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 modi®cations. 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 modi®cation 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 N 7 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 ®ndings 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 ®nely tuning the strain applied on the oxirane ring.

Clerocidin interacts with the cleavage complex of Streptococcus pneumoniae topoisomerase IV to induce selective irreversible DNA damage

“…The C12–C15 moiety of the drug is responsible for the cleavage activity; in particular, spontaneous DNA fragmentation derives from depurination at the G level caused by the electrophilic attack of the epoxide ring of CL on the N7 group of exposed Gs ( 6 ). The hemi-acetalic form of the α-ketoaldehyde function at C14–C15 enhances epoxide reactivity by applying additional strain to the oxirane ring ( 7 ). On the other hand, the diterpenoid portion of CL is dispensable for drug activity per se ; in fact the NA1 analogue of CL ( Figure 1 ), which preserves the C12–C15 ring but has the diterpenoid function substituted with naphthalene retains intrinsic DNA cleavage activity ( 7 ).…”

Differential Targeting of Unpaired Bases within Duplex DNA by the Natural Compound Clerocidin: A Valuable Tool to Dissect DNA Secondary Structure

“…The natural compound CL had been previously shown to be able to detect and induce cleavage in partially ss regions within supercoiled plasmids, whereas it resulted completely inert versus ds nucleic acids [17], [18]. Here we showed that CL could detect the presence of non-paired sequences, when at least one ss non-T base was available, in a number of non-canonical DNA conformations.…”

“…However, total synthesis of CL has been reported [38] and a structural analogue, in which the diterpenoid moiety is replaced by a naphthalene ring while preserving base selectivity and reactivity, can be easily synthesized [18], [19], [20].…”

“…In particular, CL electrophilic groups (i.e. a strained epoxy ring and an α-ketoaldehyde function in equilibrium with its hemi-acetalic form) target i) the nucleophilic N7 of guanine (G) inducing spontaneous depurination and DNA strand cleavage [17], [18], ii) the NH 2 and N3 of cytosine (C) with formation of a stable condensed ring system, which is degraded to induce DNA cleavage only after hot alkali treatment [19], and iii) the NH 2 and N1 of adenine (A) to generate an adduct that degrades upon alkali but does not result in DNA strand scission [20]. Due to lack of strong nucleophilic sites, thymine (T) does not react with CL.…”

Implementation of data‐dependent acquisitions in the study of melphalan DNA adducts by miniaturized liquid chromatography coupled to electrospray tandem mass spectrometry

“…After the initial adduct formation other processes may occur. For example, alkylation of the N7‐position in 2′‐deoxyguanosine often leads to deglycosylation11 and sometimes to imidazole ring opening 9. In order to identify all these different theoretically possible structures a classical LC/MS/MS approach is quite time‐ and sample‐consuming.…”