title: Virtual Cross-Linking of the Active Nemorubicin Metabolite PNU-159682 to Double-Stranded DNA.
abstract: The DNA alkylating mechanism of PNU-159682 (PNU), a highly potent metabolite of the anthracycline nemorubicin, was investigated by gel-electrophoretic, HPLC-UV, and micro-HPLC/mass spectrometry (MS) measurements. PNU quickly reacted with double-stranded oligonucleotides, but not with single-stranded sequences, to form covalent adducts which were detectable by denaturing polyacrylamide gel electrophoresis (DPAGE). Ion-pair reverse-phase HPLC-UV analysis on CG rich duplex sequences having a 5'-CCCGGG-3' central core showed the formation of two types of adducts with PNU, which were stable and could be characterized by micro-HPLC/MS. The first type contained one alkylated species (and possibly one reversibly bound species), and the second contained two alkylated species per duplex DNA. The covalent adducts were found to produce effective bridging of DNA complementary strands through the formation of virtual cross-links reminiscent of those produced by classical anthracyclines in the presence of formaldehyde. Furthermore, the absence of reactivity of PNU with CG-rich sequence containing a TA core (CGTACG), and the minor reactivity between PNU and CGC sequences (TACGCG·CGCGTA) pointed out the importance of guanine sequence context in modulating DNA alkylation.
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova , Via Marzolo, 5, 35131 Padova, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Scalabrin
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova , Via Marzolo, 5, 35131 Padova, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Quintieri
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova , Via Marzolo, 5, 35131 Padova, Italy.
authors.forename: Manlio
authors.initials: M
authors.lastname: Palumbo
authors.affiliation: Oncology-Chemical Core Technologies Department, Nerviano Medical Sciences , viale Pasteur 10, Nerviano, 20014 Milano, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Riccardi Sirtori
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova , Via Marzolo, 5, 35131 Padova, Italy.
authors.forename: Barbara
authors.identifier: 0000-0001-9465-6913
authors.initials: B
authors.lastname: Gatto
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry*; D018736:DNA Adducts / Q000737:chemistry; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D013056:Spectrophotometry, Ultraviolet
chemical_list: C502356:3'-deamino-3'',4'-anhydro-(2''-methoxy-3''-oxy-4''-morpholinyl)doxorubicin; D018736:DNA Adducts; D004317:Doxorubicin; D004247:DNA