Intercalative DNA binding of the marine anticancer drug variolin B.

Variolin B is a rare marine alkaloid that showed promising anti-cancer activity soon after its isolation. It acts as a cyclin-dependent kinase inhibitor, although the precise mechanism through which it exerts the cytotoxic effects is still unknown. The crystal structure of a variolin B bound to a DNA forming a pseudo-Holliday junction shows that this compound can also contribute, through intercalative binding, to either the formation or stabilization of multi-stranded DNA forms.

The inherent properties of DNA four-way junctions: comparing the crystal structures of holliday junctions.

Holliday junctions are four-stranded DNA complexes that are formed during recombination and related DNA repair events. Much work has focused on the overall structure and properties of four-way junctions in solution, but we are just now beginning to understand these complexes at the atomic level. The crystal structures of two all-DNA Holliday junctions have been determined recently from the sequences d(CCGGGACCGG) and d(CCGGTACCGG). A detailed comparison of the two structures helps to distinguish distortions of the DNA conformation that are inherent to the cross-overs of the junctions in this crystal system from those that are consequences of the mismatched dG.dA base-pair in the d(CCGGGACCGG) structure. This analysis shows that the junction itself perturbs the sequence-dependent conformational features of the B-DNA duplexes and the associated patterns of hydration in the major and minor grooves only minimally. This supports the idea that a DNA four-way junction can be assembled at relatively low energetic cost. Both structures show a concerted rotation of the adjacent duplex arms relative to B-DNA, and this is discussed in terms of the conserved interactions between the duplexes at the junctions and further down the helical arms. The interactions distant from the strand cross-overs of the junction appear to be significant in defining its macroscopic properties, including the angle relating the stacked duplexes across the junction.

DNA interaction and dual topoisomerase I and II inhibition properties of the anti-tumor drug prodigiosin.

Prodigiosin is a red pigment produced by Serratia marcescens with apoptotic activity. We examined the mechanism of action of this tripyrrole alkaloid, focusing on its interaction with DNA and its ability to inhibit both topoisomerase I and topoisomerase II. We also evaluated the DNA damage induced in cancer cell lines. Prodigiosin-DNA intercalation was analyzed using a competition dialysis assay with different DNA base sequences. Topoisomerase I and II inhibition was studied in vitro by a cleavage assay, and in cultured cells, by analysis of its ability to form covalent complexes. Furthermore, we analyzed DNA damage by pulse-field gel electrophoresis and by immunocytochemistry. Apoptosis inducing factor (AIF)/phospho-H2AX (p-H2AX) double labeling by confocal microscopy was performed to determine the possible implication of AIF in the prodigiosin-DNA damage. Finally, we studied the ability of this drug to induce copper-mediated DNA damage at different pH by a DNA cleavage assay. Our results demonstrate prodigiosin-DNA interaction in vitro and in cultured cells. It involves prodigiosin-DNA intercalation, with some preference for the alternating base pairs but with no discrimination between AT or CG sequences, dual abolition of topoisomerase I and II activity and, as consequence, DNA cleavage. Prodigiosin-DNA damage is independent of AIF. Furthermore, we found that copper-mediated cleavage activity is associated with pH (occurring at pH 6.8 rather than pH 7.4) and with the Cu(2+) ion concentration. These results indicate DNA a therapeutic target for prodigiosin and could explain the apoptosis mechanism of action induced by this antineoplastic drug.

Micro-molding with ultrasonic vibration energy: new method to disperse nanoclays in polymer matrices.

Ultrasound technology was proved as an efficient processing technique to obtain micro-molded specimens of polylactide (PLA) and polybutylene succinate (PBS), which were selected as examples of biodegradable polyesters widely employed in commodity and specialty applications. Operational parameters such as amplitude, molding force and processing time were successfully optimized to prepare samples with a decrease in the number average molecular weight lower than 6%. Ultrasonic waves also seemed an ideal energy source to provide effective disaggregation of clay silicate layers, and therefore exfoliated nanocomposites. X-ray diffraction patterns of nanocomposites prepared by direct micro-molding of PLA or PBS powder mixtures with natural montmorillonite or different organo-modified clays showed the disappearance of the 001 silicate reflection for specimens having up to 6 wt.% clay content. All electron micrographs revealed relatively homogeneous dispersion and sheet nanostructures oriented in the direction of the melt flow. Incorporation of clay particles during processing had practically no influence on PLA characteristics but enhanced PBS degradation when an organo-modifier was employed. This was in agreement with thermal stability data deduced from thermogravimetric analysis. Cold crystallization experiments directly performed on micro-molded PLA specimens pointed to a complex influence of clay particles reflected by the increase or decrease of the overall non-isothermal crystallization rate when compared to the neat polymer. In all cases, the addition of clay led to a clear decrease in the Avrami exponent.

The anticancer agent ellipticine unwinds DNA by intercalative binding in an orientation parallel to base pairs.

Ellipticine is a natural plant product that has been found to be a powerful anticancer drug. Although still unclear, its mechanism of action is considered to be mainly based on DNA intercalation and/or the inhibition of topoisomerase II. Many experimental data suggest an intercalation based on stacking interactions along the major base-pair axis, but alternative binding modes have been proposed, in particular for ellipticine derivatives. The 1.5 A resolution structure of ellipticine complexed to a 6 bp oligonucleotide unveils its mode of binding and enables a detailed analysis of the distorting effects of the drug on the DNA.

Intercalation of an acridine-peptide drug in an AA/TT base step in the crystal structure of [d(CGCGAATTCGCG)](2) with six duplexes and seven Mg(2+) ions in the asymmetric unit.

We present the crystal structure of an acridine drug derivatized at carbon 9, [N(alpha)-(9-acridinoyl)-tetraarginine], intercalated within the dodecamer [d(CGCGAATTCGCG)](2). The presence of a lateral chain at the central carbon 9 atom differentiates this compound from most acridine drugs hitherto studied, which are usually derivatized at carbon 4. The DNA:drug interaction we observe differs from that observed in previous studies, which primarily involves shorter, mainly hexameric sequences, in two important regards: the acridine intercalates within an AA/TT base step, rather than within a CG/CG base step; and the binding site is located at the center of the sequence, rather than at one end of the duplex. In addition, we observe a novel crystal packing arrangement, with six dodecamer duplexes and seven hydrated magnesium ions in the asymmetric unit of a large (66.5 x 68.4 x 77.4 A(3)) unit cell in space group P2(1)2(1)2(1). The duplexes are organized in layers parallel to the ab plane, with consecutive layers crossing each other at right angles.