Conjugation of Mono-Functional Quinone Methides to DNA Ligands for Promotion of Reversible Alkylation
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Date
2017-02-22
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Johns Hopkins University
Abstract
Quinone methides (QMs) are electrophilic intermediates that alkylate DNA through reaction with the nucleobases. An initial set of kinetic products allow for regeneration of the QM electrophiles which eventually go on to form an alternate set of thermodynamic adducts. Reversibility of QM-DNA adducts is of interest because regeneration of the reactive electrophile may facilitate evasion of DNA repair following excision of adducts. This feature extends the lifetime of a QM drug and is highly advantageous when targeting tumors because the formation of multiple lesions in series by a single molecule lowers the effective dose that is required to trigger apoptosis.
Previous studies in the Rokita lab showed that conjugation of mono-functional quinone methides (mono-QMs) to sequence directing oligonucleotides allowed for reversible alkylation of complementary strands. Although the sequence directing ligands minimizes indiscriminate reactions with endogenous nucleophiles, reversible alkylation is limited to targeted regions of a duplex and QMs are subject to loss of DNA associating affinity if oligonucleotide ligands are digested by nucleases.
Mono-quinone methides (mono-QM) have now been conjugated to acridine and ammonium ligands that allow the electrophile to move freely along duplex DNA without sequence constraints. Both acridine and diammonium conjugated mono-QMs have been synthesized and shown to alkylate dsDNA reversibly at dG-N7 via transfer of regenerated QMs. The mono-QM acridine conjugate (QMPAc) migrates along dsDNA via formation of reversible adducts and the electrophile was able to overcome kinetic barriers without stalling at nicks and bulges. On the other hand, the mono-QM diammonium conjugate (QMPDA) provided no evidence of migration. The ability of QMPAc to migrate past nicks and bulges suggest that it could also migrate along other nucleic acid structures such as tRNAs, ribozymes or DNAzymes. This may lead to applications of mono-QMs as inhibitors cellular processes of DNA and RNA synthesis.
Formation of dG-N7 adducts by an unsubstituted mono-QM partitions between regeneration of QM and deglycosylation to form G-N7. Within dsDNA, deglycosylation of dG-N7 adducts results in an abasic sites, which destabilize duplexes and may prevent transfer for regenerated QMs between complementary strands. In order to determine if a higher prevalence of deglycosylated adducts limited migration of QMPDA over QMPAc, the percentage of abasic sites generated in the presence of each mono-QM was quantified. Studies have now shown that both QMPAc and QMPDA generate similar percentages of abasic sites in ssDNA, while the percentage of abasic sites generated in dsDNA was higher in QMPAc than QMPDA. Since the QMPAc provided evidence of migration, results from this study suggest that abasic site generation may not be the only factor that impedes migration of QMPDA.
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Keywords
DNA, Alkylation