HN NH+H2

+

NNH2

+NH

HN+

DNA as anti-cancer target: structural studies by X-ray crystallographyY.Gan, C. J. Cardin. The School of Chemistry, University of Reading, RG6 6AD, UK. W.A. Denny. Auckland Cancer Society Research Centre, Faculty of Medicine and Health Science, University of Auckland, Private Bag 92019, Auckland, New Zealand.DNA as protein substrates play vital roles in enzyme functionality, by changing their conformations, the activity of some important proteins can be inhibited. Therefore

small molecules that are capable of binding to DNA and interrupting their structures have been particularly developed as potential chemotherapy agents. Among them, acridine derivatives bind to DNA through intercalation which effectively results in elongation and unwinding of these DNA molecules. One species of these

derivatives, 9-aminoacridine-4-carboxamide has demonstrated some promising antileukaemia activity in vivo, and it was also found that improved residence time greatly enhance this activity. In order to achieve that, a number of bisacridine derivatives linked at their 9th position by a flexible linker have been developed. Although

there is ample evidence to show that this activity is closely associated with the 4-carboxamide side chain and some crystallographic data illustrating the binding details of this side chain to DNA molecules1, the exact function mechanism remains unclear. Therefore, comparison experiments with unsubstituted bisacridine derivatives

have been carried out to investigate further their structural relationships with DNA molecules.

The structures of two complexes between oligonucleotide d(CGTACG) and compounds 23152, 23515 respectively have been solved and revealed two totally different structures, though the two compounds only differ slightly in their linker length and charge.

23515

NH+NNH2

+NH

HN+

H

23512

DrugDrug 2351223512 2351523515

Space-groupSpace-group P6P6552222 C222C222

Unit Cell (Å)Unit Cell (Å) a = 25.75, b = 25.75,c = 78.99a = 25.75, b = 25.75,c = 78.99 a = 29.94, b = 54.05,c = a = 29.94, b = 54.05,c = 40.0640.06

(Å)(Å) 1.0 (synchrotron)1.0 (synchrotron) 1.5418 (in-house)1.5418 (in-house)

Resolution (Å)Resolution (Å) 22.33 – 1.90 22.33 – 1.90 (2.00 – 1.90)(2.00 – 1.90)

40.06 – 2.2540.06 – 2.25(2.33 – 2.25)(2.33 – 2.25)

Completeness (%)Completeness (%) 99.3 (100)99.3 (100) 97.4 (95.9)97.4 (95.9)

Reflections – Reflections – uniqueunique 1458 (204)1458 (204) 1641 (165)1641 (165)

RedundancyRedundancy 20.1 (15.3)20.1 (15.3) 4.2 (3.9)4.2 (3.9)

Ave I/Ave I/ 49.4 (9.0)49.4 (9.0) 14.4 (2.0)14.4 (2.0)

R-merge (%)R-merge (%) 3.8 (31.2)3.8 (31.2) 5.5 (20.0)5.5 (20.0)

R-factor (%)R-factor (%) 27.227.2 21.721.7

R-free (%)R-free (%) 30.530.5 30.330.3Figures in the brackets are for the outer resolution shell

The d(CGTACG)+23512 complex:

The structure is very novel compared to the classic bis-intercalation in which compounds intercalate their dichromophores to the same DNA duplex. Instead, 23512 tethers DNA duplexes in a head to tail fashion by intercalating its acridine chromophores between guanine and cytosine base pairs belonging to different duplexes with the linker emerging from the major groove in a fully extended state. In this way, the complex of the hexamer DNA duplexes with the bisacridine becomes real polymer.

In this very condensed structure, DNA duplexes are brought close by the tricationic compound through its charge neutralisation effects, at the junction where four duplexes meet, guanine residues form two extra

hydrogen bonds with their side by side neighbouring guanine residues at the N2/N3 positions, together with the classic Watson-Crick guanine/cytosine hydrogen bonds, an enlarged guanine/cytosine quadruplex was produced.

The d(CGTACG)+23515 complex:The biological unit of this structure comprising four duplexes belonging to two columns respectively which are perpendicular to each other. These four duplexes are connected through terminal cytosine residue exchange. The compound is observed to have its chromophores intercalate between one of two G/C steps and fuse two duplexes in the same column through its polyamine linker, whilst the terminal cytosine residue is flipped out due to the cobalt and barium binding to the guanine residue.

The similar guanine / cytosine quadruplex was also observed in this structure, and chloride anions were observed close to the linker at a two fold axis countering their multi-positive charge. Most interestingly, this structure is almost identical to that of reported by Teixeira et al2. However, in that structure, the compound had its 4-carboxamide side chain making a hydrogen bond to the O6 of guanine which has been considered as a crucial element for the in vivo activities of this type of compound. Now, having the side chain free

In the close-up figure on the right, the long axis of the chromophore can be clearly seen bisecting the long axes of its stacking base pairs, and the linker is at the major groove side.

Space-groupSpace-group P2P211 P2P21122112211

Unit Cell (Å)Unit Cell (Å)a = 44.39a = 44.39b =46.39b =46.39

c = 23.87, ß=98.52c = 23.87, ß=98.52

a = 24.86a = 24.86b = 43.14b = 43.14c = 49.17c = 49.17

(Å)(Å) 1.5418 (in house)1.5418 (in house) 1.5418 (in house)1.5418 (in house)

Resolution (Å)Resolution (Å) 43.90 – 2.71 43.90 – 2.71 (2.81 – 2.71)(2.81 – 2.71)

32.43 – 2.5532.43 – 2.55(2.66 – 2.55)(2.66 – 2.55)

Completeness (%)Completeness (%) 98.5 (93.6)98.5 (93.6) 96.9 (93.8)96.9 (93.8)

Reflections – uniqueReflections – unique 2762 (278)2762 (278) 1874 (191)1874 (191)

RedundancyRedundancy 3.5 (2.7)3.5 (2.7) 4.7 (3.8)4.7 (3.8)

Ave I/Ave I/ 11.5 (2.0)11.5 (2.0) 26.1 (2.2)26.1 (2.2)

R-merge (%)R-merge (%) 6 (17.2)6 (17.2) 4.9 (19.8)4.9 (19.8)

R-factorR-factor 18.818.8 18.7218.72

R-freeR-free 27.727.7 25.425.4

compound 23515 binds to the DNA in a same way, the function or the structure and activity relationship of this side chain may need to be further investigated.

Myers et al3 have identified that the 7-nucleotide oligomer CCTCCCT is the most frequently appeared in the recombination hotspots of human by statistics calculation, but there is no any structural information available for this sequence motif so far. Therefore, the oligonucleotide d(GGCCTCCCTA) and its complementary strand have been subject to crystallography studies. Two structures have been solved in P21 and P2P21122112211 respectively and both respectively and both turned to be A-DNA.

At the guanine/cytosine terminal of both structures, the O2 of cytosine forms an extra hydrogen bond to N2 of guanine so that a G/C quadruplex is constructed.

References:

1.Denny, W. A. (2003) Current Topics in Medicinal Chemistry 3, 1349-1364.

2.Teixeira, S.C.M., Thorpe, J.H., Todd, A.K., Powell, H.R., Adams, A., Wakelin, L.P.G., Denny, W.A., Cardin, C.J. (2002), J. MOL. BIOL. 323, 167-171.

3.Myers, S., Bottolo, L., Freeman, C., McVean, G., Donnelly, P., (2005) Science, 310, 321-323.

Acknowledgments:

Yu Gan wishes to thank the University of Reading and the Overseas Research Students Award Scheme for the financial support of this study.