Acridinecarboxamide Topoisomerase Poisons: Structural and Kinetic Studies of the DNA Complexes of 5-Substituted 9-Amino-(N-(2-dimethylamino)ethyl)acridine-4-Carboxamides

Abstract

For a series of antitumor-active 5-substituted 9-aminoacridine-4-carboxamide topoisomerase II poisons, we have used X-ray crystallography and stopped-flow spectrophotometry to explore relationships between DNA binding kinetics, biological activity, and the structures of their DNA complexes. The structure of 5-F-9-amino-[N-(2-dimethylamino)ethyl]-acridine-4-carboxamide bound to d(CGTACG)(2) has been solved to a resolution of 1.55 A in space group P6(4). A drug molecule intercalates between each of the CpG dinucleotide steps, its protonated dimethylamino group partially occupying positions close to the N7 and O6 atoms of guanine G2 in the major groove. A water molecule forms bridging hydrogen bonds between the 4-carboxamide NH and the phosphate group of the same guanine. Intercalation unwinds steps 1 and 2 by 12 degrees and 8 degrees, respectively compared with B-DNA, whereas the central TpA step is overwound by 10 degrees. Nonphenyl 5-substituents, on average, decrease mean DNA dissociation rates by a factor of three, regardless of their steric, hydrophobic, H-bonding, or electronic properties. Cytotoxicity is enhanced on average 4-fold and binding affinities rise by 3-fold, thus there is an apparent association between kinetics, affinity, and cytotoxicity. Taken together, the structural and kinetic studies imply that the main origin of this association is enhanced stacking interactions between the 5-substituent and cytosine in the CpG binding site. Ligand-dependent perturbations in base pair twist angles and their consequent effects on base pair-base pair stacking interactions may also contribute to the stability of the intercalated complex. 5-Phenyl substituents modify dissociation rates without affecting affinities, and variations in their biological activity are not correlated with DNA binding properties, which suggests that they interact directly with the topoisomerase protein.

Molecular modeling study of intercalation complexes of tricyclic carboxamides with d(CCGGCGCCGG)2 and d(CGCGAATTCGCG)2

“…The sequence was selected according to the preference for intercalation at 5'-CpG-3' steps exhibited by monointercalating acridine derivatives to DNA and to modeling experience [15][16][17][30][31][32][33][34][35] with related intercalating agents.…”

“…In each case, a hydrogen-bonded water molecule bridges the NH of the carboxamide group to the guanine G2 phosphate at the intercalation site. These structures have provided a molecular rationale for understanding the structure-activity relationships for antitumor activity and enabled a mechanistic interpretation of the dependence of kinetics on ligand structure [16,17]. In particular, they have permitted the critical step in the dissociation kinetics profile that correlates with cytotoxicity and antitumor activity to be identified with the side chain-guanine interaction [15][16][17].…”

“…Some of these compounds have been determined by X-ray crystallography. In the crystal structures of 9-amino-DACA and its 5-fluoro derivative bound to the hexanucleotide d(CGTACG) 2 [16,17] , and of 6-Br-9-amino-DACA bound to the brominated hexanucleotide d(CG 5Br UACG) 2 [18], the 4-carboxamide group lies in the plane of the chromophore, and the carbonyl oxygen atom forms an internal hydrogen bond with the protonated N10 nitrogen of the acridine ring. The side chain lies in the DNA major groove with its protonated N,N-dimethylamino group forming hydrogen-bonding interactions with the O6 and N7 atoms of guanine G2.…”

“…These structures have provided a molecular rationale for understanding the structure-activity relationships for antitumor activity and enabled a mechanistic interpretation of the dependence of kinetics on ligand structure [16,17]. In particular, they have permitted the critical step in the dissociation kinetics profile that correlates with cytotoxicity and antitumor activity to be identified with the side chain-guanine interaction [15][16][17]. Attempts to crystallize DACA, its phenazine analogue and related agents with d(CGTACG) 2 have yielded an unusual quadruplex which does not add to our understanding of their complexes with duplex DNA [19][20][21], and other DNA sequences have failed to give diffracting crystals.…”

The role of topoisomerases and RNA transcription in the action of the antitumour benzonaphthyridine derivative SN 28049

“…Mithramycin A and related analogues constituted a second class of antibiotic cytotoxins that bound by a novel mechanism to GC-rich DNA [5] but clinical trials demonstrated limited clinical activity, probably because of multiple cellular actions, poor pharmacological properties and susceptibility to multidrug resistance. N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA), a compound in which the acridine moiety provides DNA intercalation ability and the carboxamide side chain provides GCselectivity, was first synthesised in this laboratory and has undergone phase I and II clinical trials [6][7][8][9][10][11][12][13], but low dose potency and an unusual form of toxicity prevented further development [14]. DACA had effects on both topoisomerase (topo) I and topo II [15,16] but a variety of evidence suggested that the principal target was topo IIα [15,17,18].…”