Narimatsu S, Kato R, Horie T, Ono S, Tsutsui M, Yabusaki Y, Ohmori S, Kitada M, Ichioka T, Shimada N, Kato R, Ishikawa T
Department of Health Chemistry, Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Chirality. 1999;11(1):1-9. doi: 10.1002/(SICI)1520-636X(1999)11:1<1::AID-CHIR1>3.0.CO;2-E.
The enantioselectivity of 4-hydroxylation of bunitrolol (BTL), a beta-adrenoceptor blocking drug, was studied in microsomes from human liver, human hepatoma (Hep G2) cells expressing CYP2D6, and lymphoblastoid cells expressing CYP2D6. Kinetics in human liver microsomes showed that the Vmax value for (+)-BTL was 2.1-fold that of (-)-BTL, and that the Km value for (+)-BTL was lower than that for the (-)-antipode, resulting in the intrinsic clearance (Vmax/Km) of (+)-BTL being 2.1-fold over its (-)-antipode. CYP2D6 (CYP2D6-met) expressed in Hep G2 cells had a methionine residue at position 373 of the amino acid sequence and a rat-type N-terminal peptide (MELLNGTGLWSM) instead of the human-type (MGLEALVPLAVIV), and showed enantioselectivity of [(+)-BTL < (-)-BTL] for the rate of BTL 4-hydroxylation. In contrast, enantioselectivity [(+)-BTL > (-)-BTL] for Hep G2-CYP2D6 (CYP2D6-val) with a human-type N-terminal peptide that had a valine residue at 374, which corresponds to the methionine of the CYP2D6-met variant, was the same as that for human liver microsomes. We further confirmed that CYP2D6-met and CYP2D6-val expressed in human lymphoblastoid cells, both of which have methionine and valine, respectively, at position 374 and a human-type N-terminal peptide, exhibited the same enantioselectivities as those obtained from CYP2D6-met and CYP2D6-val expressed in the Hep G2 cell system. These results indicate that the amino acid at 374 of CYP2D6 is one of the key factors influencing the enantioselectivity of BTL 4-hydroxylation.
对β-肾上腺素受体阻断药布尼洛尔（BTL）4-羟基化反应的对映体选择性进行了研究，研究对象为人肝微粒体、表达CYP2D6的人肝癌（Hep G2）细胞以及表达CYP2D6的淋巴母细胞。人肝微粒体中的动力学研究表明，(+)-BTL的Vmax值是(-)-BTL的2.1倍，且(+)-BTL的Km值低于其对映体(-)-BTL，这使得(+)-BTL的固有清除率（Vmax/Km）是其(-)-对映体的2.1倍。在Hep G2细胞中表达的CYP2D6（CYP2D6-met）在氨基酸序列的373位有一个甲硫氨酸残基，且其N端肽为大鼠型（MELLNGTGLWSM）而非人型（MGLEALVPLAVIV），该酶对BTL 4-羟基化反应速率表现出[(+)-BTL < (-)-BTL]的对映体选择性。相反，在374位有缬氨酸残基（对应于CYP2D6-met变体的甲硫氨酸）且N端肽为人型的Hep G2-CYP2D6（CYP2D6-val），其对映体选择性[(+)-BTL > (-)-BTL]与人肝微粒体相同。我们进一步证实，在人淋巴母细胞中表达的CYP2D6-met和CYP2D6-val，二者在374位分别有甲硫氨酸和缬氨酸且N端肽为人型，它们表现出与在Hep G2细胞系统中表达的CYP2D6-met和CYP2D6-val相同的对映体选择性。这些结果表明，CYP2D6的374位氨基酸是影响BTL 4-羟基化反应对映体选择性的关键因素之一。
