PHARMACOGENETICS OF HUMAN CARBONYL REDUCTASES

描述(申请人提供)：在临床实践中广泛使用的药理剂，如抗精神病药物氟哌啶醇和抗癌的蒽环类药物阿霉素和柔红霉素中，羰基还原酶活性占相当大的比例。在人类中，有两种羰基还原酶，即羰基还原酶1(CBR1)和羰基还原酶3(CBR3)。CBR1和CBR3的遗传变异可能是作为CBR底物的药物代谢在人与人之间的广泛差异的关键。需要一种系统的方法，将鉴定常见的CBR1和CBH3基因变异与功能研究结合起来，以关键地描述CBR1和CBR3在可变CBR介导的药物生物转化中的作用。为此，我们研究了CBR1和CBR3中单核苷酸多态(SNPs)的存在。针对CBR3中编码蛋白质氨基酸序列非同义变化的两个SNPs进行了功能鉴定研究。一个SNP编码Valine244到蛋氨酸244的变化(CBR3V244M)，而另一个SNP导致半胱氨酸4到酪氨酸4的替换(CBR3C4Y)。有趣的是，CBR3等位基因变异在不同种族中很常见。非常有希望的动力学数据表明，多态的CBR3 valine244和CBR3蛋氨酸244蛋白亚型对甲萘二酮和阿霉素具有独特的催化性能。因此，特定目标1的研究将集中在多态CBR3蛋白亚型的功能特征上。在特定目的2中，将使用200对DNA-RNA肝组织样本来研究多态CBR3对肝脏CBR活性的影响。将通过测量已知CBR3基因型的样本中的酶活性和蛋白质水平等变量来分析是否存在基因型-表型相关性。CBR个体间变异的另一个因素可能是调控CBR1和CBR3近端启动子区域存在遗传多态。在特定目标3中，将筛选来自表型CBR异常值(在AIM2中精确定位)的DNA样本，以识别迄今未发现的CBR1和CBR3近端启动子区域的新多态。在确定等位基因频率之后，将通过使用基因报告分析的DNA表达分析来检验新的等位基因变异的功能后果。总之，拟议的研究将提供关于CBR1和CBR3基因变异性对CBR介导的药物代谢的影响的基本信息。了解CBR代谢药物药效学的分子基础将有助于设计更合理的药物治疗方法。

DESCRIPTION (PROVIDED BY APPLICANT): Carbonyl reductase activity accounts for a significant fraction of the metabolism of pharmacological agents extensively used in clinical practice such as the antipsychotic haloperidol and the anticancer anthracyclines doxorubicin and daunorubicin. In humans there are two carbonyl reductases, carbonyl reductase 1 (CBR1) and carbonyl reductase 3 (CBR3). It is possible that genetic variability in CBR1 and CBR3 may be key for the wide person-to-person variation in the metabolism of drugs that are CBR substrates. A systematic approach that combines the identification of common CBR1 and CBH3 genetic variants together with functional studies will be needed to critically delineate the role of CBR1 and CBR3 in variable CBR mediated drug biotransformation. Towards this goal we investigated the presence of single nucleotide polymorphisms (SNPs) in CBR1 and CBR3. Two SNPs in CBR3 encoding for non-synonymous changes in the amino acidic sequence of the protein were pinpointed to perform functional characterization studies. One SNP encodes for a valine244 to methionine244 change (CBR3 V244M), while the other SNP results in a cysteine4 to tyrosine4 substitution (CBR3 C4Y). Interestingly, the CBR3 allelic variants are common among different ethnic groups. Very promising kinetic data suggest that the polymorphic CBR3 valine244 and CBR3 methionine244 protein isoforms have distinctive catalytic properties towards menadione and doxorubicin. Thus, studies in specific Aim 1 will focus on the functional characterization of the polymorphic CBR3 protein isoforms. In specific Aim 2 the effects of polymorphic CBR3 in hepatic CBR activity will be investigated using 200 paired DNA-RNA liver tissue samples. The presence of genotype-phenotype correlations will be analyzed by measuring variables such as enzyme activities and protein levels in samples with known CBR3 genotypes. An additional contributing factor for inter-individual CBR variability may be dictated by the presence of genetic polymorphisms in the regulatory CBR1 and CBR3 proximal promoter regions. In specific Aim 3, DNA samples from phenotypic CBR outliers (pinpointed in Aim2) will be screened to identify new polymorphisms in the thus far unexplored CBR1 and CBR3 proximal promoter regions. Subsequent to the determination of allele frequencies, the functional consequences of the novel allelic variants will be examined by DNA expression analysis using gene reporter assays. Collectively, the proposed studies will provide essential information on the impact of CBR1 and CBR3 genetic variability on CBR mediated drug metabolism. The understanding of the molecular basis that govern the pharmacodynamics of CBR metabolized drugs will assist the design of more rational pharmacological therapies.