(3)确定了儿童癌症幸存者蒽环类药物相关心脏毒性的遗传危险因素（CBR3 V244M）。基因调控研究表明，CBR1和CBR3启动子区域的特定DNA序列会影响响应各种刺激的蛋白质表达水平和活性。新的数据表明，CBR3 mRNA的表达在典型抗氧化剂叔丁基对苯二酚的存在下显著增加（8.5倍），并且CBR3启动子含有2个保守的抗氧化反应元件（AREs）。我们设计的实验将使我们能够表征AREs在抗氧化剂暴露诱导CBR3表达中的功能作用（Specific Aim 1）。这些实验还将使我们能够确定两种常见的CBR3启动子多态性（CBR3 -725T>C和CBR3 -326T>A）如何调节基因启动子活性以响应抗氧化剂。一种常见的CBR1多态性（1096G>A）决定了人肝组织中心脏毒性阿霉素的合成。我们计划进行实验，以确定CBR1 1096G>A的作用是否通过特异性microrna与多态3'-非翻译区结合而介导（specific Aim 2）。越来越多的实验证据和我们的药理学研究结果表明，CBR1和CBR3在蒽环类药物在心脏中的复杂药效学中起着至关重要的作用。CBR1和CBR3在人类心脏中的表达尚未被表征。我们计划在200个人类心肌组织样本中记录CBR1和CBR3对阿霉素和柔红霉素代谢的相对贡献（Specific Aim 3）。在这种全面的方法中，我们将使用定量实时PCR分析，纳米液相色谱耦合三重三重质谱，以及使用CBR底物（例如，阿霉素）和抑制剂（例如，心脏保护类黄酮单羟乙基芦桃苷，或mono-HER）进行酶活性测定。我们还将进行基因型-表型相关性研究，以确定CBR1和CBR3功能性多态性是否影响心脏毒性蒽环类药物代谢物的形成。从拟议的研究中收集的知识将有助于通过确定可变CBR活性的遗传决定因素来个体化抗癌治疗的发展。

DESCRIPTION (provided by applicant): Human carbonyl reductases (CBRs) catalyze the reduction of several drugs widely used in clinical practice including the anticancer anthracyclines doxorubicin and daunorubicin. The pharmacodynamics of these 2 drugs is unpredictable. We hypothesize that interindividual variability in CBR activity contributes to the unpredictable pharmacodynamic profiles for doxorubicin and daunorubicin. Therefore, our main goal is to characterize the molecular basis of variable CBR activity as a prerequisite for the design of more effective anticancer therapies. Thus far, we have (1) characterized functional allelic variants of carbonyl reductase 1 (CBR1) and carbonyl reductase 3 (CBR3); (2) documented the variability in CBR1 and CBR3 expression in hepatic tissue; and (3) identified a genetic risk factor (CBR3 V244M) for anthracycline-related cardiotoxicity in pediatric cancer survivors. Gene regulation studies indicate that specific DNA sequences in the promoter regions of CBR1 and CBR3 influence the level of protein expression and activity in response to various stimuli. New data indicate that CBR3 mRNA expression increases considerably (8.5-fold) in the presence of the prototypical antioxidant tert-butylhydroquinone and that the CBR3 promoter contains 2 conserved antioxidant response elements (AREs). We have designed experiments that will allow us to characterize the functional role of AREs in the induction of CBR3 expression in response to antioxidant exposure (Specific Aim 1). These experiments will also allow us to determine how 2 common CBR3 promoter polymorphisms (CBR3 -725T>C, and CBR3 -326T>A) modulate gene promoter activity in response to antioxidants. A common CBR1 polymorphism (1096G>A) dictates the synthesis of cardiotoxic doxorubicinol in human hepatic tissue. We have planned experiments to determine whether the effect of CBR1 1096G>A is mediated through the binding of specific microRNAs to the polymorphic 3'-untranslated region (Specific Aim 2). A growing amount of experimental evidence, together with our pharmacogenetic findings, suggests that CBR1 and CBR3 have a crucial role in the complex pharmacodynamics of anthracyclines in the heart. The expression of CBR1 and CBR3 in the human heart has not been characterized. We plan to document the relative contributions of CBR1 and CBR3 to the metabolism of doxorubicin and daunorubicin in 200 samples of human myocardial tissue (Specific Aim 3). In this comprehensive approach we will use quantitative real-time PCR analysis, nano-liquid chromatography coupled to triple quadruple mass spectroscopy, and enzyme activity assays with CBR substrates (e.g., doxorubicin) and inhibitors (e.g., the cardioprotective flavonoid monohydroxyethyl rutoside, or mono-HER). We will also conduct genotype-phenotype correlation studies to determine whether functional CBR1 and CBR3 polymorphisms affect the formation of cardiotoxic anthracycline metabolites in the heart. The body of knowledge gathered from the proposed research will contribute to the development of anticancer therapy that can be individualized by identifying the genetic determinants of variable CBR activity.