Gonzalez-Covarrubias Vanessa, Ghosh Debashis, Lakhman Sukhwinder S, Pendyala Lakshmi, Blanco Javier G
Department of Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, NY 14260-1200, USA.
Drug Metab Dispos. 2007 Jun;35(6):973-80. doi: 10.1124/dmd.107.014779. Epub 2007 Mar 7.
Human carbonyl reductase 1 (CBR1) metabolizes endogenous and xenobiotic substrates such as the fever mediator, prostaglandin E2 (PGE2), and the anticancer anthracycline drug, daunorubicin. We screened 33 CBR1 full-length cDNA samples from white and black liver donors and performed database analyses to identify genetic determinants of CBR1 activity. We pinpointed a single nucleotide polymorphism on CBR1 (CBR1 V88I) that encodes for a valine-to-isoleucine substitution for further characterization. We detected the CBR1 V88I polymorphism in DNA samples from individuals with African ancestry (p = 0.986, q = 0.014). Kinetic studies revealed that the CBR1 V88 and CBR1 I88 isoforms have different maximal velocities for daunorubicin (V(max) CBR1 V88, 181 +/- 13 versus V(max) CBR1 I88, 121 +/- 12 nmol/min . mg, p < 0.05) and PGE2 (V(max) CBR1 V88, 53 +/- 7 versus V(max) CBR1 I88, 35 +/- 4 nmol/min . mg, p < 0.01). Concomitantly, CBR1 V88 produced higher levels of the cardiotoxic metabolite daunorubicinol compared with CBR1 I88 (1.7-fold, p < 0.0001). Inhibition studies demonstrated that CBR1 V88 and CBR1 I88 are distinctively inhibited by the flavonoid, rutin (IC50 CBR1 V88, 54.0 +/- 0.4 microM versus IC50 CBR1 I88, 15.0 +/- 0.1 microM, p < 0.001). Furthermore, isothermal titration calorimetry analyses together with molecular modeling studies showed that CBR1 V88I results in CBR1 isoforms with different binding affinities for the cofactor NADPH (K(d) CBR1 V88, 6.3 +/- 0.6 microM versus K(d) CBR1 I88, 3.8 +/- 0.5 microM). These studies characterize the first functional genetic determinant of CBR1 activity toward relevant physiological and pharmacological substrates.
人类羰基还原酶1（CBR1）可代谢内源性和外源性底物，如发热介质前列腺素E2（PGE2）以及抗癌蒽环类药物柔红霉素。我们从白种人和黑种人肝脏供体中筛选了33个CBR1全长cDNA样本，并进行数据库分析以确定CBR1活性的遗传决定因素。我们在CBR1上确定了一个单核苷酸多态性（CBR1 V88I），该多态性编码缬氨酸到异亮氨酸的替换，以便进一步表征。我们在非洲裔个体的DNA样本中检测到了CBR1 V88I多态性（p = 0.986，q = 0.014）。动力学研究表明，CBR1 V88和CBR1 I88同工型对柔红霉素具有不同的最大速度（V(max) CBR1 V88为181±13，而V(max) CBR1 I88为121±12 nmol/min·mg，p < 0.05）和PGE2（V(max) CBR1 V88为53±7，而V(max) CBR1 I88为35±4 nmol/min·mg，p < 0.01）。同时，与CBR1 I88相比，CBR1 V88产生的心脏毒性代谢产物柔红霉醇水平更高（1.7倍，p < 0.0001）。抑制研究表明，黄酮类化合物芦丁对CBR1 V88和CBR1 I88有明显不同的抑制作用（IC50 CBR1 V88为54.0±0.4 microM，而IC50 CBR1 I88为15.0±0.1 microM，p < 0.001）。此外，等温滴定量热法分析以及分子模拟研究表明，CBR1 V88I导致CBR1同工型对辅因子NADPH具有不同的结合亲和力（K(d) CBR1 V88为6.3±0.6 microM，而K(d) CBR1 I88为3.8±0.5 microM）。这些研究表征了CBR1对相关生理和药理底物活性的首个功能性遗传决定因素。
