Laboratory of Physiological Chemistry, de Duve Institute & Université catholique de Louvain, B-1200 Brussels, Belgium.
Biochem Pharmacol. 2012 Jul 1;84(1):43-51. doi: 10.1016/j.bcp.2012.03.022. Epub 2012 Apr 2.
Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme in the activation of several therapeutic nucleoside analogs (NA). Its activity can be increased in vivo by Ser-74 phosphorylation, a property that could be used for enhancing NA activation and clinical efficacy. In line with this, studies with recombinant dCK showed that mimicking Ser-74 phosphorylation by a S74E mutation increases its activity toward pyrimidine analogs. However, purine analogs had not been investigated. Here, we show that the S74E mutation increased the k(cat) for cladribine (CdA) by 8- or 3-fold, depending on whether the phosphoryl donor was ATP or UTP, for clofarabine (CAFdA) by about 2-fold with both ATP and UTP, and for fludarabine (F-Ara-A) by 2-fold, but only with UTP. However, the catalytic efficiencies (k(cat)/Km) were not, or slightly, increased. The S74E mutation also sensitized dCK to feed-back inhibition by dCTP, regardless of the phosphoryl donor. Importantly, we did not observe an increase of endogenous dCK activity toward purine analogs after in vivo-induced increase of Ser-74 phosphorylation. Accordingly, treatment of CLL cells with aphidicolin, which enhances dCK activity through Ser-74 phosphorylation, did not modify the conversion of CdA or F-Ara-A into their active triphosphate form. Nevertheless, the same treatment enhanced activation of gemcitabine (dFdC) into dFdCTP in CLL as well as in HCT-116 cells and produced synergistic cytotoxicity. We conclude that increasing phosphorylation of dCK on Ser-74 might constitute a valuable strategy to enhance the clinical efficacy of some NA, like dFdC, but not of CdA or F-Ara-A.
脱氧胞苷激酶（dCK）（EC 2.7.1.74）是几种治疗性核苷类似物（NA）激活的关键酶。其活性可以通过 Ser-74 磷酸化在体内增加，这一特性可用于增强 NA 激活和临床疗效。与此一致，用重组 dCK 的研究表明，通过 S74E 突变模拟 Ser-74 磷酸化可增加其对嘧啶类似物的活性。然而，嘌呤类似物尚未被研究。在这里，我们表明 S74E 突变使 cladribine（CdA）的 k(cat)增加了 8 倍或 3 倍，具体取决于磷酸供体是 ATP 还是 UTP，对 clofarabine（CAFdA）增加了约 2 倍，无论是 ATP 还是 UTP，对 fludarabine（F-Ara-A）增加了 2 倍，但仅使用 UTP。然而，催化效率（k(cat)/Km）没有增加，或者略有增加。S74E 突变也使 dCK 对 dCTP 的反馈抑制敏感，无论磷酸供体如何。重要的是，我们没有观察到内源性 dCK 活性在 Ser-74 磷酸化增加后对嘌呤类似物的增加。因此，用aphidicolin（通过 Ser-74 磷酸化增强 dCK 活性）处理 CLL 细胞不会改变 CdA 或 F-Ara-A 转化为其活性三磷酸形式。然而，相同的治疗增强了 gemcitabine（dFdC）在 CLL 以及 HCT-116 细胞中的转化为 dFdCTP，并产生协同细胞毒性。我们得出结论，增加 dCK 上 Ser-74 的磷酸化可能是增强某些 NA（如 dFdC）但不是 CdA 或 F-Ara-A 的临床疗效的有价值策略。
