Pixantrone can be activated by formaldehyde to generate a potent DNA adduct forming agent

Abstract

Mitoxantrone is an anti-cancer agent used in the treatment of breast and prostate cancers. It is classified as a topoisomerase II poison, however can also be activated by formaldehyde to generate drug–DNA adducts. Despite identification of this novel form of mitoxantrone–DNA interaction, excessively high, biologically irrelevant drug concentrations are necessary to generate adducts. A search for mitoxantrone analogues that could potentially undergo this reaction with DNA more efficiently identified Pixantrone as an ideal candidate. An in vitro crosslinking assay demonstrated that Pixantrone is efficiently activated by formaldehyde to generate covalent drug–DNA adducts capable of stabilizing double-stranded DNA in denaturing conditions. Pixantrone–DNA adduct formation is both concentration and time dependent and the reaction exhibits an absolute requirement for formaldehyde. In a direct comparison with mitoxantrone–DNA adduct formation, Pixantrone exhibited a 10- to 100-fold greater propensity to generate adducts at equimolar formaldehyde and drug concentrations. Pixantrone–DNA adducts are thermally and temporally labile, yet they exhibit a greater thermal midpoint temperature and an extended half-life at 37°C when compared to mitoxantrone–DNA adducts. Unlike mitoxantrone, this enhanced stability, coupled with a greater propensity to form covalent drug–DNA adducts, may endow formaldehyde-activated Pixantrone with the attributes required for Pixantrone–DNA adducts to be biologically active.

Formaldehyde-Activated Pixantrone Is a Monofunctional DNA Alkylator That Binds Selectively to CpG and CpA Doublets

“…In Vitro Cross-Linking Assay. The pCC1 plasmid was linearized with HindIII and 3Ј-end-labeled as described previously (Evison et al, 2007). Multiple reactions were run in parallel, each containing end-labeled DNA (25 M bp ), 2 mM formaldehyde and either pixantrone (0-20 M) or BBR 2378 (0-100 M) in PBS, pH 7.0.…”

“…Multiple reactions were run in parallel, each containing end-labeled DNA (25 M bp ), 2 mM formaldehyde and either pixantrone (0-20 M) or BBR 2378 (0-100 M) in PBS, pH 7.0. Reactions were run at 37°C for 4 h, phenol/chloroform-extracted, and subsequently prepared for electrophoresis as described previously (Evison et al, 2007). Samples were subjected to electrophoresis through 0.8% agarose gels overnight at 30 to 40 V in 1ϫ Tris-acetate/ EDTA buffer.…”

“…Previous characterization of pixantrone-DNA adducts using an in vitro cross-linking assay established that the lesion is intrinsically unstable (Evison et al, 2007). The in vitro transcription assay provides an ideal alternative for the assessment of adduct lability at clearly defined DNA sites.…”

“…Like its parent compound mitoxantrone, pixantrone intercalates within DNA and impairs topoisomerase II activity by stimulating enzyme-mediated DNA cleavage (De Isabella et al, 1995). Formaldehydeactivated pixantrone can covalently bind to DNA more efficiently than the analogous mitoxantrone reaction (Evison et al, 2007). Pixantrone possesses primary amino functionalities in both side arms, and this seems to provide an improvement over the secondary amino functionalities inherent to mitoxantrone.…”

The Servier oncology pipeline in 2017

“…Pixantrone is an aza-athracenedione that alkylates DNA directly, forming stable DNA adducts and inducing double-strand DNA breaks, thereby inhibiting DNA replication, transcription and repair [21][22][23][24][25][26]; in addition, pixantrone-induced DNA damage also appears to impair mitosis [27,28]. Cardiotoxicity is an issue with anthracyclines, via iron-dependent formation of reactive oxygen species, and topoisomerase IIβ-mediated DNA damage response [25].…”