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7.6.6: Mapping the Major Adducts of cis- and trans-DDP on DNA; Sequence Specificity

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    As we have seen, the antitumor activity of cisplatin is most likely the result of its DNA-binding properties. But what are the adducts? The human genome has more than a billion nucleotides. Does platinum recognize any special regions of the DNA or any particular sequences? In other words, is binding simply random or is there at least a regioselectivity? In this section, we discuss the best strategies for answering these questions, strategies that evolved in pursuit of learning how cis-DDP binds to DNA. We also illustrate their power in elucidating the DNA-binding properties of other metal complexes of interest to bioinorganic chemists.

    a. Early Strategic Approaches

    The first experiments to imply the sequence preferences of cis-DDP binding to DNA employed synthetic polymers.108,109 Specifically, the buoyant density of poly(dG)•poly(dC), poly(dG•dC), and their cis-DDP adducts was studied in the analytical ultracentrifuge. The greatest shift in buoyant density was seen for the platinum adducts of poly(dG)•poly(dC), from which it was concluded that platinum forms an intrastrand crosslink between two neighboring guanosine nucleosides on the same strand. This interpretation was suggested by the known preference of metal ions, and especially platinum, for binding at the N7 position on the guanine base (Figure 9.9), information available from model studies of metal-nucleobase chemistry. Although other interpretations of the buoyant-density shift were possible, especially since the amount of platinum bound was not quantitated, the conclusion proved to be correct, as confirmed by later investigations. Interestingly, trans-DDP did not selectively increase the buoyant density of poly(dG)•poly(dC).

    Following these initial experiments, the regioselectivity of cis-DDP binding was investigated by studying the inhibition of enzymatic digestion of platinated DNA. For example, the platinum complex inhibits the cleavage of DNA by restriction enzymes that recognize specific sequences and cut both strands of the double helix.110 The resulting fragments are readily identified on electrophoresis gels. One such restriction enzyme is Bam HI. As shown by the arrows in Scheme (9.11), Bam HI cleaves a six-bp palindromic sequence at the phosphodiester bonds between two guanosine nucleosides. Formation of an intrastrand crosslink between the two adjacent guanosine nucleosides inhibits digestion by the enzyme. Another method, termed exonuclease mapping, involves digestion of the strands of duplex DNA from its 3'-ends.111,112 When the enzyme encounters a bound platinum atom, it is unable to proceed further. Analysis of the digestion products by gel electrophoresis reveals the presence of discrete bands caused by the inhibition of digestion by bound platinum at specific sequences. Results from experiments of this kind were the most definitive at this time in demonstrating the profound regioselectivity of cisplatin for adjacent guanosines, and strongly supported the earlier conclusion that the drug was making an intrastrand d(GpG) crosslink.

    clipboard_ef862873214c51b50d6850894f3fb5e30\(\tag{9.11}\)
    Figure 9.18. Platinum is first bound to a single-stranded DNA template, in this example from bacteriophage M13mp18, to which is next annealed a short, complementary oligonucleotide termed a "primer" for DNA synthesis. Addition of the large (Klenow) fragment of E. coli DNA polymerase I and deoxynucleoside triphosphates, one of which bears a 32P label, [\(\alpha\)-32P]dATP, initiates replication. When the enzyme encounters a platinum adduct, the chain is terminated. By running out the newly synthesized DNA strands on a sequencing gel, the sites of platinum binding can be detected by comparing the positions of the radiolabeled fragments with those obtained from sequencing ladders. The results of this procedure, which has been termed "replication mapping," confirmed that cis-DDP binds selectively to (dG)n (n \(\geq\) 2) sequences. In addition, they showed that trans-DDP blocks replication, in a much less regioselective manner, in the vicinity of sequences of the kind d(GpNpG), where N is an intervening nucleotide. These data afforded the first clear insight into the sequence preferences for trans-DDP on DNA. A control experiment run with DNA platinated by the monofunctional complex [Pt(dien)CI]+ gave the interesting result that DNA synthesis was virtually unaffected.
    clipboard_e44b3b2eb4dca207871b4ae7324e0c62d
    Figure 9.18 - Diagram illustrating the replication mapping experiment. To a single-stranded, platinated template is annealed a short primer for DNA synthesis using DNA polymerase I (Klenow fragment) and radiolabeled nucleotides. Sites of platinum binding are revealed as bands on gel electrophoresis where chain termination occurs (see text for details).

    In yet another approach to the problem, DNA containing cis- or trans-DDP adducts was electrostatically coupled to bovine serum albumin, to enhance its antigenicity, and injected into rabbits.113,114 The resulting antisera and antibodies were then studied for their ability to recognize and bind specifically to platinated DNAs having defined sequences, such as poly(dG)•poly(dC) and poly[d(GC)]•poly[d(GC)].

    From experiments of this kind, the major cis-DDP adduct recognized by the antibody was found to be cis-[Pt(NH3)2{d(GpG)}], in accord with the findings of the enzymatic mapping experiments. Unplatinated DNA was not recognized, nor was DNA platinated with trans-DDP. On the other hand, the antibody recognized DNA platinated with antitumor-active compounds [Pt(en)Cl2] and [Pt(DACH)(CP)], where DACH = 1,2-diaminocyclohexane and CP = 4-carboxyphthalate. This result revealed that the antibody recognized the structural change in DNA that accompanies formation of d(GpG) intrastrand crosslinks, irrespective of the diamine ligand in the coordination sphere of the platinum atom. The antibody is also capable of distinguishing adducts formed by active versus inactive platinum complexes. Most importantly, DNA isolated from the cells of mice bearing the L1210 tumor five hours after cisplatin injection, was recognized.113,115 Subsequent studies116 revealed that these antibodies could detect cisplatin-DNA adducts formed in the white blood cells of patients receiving platinum chemotherapy. Thus, the antibody work linked the regiospecificity of platination chemistry in vitro with that occurring in vivo and in a clinically relevant manner.

    Additional studies with monoclonal antibodies generated using DNA platinated with cis- or trans-DDP further confirmed and extended these results.117 This later work indicated that intrastrand crosslinked d(ApG) and d(GpG) sequences possess a common structural determinant produced by cis-DDP platination, and that carboplatin is also capable of inducing the same DNA structure. For trans-DDP-platinated DNA, a monoclonal antibody was obtained that appeared to have the intrastrand d(GpTpG) adduct as its major recognition site. In all these studies, the primary structural determinant appears to be DNA duplex opposite the site of platination, since fairly major stereochemical changes could be made in the amine ligands with no appreciable effect on antibody binding.

    b. Degradation, Chromatographic Separation, and Quantitation of DNA Adducts

    Experiments in which DNA platinated with cis-DDP is degraded to chromatographically separable, well-defined adducts have been invaluable in revealing the spectrum of products formed. In a typical experiment, platinated DNA is digested with DNAse I, nuclease P1, and alkaline phosphatase. These enzymatic digestions degrade DNA into nucleosides that can be readily separated by high-performance liquid chromatography (HPLC). Detection of the adducts can be accomplished by the UV absorption of the nucleoside bases at 260 nm or, for platinum complexes containing a radioactively labeled ligand such as [14C]ethylenediamine,118 by monitoring counts. In addition to peaks corresponding to dA, dC, dG, and dT, the chromatographic trace contains additional peaks corresponding to specific platinum nucleobase adducts such as cis-[Pt(NH3)2(dG)2]. The precise nature of these adducts was established by comparison with chemically synthesized compounds structurally characterized by NMR spectroscopy.118-121 An alternative method for identifying the adducts employed antibodies raised against specific platinum-nucleobase complexes.122

    This approach has revealed the relative amounts of various adducts formed by a variety of platinum complexes; selected results are summarized in Table 9.4. Usually, for cisplatin, the relative amounts of the various adducts formed varies according to the series cis-[Pt(NH3)2{d(pGpG)}] > cis-[Pt(NH3)2{d(pApG)}] > cis-[Pt(NH3)2{d(GMP)}2] > monofunctional adducts. Only when the total incubation time was short, less than an hour, were the monofunctional adducts more prevalent, as expected from the kinetic studies of cis-DDP binding to DNA discussed previously. It is noteworthy that no d(pGpA) adducts were detected. This result, which is consistent with information obtained by enzymatic mapping, can be understood on stereochemical grounds.123 If the guanosine nucleoside N7 position is the most-preferred binding site on DNA, closure to make an N7,N7 intrastrand crosslink between two adjacent purine nucleotides is more feasible in the 5' direction along the helix backbone (N7•••N7 distance of ≈ 3 Å) than in the 3' direction (N7•••N7 distance ≈ 5 Å). In addition, molecular-mechanics modeling studies124 indicate that a highly unfavorable steric clash occurs between the 6-amino group of the 3'-adenosine residue in a d(pGpA) crosslink and the platinum ammine ligand, whereas in the platinated d(pApG) sequence, the 6-oxo group forms a stabilizing hydrogen bond to this ligand. A 28 kJ mol-1 preference of cis-DDP for binding d(pApG) over d(pGpA) was calculated.

    Table 9.4 - Geometric features of the platinum coordination spheres of cis- [Pt(NH3)2{d(pGpG)}].

    a) Bond distances are in Angstroms and angles are in degrees.

    b) Conventions used for assigning Base/Base and Base/PtN4 dihedral angles can be found in J. D. Orbell, L. G. Marzilli, and T. J. Kistenmacher, J. Am. Chem. Soc. 103 (l981), 5126. The numbers in square brackets refer to the corresponding N(ammine)•••O6 distance, in Å (see text).

    Bond Distances and Anglesa

    Molecule 1

    Molecule 2

    Molecule 3

    Molecule 4

    Pt-N1 2.03(2) 2.01(2) 2.08(2) 2.08(2)
    Pt-N2 2.03(3) 2.09(2) 2.04(3) 2.06(3)
    Pt-N7A 2.01(2) 2.02(2) 1.91(3) 1.93(3)
    Pt-N7B 2.05(2) 1.95(3) 2.00(3) 2.06(3)
    N7A-Pt-N1 88.6(9) 90.3(9) 91.0(1) 88.4(9)
    N7A-Pt-N2 179(1) 173.3(8) 178(1) 177(1)
    N7A-Pt-N7B 89.1(9) 90.0(1) 85(1) 89(1)
    N1-Pt-N2 92(9) 90.8(9) 91(1) 93(1)
    qN1-Pt-N7B 176.5(9) 179.0(1) 173(1) 175(1)
    N2-Pt-N7B 90.3(9) 89.0(1) 93(1) 89(1)

    Dihedral Anglesb

    Molecule

    3'-Gua/5'-Gua

    5'-Gua/PtN4

    3'-Gua/PtN4

    1 76.2(5) 110.6(5) [3.30(3)] 86.1(5)
    2 81.0(5) 110.8(5) [3.49(3)] 95.5(5)
    3 86.8(6) 81.0(6) 58.0(6) [3.11(4)]
    4 80.6(5) 76.6(6) 59.6(6) [3.18(4)]

    There are two likely sources of cis-[Pt(NH3)2{d(GMP)}2] in the spectrum of adducts. This species could arise from long-range intrastrand crosslinks, where the two coordinated guanosines are separated by one or more nucleotides. In support of this possibility is the fact that digestion of chemically synthesized cis-[Pt(NH3)2{d(GpNpG)}], where N = C or A, led to cis-[Pt(NH3)2{d(Gua)}-{d(GMP)}] and mononucleotides.118,119,121 The other source of this product is interstrand crosslinked DNA, known to occur from the alkaline elution studies.

    As indicated in Table 9.4, in all the experiments there was platinum that was unaccounted for in the quantitation procedures, which employed either antibodies, platinum atomic absorption spectroscopy, or a radiolabeled ethylenediamine ligand. Some of this material was assigned to oligonucleotides having high platinum content, resistant to enzymatic degradation.

    Two important points emerge from the quantitation of adducts by this method. One is that intrastrand d(GpG) and d(ApG) crosslinks constitute the major adducts (>90 percent of total platination) made by cisplatin on DNA in vivo. Because they were identified by an antibody specific for their structures, no chemical change brought about by cellular metabolism has occurred. Secondly, the preponderance of these adducts far exceeds the frequency of adjacent guanosine or guanosine/adenosine nucleosides in DNA. This latter result implies a kinetic preference for, or recognition of, d(pGpG)- and d(pApG)-containing sequences by cisplatin.

    c. Postscript: A Comment on Methodologies

    With few exceptions, none of the experimental studies described in this section could have been carried out in 1969, when Rosenberg first demonstrated the anticancer activity of cis-DDP. The techniques of DNA sequencing, monoclonal antibody formation, oligonucleotide synthesis, HPLC, FPLC, and many of the higher resolution gel electrophoresis methodologies employed were the result of later developments driven by rapid advances in the fields of molecular biology and immunology. Future progress in elucidating the molecular mechanisms of action of cisplatin and other inorganic pharmaceuticals will no doubt benefit from new technological discoveries and inventions of this kind yet to come.


    7.6.6: Mapping the Major Adducts of cis- and trans-DDP on DNA; Sequence Specificity is shared under a not declared license and was authored, remixed, and/or curated by LibreTexts.

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