DNA Sequence Recognition by Bispyrazinonaphthalimides Antitumor Agents^†

Bifunctional DNA intercalating agents have long attracted considerable attention as anticancer agents. One of the lead compounds in this category is the dimeric antitumor drug elinafide, composed of two tricyclic naphthalimide chromophores separated by an aminoalkyl linker chain optimally designed to permit bisintercalation of the drug into DNA. In an effort to optimize the DNA recognition capacity, different series of elinafide analogues have been prepared by extending the surface of the planar drug chromophore which is important for DNA sequence recognition. We report here a detailed investigation of the DNA sequence preference of three tetracyclic monomeric or dimeric pyrazinonaphthalimide derivatives. Melting temperature measurements and surface plasmon resonance (SPR) studies indicate that the dimerization of the tetracyclic planar chromophore considerably augments the affinity of the drug for DNA, polynucleotides, or hairpin oligonucleotides and promotes selective interaction with G·C sites. The (CH₂)₂NH(CH₂)₃NH(CH₂)₂ connector stabilizes the drug−DNA complexes. The methylation of the two nitrogen atoms of this linker chain reduces the binding affinity and increases the dissociation rates of the drug−DNA complexes by a factor of 10. DNase I footprinting experiments were used to investigate the sequence selectivity of the drugs, demonstrating highly preferential binding to G·C-rich sequences. It also served to select a high-affinity site encompassing the sequence 5‘-GACGGCCAG which was then introduced into a biotin-labeled hairpin oligonucleotide to accurately measure the binding parameters by SPR. The affinity constant of the unmethylated dimer for this sequence is 500 times higher than that of the monomer compound and ∼10 times higher than that of the methylated dimer. The DNA groove accessibility was also probed with three related oligonucleotides carrying G → c⁷G, G → I, and C → M substitutions. The level of drug binding to the two hairpin oligonucleotides containing 7-deazaguanine (c⁷G) or 5-methylcytosine (M) residues is unchanged or only slightly reduced compared to that of the unmodified target. In contrast, incorporation of inosine (I) residues considerably decreases the extent of drug binding or even abolishes the interaction as is the case with the monomer. The pyrazinonaphthalimide derivatives are thus much more sensitive to the deletion of the exocyclic guanine 2-amino group exposed in the minor groove of the duplex than to the modification of the major groove elements. The complementary SPR footprinting methodology combining site selection and quantitative DNA affinity analysis constitutes a reliable method for dissecting the DNA sequence selectivity profile of reversible DNA binding small molecules.