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.


Results and Discussion
In the final model for the DNA-variolin B complex the DNA adopts a B-form conformation only along the four central base pairs of the d(CGTACG) 2 duplex (Fig. 1b). As in previously reported isomorphous structures including Co 2+ ions [14][15][16][17][18] , the strands at both ends of the DNA duplex are distorted and interact with symmetry-related DNA molecules. At one end cytosine C1 is disordered and the complementary guanine G12 is lying in the minor groove of a neighboring duplex. At the other end, cytosine C7 is rotated so that it pairs the guanine G6 of a symmetry-related duplex, forming a pseudo-Holliday junction 19 that involves four DNA duplexes (Fig. 1c). Such association generates an intercalation site between the interduplex G6 · C7 and the intraduplex C5 · G8 base pairs (Fig. 2). While this particular crystal packing is caused mainly by DNA-DNA and DNA-ion interactions, a DNA intercalator is the keystone required to fix the whole structure.
The electron density for the intercalator is strong but does not allow to define a single orientation of the compound. Instead, the final model shows two drug molecules in different orientations, each with half occupancy.
One of the orientations enables a bifurcated hydrogen bond between the −NH 2 group of the tricyclic moiety of the drug and the guanine G6 phosphate.
Once intercalated, variolin B adopts a quasi-planar conformation (Fig. 2). The 2-aminopyrimidine ring is only 10.5° out of the pyridopyrrolopyrimidine mean plane in one drug molecule, whereas in the other this angle is 8.4°. Both conformations are consequently flatter than the one shown in the crystallographic structure of this small molecule alone 2 , where the external ring is 23.8° out of the heterocyclic core plane. Interestingly, in the structure of the CDK2/Cyclin A/variolin B complex 13 , the 2-aminopyrimidine ring of the drug is almost in plane with the heterocyclic system but flipped 180°. The interaction with CDK2, which probably determines this torsion, is based on three direct hydrogen bonds between variolin B and the ATP-binding site of the protein (residues Glu81, Leu83 and Ile10) and on several water-mediated hydrogen bonds.
This adaptability to several targets, either proteins or DNA, is rare but not unprecedented among similar natural alkaloids. Ellipticine, a proven DNA intercalator and topoisomerase II poison that is used in ovarian and breast cancer treatment, was also found to bind and inhibit protein kinase CK2 through competition with ATP 20 . In spite of this coincidence, the variolin B mode of binding to DNA cannot be paralleled to that of ellipticine 10 , as had been proposed 2 . Deoxyvariolin B affects the topoisomerase I-mediated unwinding of supercoiled DNA, but neither deoxyvariolin B nor variolin B increase the number of DNA strand breaks in a Comet assay with cells 3 . Hence, neither of these compounds seem to exert their cytotoxicity through DNA intercalation-mediated poisoning of topoisomerase I or II.
Variolin B is too insoluble to be reliably tested for its ability to bind DNA using absorption spectroscopy or surface plasmon resonance. However, its closely-related analogue deoxyvariolin B showed positive, albeit modest, binding to DNA using these techniques 3 . Variolin B also behaved as a weak DNA binder during our crystallization trials. After testing several oligonucleotides typically used to crystallize DNA-intercalator complexes, we observed variolin B only when binding to DNA folded into the intermolecular secondary structure described above. Taken together, these results suggest that variolins bind to DNA only under certain conditions, possibly involving the formation of non-B-form secondary structures. Variolin B intercalation stabilizes the unwinding of the DNA helix and the separation of the bases of the CpG dinucleotides, and also promotes the formation of the interduplex G6 · C7 base pair. The insertion of the drug is aided by the formation of a hydrogen bond between one of the NH2 groups of variolin B and the G6 phosphate. As a result of the rearrangement, the N7 atoms of guanines G6 and G8 are no longer able to interact with Co 2+ ions 17 , which would probably impair the formation of the observed multi-stranded DNA structure. As we have shown, variolin B is fully capable of disturbing B-DNA, promoting the formation of stable inter-helix junctions, just as other potent intercalators do 14 . This finding supports the notion that variolin B is a dual-action drug and suggests that its ability to stabilize nucleic acid junctions (either of DNA or RNA) might be further exploited 21,22 .

Conclusions
Our structure confirms that variolin B is indeed a DNA intercalator. Therefore, a dual action of variolin B on both proteins and nucleic acids is most likely to occur. The cytotoxic effects of variolin B would then arise from a combination of its capacity to interact with crucial ATP-binding proteins and its ability to promote the formation of high-order nucleic acid systems in the cell environment.
Structure solution and refinement. The structure was solved by molecular replacement with Phaser 25 using the DNA coordinates from the structure of 9-amino-[N-(2-dimethylamino)propyl]-acridine-4-carboxamide bound to d(CGTACG) 2 (PDB entry 1RQY) 14 as a search model. Refinement followed with REFMAC5 26 . The optimum orientations of the intercalated variolin B were identified by placing the drug in each of the four possible positions and refining until the best fit and corresponding best R-factor and R-free were found. At this stage, an iterative refinement procedure was carried out using REFMAC5, interspersed with inspection of electron-density maps, water positioning, and manual model rebuilding with COOT 27 . All data were used (40.0-1.4 Å) with no resolution or σ cut-off. The final refinement statistics are shown in Table 1.