Monica Cacho | PubMed Health

PubMed2003

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 (CH(2))(2)NH(CH(2))(3)NH(CH(2))(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 approximately 10 times higher than that of the methylated dimer. The DNA groove accessibility was also probed with three related oligonucleotides carrying G --> c(7)G, G --> I, and C --> M substitutions. The level of drug binding to the two hairpin oligonucleotides containing 7-deazaguanine (c(7)G) 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.

Adenine↗

PubMed2003

Chromophore-modified bisnaphthalimides: DNA recognition, topoisomerase inhibition, and cytotoxic properties of two mono- and bisfuronaphthalimides.

Bisnaphthalimides represent a promising group of DNA-targeted anticancer agents. In this series, the lead compounds elinafide and bisnafide have reached clinical trials, and the search for more potent analogues remains a priority. In the course of a medicinal chemistry program aimed at discovering novel antitumor drugs based on the naphthalimide skeleton, different dimeric molecules containing two tetracyclic neutral DNA intercalating chromophores were synthesized. The naphthalimide unit has been fused to a benzene ring (azonafide derivatives), an imidazole, a pyrazine, or, as reported here, a furan ring which increases the planar surface of the chromophore and enhances its stacking properties. We report a detailed investigation of the DNA binding capacity of the dimeric molecule MCI3335 composed of two furonaphthalimide units connected by a 12 A long amino alkyl linker [(CH(2))(2)-NH-(CH(2))(3)-NH-(CH(2))(2)] identical to that of elinafide. Qualitative and quantitative binding studies, in particular using surface plasmon resonance, establish that the dimer binds considerably more tightly to DNA (up to 1000 times) than the corresponding monomer and exhibits a higher sequence selectivity for GC-rich sequences. DNase I footprinting experiments attest that the dimer, and to a lesser extent the monomer, preferentially intercalate at GC sites. The strong binding interaction between the drugs and DNA perturbs the relaxation of supercoiled DNA by topoisomerases, but the test compounds do not promote DNA cleavage by topoisomerase I or II. Despite the lack of poisoning effect toward topoisomerase II, MCI3335 displays a very high cytotoxicity toward CEM human leukemia cells, with an IC(50) in the low nanomolar range, approximately 4 times inferior to that of the reference drug elinafide. Confocal microscopy observations indicate that the monomer shows a stronger tendency to accumulate in the cell nuclei than the dimer. The extremely high cytotoxic potential of MCI3335 is attributed to its enhanced capacity to bind to DNA and to inhibit DNA synthesis, as evidenced by flow cytometry experiments using the BrdU assay. The results provide novel mechanistic information that furthers the understanding of the structure-activity relationships in the bisnaphthalimide series and identify MCI3335 as a novel lead compound for further preclinical investigations.

Base Sequence↗