title: Clerocidin alkylates DNA through its epoxide function: evidence for a fine tuned mechanism of action.
abstract: Clerocidin (CL) is an effective topoisomerase II-poison, which has been shown to produce DNA depurination and strand breaks per se at the guanine (G) level. To elucidate the roles played by the different functional groups of CL in the reactivity towards nucleic acids, we investigated CL derivatives with key structural modifications. The derivatives were reacted with plasmid, single-/double-stranded DNA and isolated 2'-deoxy-guanosines (dG). We show here that an intact oxirane ring is essential to achieve DNA modification and depurination. Through HPLC/MS and MS/MS techniques we were able to unambiguously characterize adducts obtained by reacting isolated dG and single-/double-stranded DNA with the drugs, indicating beyond reasonable doubt that the structure of a typical adduct is formed by epoxide alkylation at N7 of G with subsequent loss of the pentose unit. Further, we showed that reduction of vicinal carbonyl functions affect drug activity to a large extent. Our findings demonstrate that the characteristic DNA-alkylating properties of CL arise from mutual action of the functional groups present in this molecule. Its oxidation state seems crucial to modulate the rates of reactivity by finely tuning the strain applied on the oxirane ring.
authors.affiliation: Department of Pharmaceutical Sciences, University of Padova, via Marzolo 5, 35131 Padova, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Richter
authors.forename: Barbara
authors.initials: B
authors.lastname: Gatto
authors.forename: Daniele
authors.initials: D
authors.lastname: Fabris
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Takao
authors.forename: Susumu
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Manlio
authors.initials: M
authors.lastname: Palumbo
mesh_terms: D000477:Alkylating Agents / Q000737:chemistry* / Q000494:pharmacology; D000478:Alkylation; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D018736:DNA Adducts / Q000187:drug effects / Q000378:metabolism; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000187:drug effects / Q000378:metabolism; D004224:Diterpenes / Q000737:chemistry* / Q000494:pharmacology; D004852:Epoxy Compounds / Q000737:chemistry*; D006147:Guanine / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D015394:Molecular Structure; D010957:Plasmids / Q000187:drug effects / Q000378:metabolism
chemical_list: D000477:Alkylating Agents; D018736:DNA Adducts; D004277:DNA, Single-Stranded; D004224:Diterpenes; D004852:Epoxy Compounds; D006147:Guanine; C039027:clerocidin; D004247:DNA