Formaldehyde-activated WEHI-150 induces DNA interstrand crosslinks with unique structural features
Graphical abstract
Introduction
Small molecular weight agents that covalently modify DNA have long been applied in the treatment of human cancers.1 Covalent bonding compounds can be classified into those that (i) are bifunctional cross-linking agents; (ii) result in monofunctional covalent bonding of the compound to one site of the DNA; and (iii) bind covalently to DNA via secondary endogenous bridging compounds (e.g. anthracyclines which bind covalently to DNA via formaldehyde).2
The identification of this final sub-group of covalent bonding by X-ray diffraction studies3 has shed further light on this particular mechanism of activity of anticancer agents. Double stranded DNA sequences that serve as cross-linking sites for atomic interaction with drugs tend to occur preferentially at locations that minimally distort the conformation of B-DNA.4 Anthracenediones, the synthetic analogues of anthracyclines, also have the potential to induce anticancer activity via this same mechanism and utilize a bridging compound for activation.5, 6 By forming functional cross-links with DNA, this group of drugs interferes with basic cellular processes such as transcription, replication and DNA repair, and subsequently culminate in apoptosis.2, 7 This exceptional benefit has been potentiated by the availability of the methylene-donating molecule, formaldehyde, which can be made bio-available by formaldehyde-releasing prodrugs8 or by the synthesis of formaldehyde-preactivated compounds such as doxoform and doxazolidine.9 For anthracyclines and anthracenediones, formaldehyde is an absolute requirement for DNA adduct formation.3, 10 Formaldehyde-mediated covalent adduct formation by the anthracenedione parent compound mitoxantrone (Fig. 1) provides a possible basis for its anticancer activity against leukaemias which have higher than usual inherent formaldehyde levels.11
Pixantrone (Fig. 1) is a 2-aza-anthracenedione with terminal primary amino moieties in its side chain was approved as a monotherapy for Non-Hodgkin’s lymphoma in Europe in 2012, and has shown improved adduct forming potential relative to mitoxantrone.12, 13 Both mitoxantrone and pixantrone have demonstrated preferential adduct formation at CpG and CpA sites in contrast to the GpC sequence specificity displayed by the anthracyclines.6, 14, 15 Moreover, the specific epigenetic modification imparted by methylation of CpG sequences has been shown to result in greatly enhanced adduct formation by both drugs.16, 17 While pixantrone is superior in terms of DNA adduct formation, mitoxantrone displays additional initial modes of non-covalent DNA interactions.18 Previous studies such as these indicated that inclusion of the mitoxantrone chromophore along with long side chain-length terminating in primary amino groups were important structural considerations for an ideal anthracenedione with the potential for not only forming high levels of DNA adducts, but also highly stable adducts. To this end, hybrid compounds containing a mitoxantrone chromophore combined with the terminal primary amino groups of pixantrone were synthesised with symmetrical carbon side chains of varying lengths and analysed for their DNA interacting properties.19, 20
Further refinement of anthracenedione side chain chemistry led to the synthesis of WEHI-150 in which the terminal hydroxyl group of mitoxantrone has been replaced with an amine group (Fig. 1).21, 22 HPLC was used to investigate the interaction of WEHI-150 with oligonucleotides containing CpG or methylated CpG sequences. Like mitoxantrone and pixantrone, it was shown that WEHI-150 forms covalent adducts at CpG sequences and exhibits a preference for methylated CpG sites.21 Intriguingly, mass spectral analysis revealed that WEHI-150 was able to form up to three formaldehyde-mediated linkages with oligonucleotide substrates containing CpG repeats, as distinct from mitoxantrone and pixantrone that could only form single covalent linkages.21 NMR analysis of the major covalent adduct between WEHI-150 and a methylated CpG repeat hexanucleotide revealed only one covalent bond with the N-2 of guanine, yet demonstrated that one side chain is in the major groove, with the other in the minor groove. Since WEHI-150 exhibits unique properties with respect to covalent adduct formation, it is expected to possess unique biological activities. It is hypothesised that WEHI-150 can react with multiple bases at multiple DNA sites, with a portion of these adducts comprising crosslinks. Here we used a series of related compounds to investigate the structural requirements for covalent adduct formation, and in particular crosslink formation, on a series of heterogeneous and base-modified DNA substrates. A major focus was to examine the DNA sequence specificity of WEHI-150 in order to examine the impact of multiple covalent linkages to DNA bases.
Section snippets
Materials
Formaldehyde solution (40% v/v) and chloroform were obtained from Merck (Darmstadt, Germany). Calf thymus DNA was purchased from Worthington Biochemical Corporation (Lakewood NJ, USA). A Maxi Plasmid Purification Kit was purchased from Qiagen (Valencia CA, USA). Plasmid pCC1 containing the lac UV5 promoter sequence has been previously described.23 Ultra-pure buffered phenol was purchased from Invitrogen (Carlsbad CA, USA). Ultra-pure dNTPs, [α32P]-dATP (3000 Ci/mmol), [γ32P]-dATP (3000 Ci/mmol)
Results
To undertake a systematic approach in exploring the unique properties of WEHI-150, various control compounds were utilised in the study for comparison as presented in Fig. 1. Since mitoxantrone is known to link to DNA via secondary amino groups located on its side arms, the adducts formed are inherently unstable.10, 13, 20 However, Mito2 exhibits the same DNA sequence specificity as mitoxantrone20 while incorporating side arm primary amino groups like WEHI-150 (Fig. 1) that endow Mito2 adducts
Discussion
The anthracycline and anthracenedione families of anticancer agents are known to mainly exert their biological activities through DNA intercalation and subsequent poisoning of topoisomerases. However, in the presence of formaldehyde, they can bind covalently to DNA and thus work through an alternative mechanism of action. This has been well documented for doxorubicin whereby the formaldehyde-releasing prodrug AN-9 facilitates doxorubicin DNA adducts, with combined doxorubicin and AN-9 treatment
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by grants from the NHMRC, ARC and the CASS Foundation (SMC and DRP), an AusAID ADS Scholarship and also support from the La Trobe University Postgraduate Write-up Award (PWA). BES is a Corin Centenary Fellow. BJE is an employee of Nyrada Inc and declares no conflict of interest.
References (37)
- et al.
Chemical cross-linking of drugs to DNA
MethodsEnzymol
(2001) Sequence preferences of DNA interstrand cross-linking agents – importance of minimal DNA structural reorganization in the cross-linking reactions of mechlorethamine, cisplatin, and Mitomycin-C
Tetrahedron
(1991)A molecular understanding of mitoxantrone-DNA adduct formation: effect of cytosine methylation and flanking sequences
J Biol Chem
(2004)- et al.
Cytosine methylation enhances mitoxantrone-DNA adduct formation at CpG dinucleotides
J Biol Chem
(2001) - et al.
Correlation of DNA reactivity and cytotoxicity of a new class of anticancer agents: aza-anthracenediones
CancerLett
(1995) M2, a novel anthracenedione, elicits a potent DNA damage response that can be subverted through checkpoint kinase inhibition to generate mitotic catastrophe
Biochem Pharmacol
(2011)Isolation and structural analysis of the covalent adduct formed between a bis-amino mitoxantrone analogue and DNA: a pathway to major-minor groove cross-linked adducts
Org Biomol Chem
(2016)Reversible and formaldehyde-mediated covalent binding of a bis-amino mitoxantrone analogue to DNA
Org Biomol Chem
(2016)Mass spectrometric investigation of the DNA-binding properties of an anthracycline with two trisaccharide chains
Arch Biochem Biophys
(2008)Binding of pixantrone to DNA at CpA dinucleotide sequences and bulge structures
Org Biomol Chem
(2015)