Pharmacogenetics of Human Carbonyl Reductases

描述(申请人提供)：人羰基还原酶(CBRs)催化几种在临床实践中广泛使用的药物的还原，包括抗癌的蒽环类药物、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阿霉素和柔红霉素的不可预测的药效学特征。因此，我们的主要目标是表征CBR活性可变的分子基础，作为设计更有效的抗癌治疗的先决条件。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)的功能等位基因变体；(2)记录了CBR1和CBR3在肝组织中表达的差异性；(3)确定了导致儿童癌症幸存者与蒽环类药物相关的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序列影响蛋白质的表达水平和活性，以响应各种刺激。新的数据表明，在典型抗氧化剂叔丁基对苯二酚存在下，CBR3mRNA的表达显著增加(8.5倍)，并且CBR3启动子包含两个保守的抗氧化反应元件(ARs)。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(特定目标1)。这些实验还将使我们能够确定两个常见的CBR3启动子多态(CBR3-725T&gt；C和CBR3-326T&gt；A)如何调节基因启动子的活性，以响应抗氧化剂。一个常见的CBR1多态(1096G&gt；A)决定了人类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CBR11096G&gt；A的作用是否通过特定的microRNAs与多态的3‘-非翻译区结合而介导(特定目标2)。越来越多的实验证据，加上我们的药物遗传学发现，表明CBR1和CBR3在心脏中蒽环类药物的复杂药效中发挥着至关重要的作用。CBR1和CBR3在人心脏中的表达尚未确定。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(特定目标3)。在这一综合方法中，我们将使用实时定量聚合酶链式反应分析、纳米液相色谱与三重四重质谱联用，以及使用CBR底物(例如阿霉素)和抑制剂(例如心脏保护性黄酮单羟乙基芦丁苷，或Mono-HER)进行酶活性分析。我们还将进行基因-表型相关性研究，以确定功能性CBR1和CBR3多态是否会影响心脏中对心脏有害的蒽环类代谢物的形成。从拟议的研究中收集的知识体系将有助于抗癌治疗的发展，通过识别不同CBR活性的遗传决定因素，可以使抗癌治疗个体化。

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.