Phosphodiester-Mediated Reaction of Cisplatin with Guanine in Oligodeoxyribonucleotides

Abstract

The cancer chemotherapeutic agent cis-diamminedichloroplatinum(II) or cisplatin reacts primarily with guanines in DNA to form 1,2-Pt-GG and 1,3-Pt-GNG intrastrand cross-links, and to a lesser extent, G-G interstrand cross-links. Recent NMR evidence has suggested that cisplatin can also form a coordination complex with the phosphodiester internucleotide linkage of DNA. We have examined the effects of the phosphodiester backbone on the reactions of cisplatin with oligodeoxyribonucleotides that lack or contain a -GTG-sequence. Cisplatin forms a stable adduct with TpT that can be isolated by reversed phase HPLC. The cis-Pt-TpT adduct contains a single Pt, as determined by atomic absorption spectroscopy (AAS) and by electrospray ionization mass spectrometry (ESI-MS), and is resistant to digestion by snake venom phosphodiesterase. Treatment of the adduct with sodium cyanide regenerates TpT. Similar adduct formation was observed when T(pT) 8 was treated with cisplatin, but not when the phosphodiester linkages of T(pT) 8 were replaced with methylphosphonate groups. These results suggest that the platinum may be coordinated with the oxygens of the thymine and possibly with those of the phosphodiester group. As expected reaction of a 9-mer containing a -GTG-sequence with cisplatin yielded an adduct that contained a 1,3-Pt-GTG intrastrand cross-link. However, we found that the number and placement of phosphodiesters surrounding a -GTG-sequence significantly affected intrastrand cross-link formation. Increasing the number of negatively charged phosphodiesters in the oligonucleotide, increased the amount of -GTGplatination. Surrounding the -GTG-sequence with non-ionic methylphosphonate linkages reduced or eliminated cross-link formation. These observations suggest that interactions between cisplatin and the negatively charged phosphodiester backbone may play an important role in facilitating platination of guanine nucleotides in DNA.Reactions of the chemotherapeutic drug cis-diamminedichloroplatinum(II) (cisplatin or cis-DDP) with DNA have been studied for many years. Although it is widely accepted that the therapeutically relevant lesion is a 1,2-Pt-GG intrastrand cross-link, which links the N7s of the two guanines (1-3), the precise pathway leading to formation of this cross-link, as well as to 1,3-Pt-GNG intrastrand and G-G interstrand cross-links, is still under investigation. It is thought that cis-DDP is converted to cis-diamminediaquaplatinum(II) upon leaving the high concentration of chloride in the blood and entering the low concentration of chloride in the cell (3)(4)(5). This dicationic, aquated species then reacts with the DNA bases and other nucleophiles within the cell. While the reactions of cisplatin with nucleobases have been studied extensively, interactions with the DNA backbone have been largely overlooked. Platinum, a "soft" metal, preferentially binds to other "soft" ligands, such as amines and thiols (6,7). This binding preference does not, however, exclude the possibility that platinum can for...

Enzymatic Processing of Platinated RNAs

“…In biologically relevant pH ranges, the potential N3 amine ligands in uridine (and thymidine) nucleobases are not typically considered to be targets for cisplatin because they are protonated at pH values below 10 . Platination of thymine and uracil have, however, both been observed. , In addition, characterization of a platinated d(TpG) DNA dinucleotide by NMR spectroscopy found evidence for solvent-dependent Pt(II) coordination to either the N3 of a neighboring pyrimidine base in aqueous solution, or alternatively, interaction with nonbridging oxygens in the phosphodiester backbone in less polar solvents . The multiple products resulting from nuclease digestion of the platinated 5′-(U) 6 -GU-(U) 5 RNA may reflect a combination of these types of Pt(II) interactions.…”

Chemical synthesis of RNA with site-specific methylphosphonate modifications

“…Thus, great potential was seen for their use as regulators of gene expression by the antisense approach where antisense oligonucleotides inhibit translation by binding to mRNA [ 4 , 7 , 24 , 25 ]. Other studies included the investigation of B to Z-DNA transition using oligonucleotides containing a mP modification of distinct stereochemistry [ 11 ], footprinting of protein-DNA backbone contacts [ 26 ], DNA bending upon phosphate neutralisation [ 27 ], or an examination of the coordination of cis-plastin to the DNA phosphate backbone [ 28 ].…”

Nucleotides and Nucleic Acids; Oligo- and Polynucleotides

“…28 The importance of the phosphate group has also been shown for the formation of cisplatin adducts at TpT sites, as replacement of phosphate by methylphosphonate leads to no adduct formation. 29 Similarly, the double-stranded break repair protein Ku shows a 280-fold reduction in binding affinity when phosphate is replaced by methylphosphonate. 30 The other major backbone modification to have been studied is the phosphoramidate, in which one of the phosphodiester oxygen atoms is replaced by an amino group.…”

“…This complex clearly coordinates through the phosphodiester backbone, as replacement by methylphosphonate prevents the complex forming. 29 The target for cisplatin is nuclear DNA, which is packaged in nucleosomes. Treatment of nuclear DNA with cisplatin alters the rotational setting of the DNA on the histone core by between 20-401, with the lesion facing into the core, though the effect is very sequence dependent.…”