PHARMACOGENETICS OF HUMAN CARBONYL REDUCTASES

描述（由申请人提供）：羰基还原酶活性在临床实践中广泛使用的药物代谢中占有重要的一部分，如抗精神病药氟哌啶醇和抗癌蒽环类药物阿霉素和柔红霉素。人类有两种羰基还原酶，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底物的药物代谢中广泛的人与人差异的关键。需要一种系统的方法，将常见CBR1和CBR3遗传变异的鉴定与功能研究结合起来，以严格描述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中单核苷酸多态性（snp）的存在。CBR3中编码蛋白质氨基酸序列非同义变化的两个snp被确定进行功能表征研究。一个SNP编码缬氨酸244到蛋氨酸244的变化（CBR3 V244M），而另一个SNP导致半胱氨酸4到酪氨酸4的替换（CBR3 C4Y）。有趣的是，CBR3等位基因变异在不同的种族群体中很常见。非常有希望的动力学数据表明，多态性CBR3缬氨酸244和CBR3蛋氨酸244蛋白异构体对甲萘醌和阿霉素具有独特的催化性能。因此，特定Aim 1的研究将集中在多态性CBR3蛋白亚型的功能表征上。在特定的目的2中，将使用200个配对的DNA-RNA肝组织样本研究多态性CBR3对肝脏CBR活性的影响。将通过测量已知CBR3基因型样品中的酶活性和蛋白质水平等变量来分析基因型-表型相关性的存在。个体间CBR变异的另一个因素可能是CBR1和CBR3近端启动子区域遗传多态性的存在。在特定的Aim 3中，将筛选来自表型CBR异常值（在Aim2中精确定位）的DNA样本，以确定迄今未开发的CBR1和CBR3近端启动子区域的新多态性。在确定等位基因频率之后，新的等位基因变异的功能后果将通过使用基因报告基因的DNA表达分析来检验。总的来说，拟议的研究将提供CBR1和CBR3遗传变异对CBR介导的药物代谢影响的重要信息。了解控制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.