ENZYMATIC OXIDATION OF DRUGS TO TOXIC AND CARCINOGENIC METABOLITES

致癌多环芳烃的湾区二醇环氧化物(DE)代谢物被认为是通过DNA的共价修饰来启动细胞转化的。两个非对映异构体在哺乳动物中以一对对映体的形式在代谢中形成：De-1，其中苄基羟基和环氧基是顺式的；DE-2，其中这些取代基是反式的。脱氧鸟苷(Dg)和脱氧腺苷(Da)的外环N-2和N-6氨基分别是脱氧核糖核酸的主要靶点。我们研究的目的是阐明特定DES形成的DNA加合物的物理结构与其在纯化的酶和完整细胞中的生化过程之间的关系。实验室今年合成工作的一个亮点是开发了改进的方法来制备DG的N-2位的顺式和反式开环DE加合物作为它们的亚氨基，使用一种新的DG的O-6封闭/解封协议。以前，这些反式打开的DG加合物亚磷酰胺需要更费力的、多步骤的路线，而它们的顺式打开的对应物基本上是无法合成的。构建具有特定加合物立体化学和核苷酸序列的DE加合物寡核苷酸的方法的可用性使系统地研究其结构特征对加合物外DNA复制所导致的突变的影响成为可能。去年，我们报道了对M13mp7L2噬菌体进行定点突变实验的结果，该实验使用了8个16聚体寡核苷酸，其中包含与湾区DE-1和DE-2的每个对映体从苯并[a]芘(BaP)中反式打开相对应的da加合物。这些加合物被结合在来源于supF基因的两个不同序列上下文中的括号位置：上下文I，5-TTT[A]GAGTCTGCTCCC-3和上下文II，5-CAG[A]TTTAGAGTCTGC-3。我们现在已经将这些突变研究扩展到顺式开放的Bap DE da加合物，以及相同序列中类似的顺式和反式开放的Fjord区苯并[c]菲(BcPh)DE da加合物。在给定的序列背景下，各个BaP加合物异构体之间的总体致突变性差异相对较小，从该碳氢化合物衍生的加合物的立体化学和致突变性之间没有明确的关系。相反，每个BcPh DE异构体与da的N-6的结合部位的构型对致突变性有显著影响，并构成了影响这些加合物诱变活性的主要立体化学特征。在环境I中，所有在烃与N-6的结合点具有R构型的BcPh加合物都是非常弱的诱变剂(突变率小于1%)，而在该碳原子上具有S构型的加合物在该序列中的突变率为10-32%。尽管这种立体化学效应在上下文II中不那么明显，但值得注意的是，具有S构型的加合物是两个序列中所有BcPh DE da加合物中最具突变性的(突变频率在上下文I中为32%，在上下文II中为57%)。拓扑异构酶是一种DNA加工酶，通过形成可逆的DNA裂解复合体来松弛超螺旋和减轻扭转应变，其中酶中的酪氨酸与3-磷酸基团共价键合，并在裂解部位产生新的5-羟基。某些抗癌药物，如喜树碱，通过捕获裂解复合体和阻止裂解的DNA的宗教来使拓扑异构酶I(TOP1)失活。其中许多药物被认为是次要的沟槽粘结剂。我们研究了Bap DE-2的(+)-和(-)-对映体的顺式和反式开环N-2DG加合物在作为Top1底物的短双链寡核苷酸(22-mer)中的作用。这些烃类加合物为研究Top1的抑制机制提供了独特的探针，因为与喜树碱不同，它们是共价结合在DNA上的，并且它们在DNA中的构象可以从先前的核磁共振研究中预测到：反式开环的DG加合物的芳香部分取向于小凹槽，而顺式开环的DG加合物的芳香部分插入螺旋中。当这些Bap DE加合物位于DG上时，DG在断裂通常由top1裂解的磷酸二酯键后具有5-羟基，它们抑制该键的断裂，并诱导DNA在序列中的其他磷酸二酯键上裂解。反式(小槽)加合物比顺式(插层)加合物更有效地诱导了在加合物的第三个磷酸二酯键5上的新的裂解位置。在所研究的四个加合物中，来自(+)-bap DE-2的反式开放的DG加合物在诱导top1切割方面最有活性。据我们所知，这是在分子水平上的第一个例子，与其他三个立体异构体BaP DE-2 DG加合物相比，这种最具致癌性的BaP DE异构体的主要加合物明显增强了潜在的DNA损伤。

Bay-region diol epoxide (DE) metabolites of carcinogenic polycyclic aromatic hydrocarbons are believed to initiate cell transformation by covalent modification of DNA. Two diastereomeric DEs, each of which exists as a pair of enantiomers, are formed metabolically in mammals: DE-1, in which the benzylic hydroxyl group and epoxide oxygen are cis, and DE-2, in which these substituents are trans. The primary targets in DNA for these DEs are the exocyclic N-2 and N-6 amino groups of deoxyguanosine (dG) and deoxyadenosine (dA), respectively. The goal of our research is to elucidate the relationships between the physical structures of DNA adducts formed from specific DEs and their biochemical processing with purified enzymes and in intact cells. A highlight of the laboratorys synthetic work this year has been the development of improved methods for the preparation of both cis and trans opened DE adducts at N-2 of dG as their phosphoramidites, using a novel blocking/deblocking protocol for O-6 of dG. Previously, these trans opened dG adduct phosphoramidites had required a more laborious, multi-step route, and their cis opened counterparts were essentially inaccessible synthetically. Availability of methods for the construction of DE-adducted oligonucleotides of defined adduct stereochemistry and nucleotide sequence makes possible the systematic study of effects of their structural features on mutations that result from DNA replication past the adducts. Last year, we reported the results of site-specific mutagenesis experiments in M13mp7L2 bacteriophage using eight 16-mer oligonucleotides containing dA adducts corresponding to trans opening of each enantiomer of the bay-region DE-1 and DE-2 from benzo[a]pyrene (BaP). These adducts were incorporated at the bracketed positions in two different sequence contexts derived from the supF gene: Context I, 5-TTT[A]GAGTCTGCTCCC-3, and Context II, 5-CAG[A]TTTAGAGTCTGC-3. We have now extended these mutagenesis studies to the cis opened BaP DE dA adducts, as well as the analogous cis and trans opened fjord-region benzo[c]phenanthrene (BcPh) DE dA adducts in the same two sequences. In a given sequence context, differences in overall mutagenicity between individual BaP adduct isomers were relatively small, and there was no clear relationship between stereochemistry and mutagenicity for adducts derived from this hydrocarbon. In contrast, the configuration at the site of attachment of each BcPh DE isomer to N-6 of dA had marked effects on mutagenicity, and constituted the dominant stereochemical feature influencing the mutagenic activity of these adducts. All the BcPh adducts with R configuration at the point of attachment of the hydrocarbon to N-6 were very poor mutagens in Context I (mutation frequencies less than 1%), whereas the adducts with S configuration at this carbon atom exhibited mutation frequencies from 10-32% in this sequence. Although this stereochemical effect was less pronounced in Context II, it is notable that an adduct with the S configuration was the most mutagenic of all the BcPh DE dA adducts in both sequences (mutation frequency 32% in Context I and 57% in Context II). Topoisomerases are DNA processing enzymes that relax supercoiling and relieve torsional strain by forming reversible DNA cleavage complexes in which a tyrosine in the enzyme becomes covalently bonded to a 3-phosphate group, and a new 5-hydroxyl group is generated at the cleavage site. Certain anticancer drugs such as the camptothecins inactivate topoisomerase I (top1) by trapping the cleavage complex and preventing religation of the cleaved DNA. Many of these drugs are thought to be minor groove binders. We have investigated the effects of cis and trans opened N-2 dG adducts of the (+)- and (-)-enantiomers of BaP DE-2 in a short duplex oligonucleotide (22-mer) that is a substrate for top1. These hydrocarbon adducts provide unique probes for investigating the mechanism of top1 inhibition, since, unlike the camptothecins, they are covalently bound to the DNA, and their conformations in DNA are predictable from prior NMR studies: the trans opened dG adducts have the aromatic moiety oriented in the minor groove, whereas the cis opened dG adducts have the aromatic portion intercalated into the helix. These BaP DE adducts, when located on the dG that would have a 5-hydroxyl group after breaking the phosphodiester bond normally cleaved by top1, inhibit cleavage of this bond and induce DNA cleavage at other phosphodiester bonds in the sequence. The trans (minor groove) adducts were more effective than the cis (intercalated) adducts as inducers of a new cleavage site at the third phosphodiester bond 5 to the adduct. Of the four adducts studied, the trans opened dG adduct derived from (+)-BaP DE-2 was the most active in inducing cleavage by top1. To our knowledge, this constitutes the first example at the molecular level of a clear enhancement of potential DNA damage by this major adduct of the most carcinogenic BaP DE isomer as compared with the other three, stereoisomeric BaP DE-2 dG adducts.