El Omari Kamel, Liekens Sandra, Bird Louise E, Balzarini Jan, Stammers David K
Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.
Mol Pharmacol. 2006 Jun;69(6):1891-6. doi: 10.1124/mol.106.023002. Epub 2006 Mar 23.
Varicella zoster virus encodes a thymidine kinase responsible for the activation of antiherpetic nucleoside prodrugs such as acyclovir. In addition, herpes virus thymidine kinases are being explored in gene/chemotherapy strategies aimed at developing novel antitumor therapies. To investigate and improve compound selectivity, we report here structure-based site-directed mutagenesis studies of varicella zoster virus thymidine kinase (VZVTK). Earlier reports showed that mutating residues at the core of the VZVTK active site invariably destroyed activity; hence, we targeted more distal residues. Based on the VZVTK crystal structure, we constructed six mutants (E59S, R84V, H97Y/A, and Y21H/E) and tested substrate activity and competitive inhibition for several compound series. All VZVTK mutants tested retained significant phosphorylation activity with dThd as substrate, apart from Y21E (350-fold diminution in the k(cat)/K(m)). Some mutations give slightly improved affinities: bicyclic nucleoside analogs (BCNAs) with a p-alkyl-substituted phenyl group seem to require aromatic ring stacking interactions with residue 97 for optimal inhibitory effect. Mutation Y21E decreased the IC(50) value for the BCNA 3-(2'-deoxy-beta-D-ribofuranosyl)-6-octyl-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (Cf1368) 4-fold, whereas mutation Y21H increased the IC(50) value by more than 15-fold. These results suggest that residue 21 is important for BCNA selectivity and might explain why HSV1TK is unable to bind BCNAs. Other mutants, such as the E59S and R84V thymidine kinases, which in wild-type VZVTK stabilize the dimer interface, give opposite results regarding the level of sensitivity to BCNAs. The work described here shows that distal mutations that affect the VZVTK active-site may help in the design of more selective substrates for gene suicide therapy or as anti-varicella zoster virus drugs.
水痘带状疱疹病毒编码一种胸苷激酶，该激酶负责激活如阿昔洛韦等抗疱疹核苷前药。此外，疱疹病毒胸苷激酶正在基因/化疗策略中进行探索，旨在开发新型抗肿瘤疗法。为了研究并提高化合物的选择性，我们在此报告基于结构的水痘带状疱疹病毒胸苷激酶（VZVTK）定点诱变研究。早期报告显示，突变VZVTK活性位点核心处的残基总会破坏活性；因此，我们将目标转向更外围的残基。基于VZVTK晶体结构，我们构建了六个突变体（E59S、R84V、H97Y/A和Y21H/E），并测试了几种化合物系列的底物活性和竞争性抑制作用。除Y21E外（催化常数/米氏常数降低350倍），所有测试的VZVTK突变体以dThd为底物时均保留显著的磷酸化活性。一些突变使亲和力略有提高：具有对烷基取代苯基的双环核苷类似物（BCNAs）似乎需要与残基97进行芳环堆积相互作用以获得最佳抑制效果。突变Y21E使BCNA 3-(2'-脱氧-β-D-呋喃核糖基)-6-辛基-2,3-二氢呋喃并[2,3-d]嘧啶-2-酮（Cf1368）的半数抑制浓度（IC50）值降低4倍，而突变Y21H使IC50值增加超过15倍。这些结果表明，残基21对BCNA的选择性很重要，这可能解释了为什么单纯疱疹病毒1型胸苷激酶（HSV1TK）无法结合BCNAs。其他突变体，如野生型VZVTK中稳定二聚体界面的E59S和R84V胸苷激酶，在对BCNAs的敏感程度上给出了相反的结果。此处描述的工作表明，影响VZVTK活性位点的外围突变可能有助于设计用于基因自杀疗法的更具选择性的底物或作为抗水痘带状疱疹病毒药物。
