Pharmacogenomics of Gemcitabine Metabolism: Functional Analysis of Genetic Variants in Cytidine Deaminase and Deoxycytidine Kinase
Pharmacogenomics of Gemcitabine Metabolism: Functional Analysis of Genetic Variants in Cytidine Deaminase and Deoxycytidine Kinase
复制标题
DOI：
10.1124/dmd.112.048769
复制
发表时间：
2013-03-01
影响因子：
3.9
通讯作者：
Guo, Yingying
中科院分区：
文献类型：
--
作者：
Baker, Jessica A. Roseberry;Wickremsinhe, Enaksha R.;Guo, Yingying
吉西他滨（dFdC，2 '，2'-二氟脱氧胞苷）通过胞苷脱氨酶（CDA）和脱氧胞苷激酶（DCK）代谢，但这些酶的遗传变异对在癌症患者中观察到的全身暴露和应答变异性的贡献尚不清楚。在大肠杆菌中表达并纯化CDA（Lys 27 Gln和Ala 70 Thr）和DCK（Ile 24Val、Ala 119 Gly和Pro 122 Ser）的野生型酶和变体，并估计阿糖胞苷（Ara-C）、dFdC及其代谢物2 '，2'-二氟脱氧尿苷（dFdU）作为底物的酶动力学参数。所有三种CDA蛋白对于Ara-C和dFdC脱氨基作用显示出相似的K-m和V-max，除了CDA 70 Thr，其对于Ara-C脱氨基作用具有低2.5倍的K-m和低6倍的V-max。所有四种DCK蛋白质对Ara-C和dFdC单磷酸化产生相当的代谢活性，除了DCK 24 Val，其证明由于K-m降低40%（P < 0.05），dFdC单磷酸化的内在清除率增加约2倍（P < 0.05）。DCK对dFdU单磷酸化没有显著贡献。总之，Lys 27 Gln取代不会显著调节CDA对dFdC的活性，因此不会导致吉西他滨应答的个体间变异性。DCK 24 Val对dFdC单磷酸化的较高体外催化效率可能与dFdC临床反应相关。首次观察到CDA 70 Thr和DCK 24Val体外活性的底物依赖性改变，并证明这些遗传变异的体内后果不应从这些酶的一种底物外推到另一种底物。
Gemcitabine (dFdC, 2',2'-difluorodeoxycytidine) is metabolized by cytidine deaminase (CDA) and deoxycytidine kinase (DCK), but the contribution of genetic variation in these enzymes to the variability in systemic exposure and response observed in cancer patients is unclear. Wild-type enzymes and variants of CDA (Lys27Gln and Ala70Thr) and DCK (Ile24Val, Ala119Gly, and Pro122Ser) were expressed in and purified from Escherichia coli, and enzyme kinetic parameters were estimated for cytarabine (Ara-C), dFdC, and its metabolite 2',2'-difluorodeoxyuridine (dFdU) as substrates. All three CDA proteins showed similar K-m and V-max for Ara-C and dFdC deamination, except for CDA70Thr, which had a 2.5-fold lower K-m and 6-fold lower V-max for Ara-C deamination. All four DCK proteins yielded comparable metabolic activity for Ara-C and dFdC monophosphorylation, except for DCK24Val, which demonstrated an approximately 2-fold increase (P < 0.05) in the intrinsic clearance of dFdC monophosphorylation due to a 40% decrease in K-m (P < 0.05). DCK did not significantly contribute to dFdU monophosphorylation. In conclusion, the Lys27Gln substitution does not significantly modulate CDA activity toward dFdC, and therefore would not contribute to interindividual variability in response to gemcitabine. The higher in vitro catalytic efficiency of DCK24Val toward dFdC monophosphorylation may be relevant to dFdC clinical response. The substrate-dependent alterations in activities of CDA70Thr and DCK24Val in vitro were observed for the first time, and demonstrate that the in vivo consequences of these genetic variations should not be extrapolated from one substrate of these enzymes to another.