PHARMACOGENETICS OF HUMAN CARBONYL REDUCTASES

性状（申请人提供）：羰基还原酶活性在临床实践中广泛使用的药物（如抗精神病药氟哌啶醇和抗癌蒽环类药物多柔比星和柔红霉素）的代谢中占很大比例。在人类中有两种羰基还原酶，羰基还原酶1（CBR 1）和羰基还原酶3（CBR 3）。CBR 1和CBR 3的遗传变异可能是CBR底物药物代谢中广泛的人与人之间差异的关键。将需要一种系统的方法，结合常见的CBR 1和CBH 3遗传变异的鉴定与功能研究，以严格描述CBR 1和CBR 3在可变CBR介导的药物生物转化中的作用。为此，我们研究了CBR 1和CBR 3中单核苷酸多态性（SNP）的存在。CBR 3中编码蛋白质氨基酸序列中非同义变化的两个SNP被精确定位以进行功能表征研究。一个SNP编码缬氨酸244到甲硫氨酸244的变化（CBR 3 V244 M），而另一个SNP导致半胱氨酸4到酪氨酸4的取代（CBR 3 C4 Y）。有趣的是，CBR 3等位基因变体在不同种族群体中很常见。非常有希望的动力学数据表明，多晶型CBR 3缬氨酸244和CBR 3蛋氨酸244蛋白异构体对甲萘醌和多柔比星具有独特的催化特性。因此，特定目标1的研究将集中于多态性CBR 3蛋白同种型的功能表征。在具体目标2中，将使用200个配对的DNA-RNA肝组织样本研究多态性CBR 3对肝CBR活性的影响。将通过测量已知CBR 3基因型样本中的酶活性和蛋白质水平等变量，分析基因型-表型相关性的存在。个体间CBR变异性的另一个影响因素可能是调节性CBR 1和CBR 3近端启动子区存在遗传多态性。在特定目标3中，将筛选来自表型CBR离群值（在Aim 2中精确定位）的DNA样本，以鉴定迄今为止未探索的CBR 1和CBR 3近端启动子区域中的新多态性。在确定等位基因频率之后，将使用基因报告基因测定通过DNA表达分析来检查新等位基因变体的功能后果。总体而言，拟定研究将提供关于CBR 1和CBR 3遗传变异性对CBR介导的药物代谢影响的基本信息。对CBR代谢药物药效学的分子基础的理解将有助于设计更合理的药理学治疗。

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.