Enzymatic Oxidation Of Drugs To Toxic And Carcinogenic M

致癌的多环芳烃如苯并[a]芘（BaP）的海湾区二醇环氧化物（DE）代谢物被认为通过形成共价DNA加合物来启动细胞转化。哺乳动物代谢的烃得到两个非对映体DE，其中每一个存在的对映体，其中的苄基羟基和环氧氧是顺式或反式。BaP DE加合物在本报告中描述的是来自后者的非对映体的两个对映体。由这些DE在DNA中形成的加合物分别由脱氧鸟苷（dG）和脱氧腺苷（dA）的环外N-2和N-6氨基顺式和反式打开环氧环而产生。我们已经开发了强大的新的合成方法来制备位点和立体特异性DE-DNA加合物，用于探测DNA加工所需的酶的结构生物学和机械酶学，如HIV-1整合酶，解旋酶，拓扑异构酶和DNA聚合酶。过去一年的研究集中在拓扑异构酶上，拓扑异构酶通过使单链或双链短暂断裂，分别允许单链或双链DNA通过断裂，然后使切割的末端重新连接，从而放松DNA的扭转应变。拓扑异构酶在DNA复制、转录和重组中起关键作用，并且是广泛使用的抗菌药物如环丙沙星和抗肿瘤药物如喜树碱的靶标。在病毒、原核和真核系统中，拓扑异构酶有几个不同的机制家族。人拓扑异构酶（top1）和牛痘拓扑异构酶都通过在DNA中产生可逆的单链断裂来发挥作用，以在酶和DNA 3 '-磷酸基团之间在切割位点产生磷酸酪氨酰键，并排出含有游离5'-羟基的DNA片段。我们以前曾报道，小沟结合dG加合物来自反式开放的BaP DE，当位于或附近的切割位点为人类top1，抑制在这个网站上的切割，并诱导新的切割在几个位置远离加合物。我们现在已经检查了这些dG加合物对牛痘病毒拓扑异构酶的影响，与人酶不同，牛痘病毒拓扑异构酶对5 '-CCCTT-XXX靶序列处的切割具有高度特异性，并且当该位点被阻断时不显示任何切割。我们已经映射的小沟和牛痘拓扑异构酶的活性位点之间的相互作用，通过测量的网站和立体特异性BaP DE-dG加合物的切割步骤的速率的影响。当加合物位于紧邻切割位点侧翼碱基的3'（下游）位置或位于切割位点5'（上游）的4个碱基位置时，在不可切割链上存在dG加合物的情况下，切割速率（相对于未加合的对照）相对不受影响。与此相反，近2000倍的抑制观察到dG加合物2或3个碱基上游的切割位点。因此，确定了DNA底物的特定区域，其中小沟与酶的相互作用是关键的。我们还研究了牛痘拓扑异构酶与BaP DE-dA加合物的位点特异性相互作用，当dA的N-6连接点的构型分别为R或S时，所述加合物插入5'或3'修饰的腺嘌呤。当烃插入在切割的（T-X）键两侧的两个碱基之间或插入在位点的5 ′侧上的紧邻碱基之间时，切割速率显著延迟（2500- 16，000倍）。这种延迟显着降低时，插层的烃被放置远离裂解键。与人类top1和牛痘不同，人类拓扑异构酶II α（top2）是一种同源二聚体酶，在双链DNA中产生两个断裂，由四个碱基对交错。嵌入BaP DE-dA加合物抑制top2裂解时，它们位于外部的四个碱基对交错或在中间的交错。然而，当烃嵌入在交错切割位点中的任一个处或紧邻交错切割位点时，DNA被切割，但是所得到的top2-DNA共价复合物通过抑制再连接而被捕获，从而使酶中毒。

Bay-region diol epoxide (DE) metabolites of carcinogenic polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) are believed to initiate cell transformation by formation of covalent DNA adducts. Mammalian metabolism of the hydrocarbon gives two diastereomeric DEs, each of which exists as a pair of enantiomers, in which the benzylic hydroxyl group and epoxide oxygen are either cis or trans. BaP DE adducts described in the present report are derived from the two enantiomers of the latter diastereomer. Adducts formed in DNA by these DEs result from cis and trans opening of the epoxide ring by the exocyclic N-2 and N-6 amino groups of deoxyguanosine (dG) and deoxyadenosine (dA), respectively. We have developed powerful new synthetic methods to prepare site- and stereospecific DE-DNA adducts for use in probing the structural biology and mechanistic enzymology of enzymes required for DNA processing, such as HIV-1 integrase, helicases, topoisomerases and DNA polymerases. Studies during the past year have focused on topoisomerases, which relax torsional strain in DNA by making transient single- or double-strand breaks that permit the passage of single or double stranded DNA, respectively, through the break, followed by rejoining of the cleaved ends. Topoisomerases play critical roles in DNA replication, transcription and recombination and are targets for widely used antibacterial drugs such as ciprofloxacin and antitumor drugs such as camptothecin. Several mechanistically distinct families of topoisomerases have been characterized in viral, procaryotic and eucaryotic systems. Both human topoisomerase (top1) and vaccinia topoisomerase function by making reversible single-strand breaks in DNA to give a phosphotyrosyl bond between the enzyme and a DNA 3'-phosphate group at the cleavage site, with expulsion of a DNA fragment containing a free 5'-hydroxyl group. We have previously reported that minor-groove bound dG adducts derived from trans opening of BaP DE, when located at or near a cleavage site for human top1, inhibit cleavage at this site and induce new cleavages at several positions remote from the adduct. We have now examined the effects of these dG adducts on vaccinia virus topoisomerase, which unlike the human enzyme, is highly specific for cleavage at a 5'-CCCTT-XXX target sequence and does not exhibit any cleavage when this site is blocked. We have mapped interactions between the minor groove and the active site of vaccinia topoisomerase by measuring the effect of site- and stereospecific BaP DE-dG adducts on the rates of the cleavage step. The cleavage rate (relative to unadducted control) in the presence of a dG adduct on the non-scissile strand was relatively unaffected when the adduct was at the position immediately 3' (downstream) to a base flanking the cleavage site or 4 bases 5' (upstream) from the site. In contrast, nearly 2000-fold inhibition was observed with dG adducts 2 or 3 bases upstream from the cleavage site. Thus a specific region of the DNA substrate was identified where minor-groove interactions with the enzyme are critical. We have also examined site-specific interactions vaccinia topoisomerase with BaP DE-dA adducts, which intercalate either 5' or 3' to the modified adenine when the configuration at the point of attachment to N-6 of dA is R or S, respectively. When the hydrocarbon is intercalated between the two bases flanking the cleaved (T-X) bond or between the immediately adjacent bases on the 5'-side of the site, the cleavage rate is dramatically retarded (2500-16,000-fold). This retardation decreases markedly when intercalated hydrocarbon is placed farther away from the cleaved bond. Unlike human top1 and vaccinia, human topoisomerase II alpha (top2) is a homodimeric enzyme that makes two breaks, staggered by four base pairs, in double-stranded DNA. Intercalated BaP DE-dA adducts inhibit top2 cleavage when they are located either outside the four base-pair stagger or in the middle of the stagger. However, when the hydrocarbon is intercalated at or immediately adjacent to either of the staggered cleavage sites, the DNA is cleaved, but the resultant top2-DNA covalent complex is trapped by inhibition of religation, thus poisoning the enzyme.