Proteins that discriminate between cisplatin-DNA adducts and oxaliplatin-DNA adducts are thought to be responsible for the differences in tumor range, toxicity, and mutagenicity of these two important chemotherapeutic agents. However, the structural basis for differential protein recognition of these adducts has not been determined and could be important for the design of more effective platinum anticancer agents. We have determined high-resolution NMR structures for cisplatin-GG and undamaged DNA dodecamers in the AGGC sequence context and have compared these structures with the oxaliplatin-GG structure in the same sequence context determined previously in our laboratory. This structural study allows the first direct comparison of cisplatin-GG DNA and oxaliplatin-GG DNA solution structures referenced to undamaged DNA in the same sequence context. Non-hydrogen atom rmsds of 0.81 and 1.21 were determined for the 15 lowest-energy structures for cisplatin-GG DNA and undamaged DNA, respectively, indicating good structural convergence. The theoretical NOESY spectra obtained by back-calculation from the final average structures showed excellent agreement with the experimental data, indicating that the final structures are consistent with the NMR data. Several significant conformational differences were observed between the cisplatin-GG adduct and the oxaliplatin-GG adduct, including buckle at the 5′ G6•C19 base pair, opening at the 3′ G7•C18 base pair, twist at the A5G6•T20C19 base pair step, slide, twist, and roll at the G6G7•C19C18 base pair step, slide at the G7C8•C18G17 base pair step, G6G7 dihedral angle, and overall bend angle. We hypothesize that these conformational differences may be related to the ability of various DNA repair proteins, DNA binding proteins, and DNA polymerases to discriminate between cisplatin-GG and oxaliplatin-GG adducts. † This work was supported by NIH Grant CA8440 (to S.G.C.) and NIEHS Grant P30ES10126 (to J.A.S.). ‡ Coordinates are available from the Protein Data Bank (PDB): 2NPW for the averaged structure of the CP-GG adduct and 2NQ0 for the family of the 15 best structures, 2NQ1 for the averaged structure of undamaged DNA in the AGGC sequence context, and 2NQ4 for the family of the 15 best structures.

Platinum chemotherapeutic agents have been widely used in the treatment of cancer. Cisplatin was the first of the platinum based chemotherapeutic agents and therefore has been extensively studied as an anti-tumor agent since the late 1960s. Because this agent forms several DNA adducts, a highly sensitive and specific quantitative assay is needed to correlate the molecular dose of individual adducts with the effects of treatment. An ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for quantification of 1,2 guanine-guanine intrastrand cisplatin adducts [CP-d(GpG)], using 15 N 10 CP-d(GpG) as an internal standard, was developed. The internal standard was characterized by MS/MS and its concentration was validated by ICP-MS. Samples containing CP-d(GpG) in DNA were purified by enzyme hydrolysis , centrifugal filtration and HPLC with fraction collection prior to quantification by UPLC-MS/MS in the selective reaction monitoring (SRM) mode (m/z 412.5→248.1 for CP-d(GpG); m/z 417.5→253.1 for [ 15 N 10 ] CP-d(GpG)). Recovery of standards was >90% and quantification was unaffected by increasing concentrations of calf thymus DNA. This method utilizes 25 μg of DNA per injection. The limit of quantification was 3 fmol or 3.7 adducts per 10 8 nucleotides, which approaches the sensitivity of the 32 P postlabeling method for this adduct. These data suggested that this method is suitable for in vitro and in vivo assessment of CP-d(GpG) adducts formed by cisplatin and carboplatin. Subsequently the method was applied to studies using ovarian carcinoma cell lines and C57/BL6 mice to illustrate that this method is capable of quantifying CP-d(GpG) adducts using biologically relevant systems and doses. The development of biomarkers to determine tissue-specific molecular dosimetry during treatment will lead to a more complete understanding of both therapeutic and adverse effects of cisplatin and carboplatin. This will support the refinement of therapeutic regimes and appropriate individualized treatment protocols.

The differences in efficacy and molecular mechanisms of platinum anti-cancer drugs cisplatin (CP) and oxaliplatin (OX) are thought to be partially due to the differences in the DNA conformations of the CP and OX adducts that form on adjacent guanines on DNA, which in turn influence the binding of damage-recognition proteins that control downstream effects of the adducts. Here we report a comprehensive comparison of the structural distortion of DNA caused by CP and OX adducts in the TGGT sequence context using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations. When compared to our previous studies in other sequence contexts, these structural studies help us understand the effect of the sequence context on the conformation of Pt-GG DNA adducts. We find that both the sequence context and the type of Pt-GG DNA adduct (CP vs. OX) play an important role in the conformation and the conformational dynamics of Pt-DNA adducts, possibly explaining their influence on the ability of many damage-recognition proteins to bind to Pt-DNA adducts.

Purpose To characterize the cellular action mechanism of Debio 0507, we compared the major DNA adducts formed by Debio 0507- and oxaliplatin-treated HCT116 human colon carcinoma cells by a combination of inductively coupled plasma mass spectrometry (ICP-MS) and ultra-performance liquid chromatography mass spectrometry (UPLC-MS/MS). Methods HCT116 cells were treated with IC50 doses of Debio 0507 or oxaliplatin for 3 days. Total cellular Pt–DNA adducts were determined by ICP-MS. The DNA was digested, and the major Pt–DNA adducts formed by both drugs were characterized by UPLC/MS/MS essentially as described previously for cisplatin (Baskerville-Abraham et al. in Chem Res Toxicol 22:905–912, 2009). Results The Pt level/deoxynucleotide was 7.4/104 for DNA from Debio 0507-treated cells and 5.5/104 for oxaliplatin-treated cells following a 3-day treatment at the IC50 for each drug. UPLC-MS/MS in the positive ion mode confirmed the major Pt–DNA adducts formed by both drugs were dach-Pt-d(GpG) (904.2 m/z → 610 m/z and 904.2 m/z → 459 m/z) and dach-Pt-d(ApG) (888.2 m/z → 594 m/z and 888.2 m/z → 459 m/z). Conclusions These data show that the major DNA adducts formed by Debio 0507 are the dach-Pt-d(GpG) and dach-Pt-d(ApG) adducts and at equitoxic doses Debio 0507 and oxaliplatin form similar levels of dach-Pt-d(GpG) and dach-Pt-d(ApG) adducts. This suggests that the action mechanisms of Debio 0507 and oxaliplatin are similar at a cellular level.

The major DNA adducts formed by Debio 0507 in HCT116 human colon carcinoma cells were determined by a combination of inductively coupled mass spectrometry (ICP-MS) and ultraperformance liquid chromatography mass spectrometry (UPLC-MS/MS). Debio 0507 is a micellular copolymer of trans-RR-diaminocyclohexane-platinum(II) (DACH-Pt) with polyethylene-glycol (PEG)-polyglutamate (pGlu). Debio 0507 is long lasting compared to oxaliplatin (platinum is detectable in plasma and tumor for up to 14 days) and has shown anti-tumor activity and low toxicity in several tumor models. Debio 0507 has been shown to accumulate in tumors, but the intracellular activation mechanism and type of DNA adducts formed were not previously known. We have previously reported an UPLC-MS/MS method for the identification and quantification of the intrastrand GG DNA adducts formed by cisplatin (Baskerville-Abraham et al, Chem. Res. Toxicol., 22: 905-912, 2009). The limit of quantification of this assay was 3 fmol or 3.7 adducts per 108 nucleotides. This assay was modified slightly to identify the major DNA adducts formed by Debio 0507 and to compare them to the major DNA adducts formed by oxaliplatin. HCT116 cells were incubated with equitoxic doses of Debio 0507 (0.014 ug Pt/ml) and oxaliplatin (0.0006 ug Pt/ml) for 3 days. DNA was isolated from the cells and digested as described previously for cisplatin-treated DNA. ICP-MS was used to quantify Pt adducts in the DNA digests. The Pt adducts were 7.4/104 deoxynucleosides for Debio 0507-treated cells and 5.5/104 deoxynucleosides for oxaliplatin-treated cells following the DNA digestion step. DACH-Pt-d(GpG) and DACH-Pt-d(ApG) adducts were then separated from deoxynucleosides by HPLC. Following HPLC purification, the total Pt DNA adducts recovered were 63 pmoles (29% recovery) for Debio 0507 and 58 pmoles (38% recovery) for oxalipltin by ICP-MS, with approximately equal amounts of DACH-Pt-d(GpG) and DACH-Pt-d(ApG) adducts recovered at this stage (The DACH-Pt-d(GpG) peak overlapped with the deoxythymidine peak, which diminished the recovery of DACH-Pt-d(GpG)). Finally, UPLC-MS/MS in the positive ion mode was used to confirm the identity of the DACH-Pt-d(GpG) (904.2 m/z→459 m/z and 904.2 m/z→610 m/z) and DACH-Pt-d(ApG) (888.2 m/z→459 m/z and 888.2 m/z→594 m/z). These data show that the major DNA adducts formed by Debio 0507 are the DACH-Pt-GG and DACH-Pt-AG adducts and at equitoxic doses Debio 0507 and oxaliplatin form similar levels of DACH-Pt-GG and DACH-Pt-AG adducts which makes it unlikely that Debio 0507 forms significant amounts of other DNA adducts of equal or greater cytotoxicity. (Supported by Research Contract Debio 0507-069 with Debiopharm SA and P30-ES10126) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3504.