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X-Ray diraction analyses of the natural isoquinoline alkaloids Berberine and Sanguinarine complexed with double helix DNA d(CGTACG)w
Marta Ferraroni,a Carla Bazzicalupi,*a Anna Rita Biliab and Paola Gratteri*bc
Downloaded by Ajou University Central Library on 02 July 2011 Published on 22 March 2011 on http://pubs.rsc.org | doi:10.1039/C1CC10971E

Received 18th February 2011, Accepted 4th March 2011 DOI: 10.1039/c1cc10971e The rst crystal structures of Berberine and Sanguinarine intercalated with a d(CGTACG)2 DNA sequence were obtained by X-ray diraction analysis at 2.3 A resolution. Both drugs join the end of two two-molecules DNA units, stacked in a nonclassic intercalation site formed by six bases. Sanguinarine interacts with d(CGTACG)2 DNA in its iminium form. Alkaloids are an important class of natural products. Among them, isoquinoline derivatives belong to one of the most widespread building blocks and possess striking biological and pharmacological activities, with a role of remarkable importance in the contemporary biomedical research and drug discovery programs.13 Berberine (benzo[g]-1,3-benzodioxolo [5,6a]quinolizinium, 5,6-dihydro-9,10-dimethoxy) and Sanguinarine (13-methyl[1,3]benzodioxolo[5,6-c]-1,3-dioxolo[4,5-i]phenanthridinium) (Scheme 1) are the most representative compounds among the isoquinoline alkaloids and belong to the protoberberine and benzophenanthridine group, respectively. Berberine and Sanguinarine are constituted by a fused ring system featuring dierent degrees of double bond delocalization. Both have been and still are intensively investigated, and their antimicrobial, anti-inammatory, anti-oxidative and antitumor activities have been reported.110 The anticancer properties derive certainly from their ability to form complexes with DNA and RNA which give rise to additional eects such as telomerase and topoisomerase inhibition. In addition, other dierent mechanisms are also responsible for their antitumor properties, i.e. antimicrobial eects on tumorigenic microorganisms and regulation of oncogene and carcinogenesisrelated genes expression.10 A large number of studies have been recently published on their biological activity against DNA related diseases,11,12 as well as on the chemicalphysical aspects of their DNA binding behaviour.13 Several studies described the complexation of Berberine and Sanguinarine with DNA. Physicochemical and biochemical investigations combined with modeling simulations have pointed out a mixed mode DNA binding model for Berberine, that exhibits both intercalation and minor groove interactions,1416 although spectroscopic studies with calf thymus DNA17 and high-resolution 1H-NMR18 carried out on Berberine complexed with various short oligonucleotide duplexes highlighted a preference for minor groove binding geometry. In spite of the intense interest toward this kind of alkaloids and their DNA binding properties, no direct structural data have been till now reported, but a crystal structure of the adduct of the indoloquinoline alkaloid cryptolepine with d(CCTAGG) double stranded DNA.19 As a consequence, we have tried a systematic screening for the crystallization of both Berberine and Sanguinarine with several DNA polymers. Here we report the rst crystal structures of Berberine or Sanguinarine intercalated with the d(CGTACG)2 DNA sequence, both obtained by X-ray diraction analysis at 2.3 A resolution.z The complexes crystallize in the P3221 space group, and show similar crystal packings: the asymmetric unit contains four DNA strands, arranged in a two-molecules unit, a drug molecule, few cocrystallized water molecules, and a Ca2+ ion. The overall conformation assumed by each DNA duplex in these structures has been previously seen for the CGTACG hexamer, even if the reported crystal structures are characterized by a dierent space group and a dierent crystal packing.20
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Scheme 1
a

Department of Chemistry Ugo Schi, University of Firenze, Via della Lastruccia 3-13, I-50019 Sesto Fiorentino, Firenze, Italy. E-mail: carla.bazzicalupi@uni.it; Fax: +39 0554573385; Tel: +39 0554573286 b Department of Pharmaceutical Sciences, University of Firenze, Via Ugo Schi 6, I-50019 Sesto Fiorentino, Firenze, Italy c Laboratory of Molecular Modeling Cheminformatics & QSAR, Department of Pharmaceutical Sciences, University of Firenze, Via Ugo Schi 6, I-50019 Sesto Fiorentino, Firenze, Italy. E-mail: paola.gratteri@uni.it; Fax: +39 0554573380; Tel: +39 0554573702 w Electronic supplementary information (ESI) available: Crystallization and data collection details and gures showing the Berberine/ dCGTACG adduct. See DOI: 10.1039/c1cc10971e

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Fig. 1 DNA helices and stacked Sanguinarine molecules. DNA chains color scheme: A = orange, B = yellow, C = green, D = blue.

In the present structures, the gross crystal organization features DNA helices stacked on each other, giving rise to continuous columns growing in the (100), (010) or (110) symmetry related directions. As shown in Fig. 1 for the Sanguinarine/CGTACG complex (for the Berberine/CGTACG complex see Fig. S1, ESIw), in the two molecule unit, the central 4 base pairs of each DNA helix give rise to a continuous array of 8 stacked base pairs adopting a B-like motif, while the terminal bases of each helix are involved in dierent kinds of contacts. In particular, at the central junction of the two molecule unit, the terminal 3 0 -Gs lie in the minor groove of the next duplex in the column and are well localized in the electron density map, while the 5 0 -Cs are disordered and have not been detected, analogously to the 5 0 -Cs residues in the above mentioned CGTACG crystal structures.20 In both Berberine and Sanguinarine structures, the drug molecule is found at the interface that joins two twomolecules DNA units, located in an intercalation site formed

by six bases. Actually, as shown in Fig. 2 for Sanguinarine (for Berberine see Fig. S2, ESIw), two 3 0 -GpC dinucleotides from chains A and C of adjacent units dene a non-classic intercalation gap and the overall binding site is completed by two more guanine residues of the 5 0 -CpG end, the cytosine groups being ipped out from the duplex. In the CGTACG crystal structures reported by Valls et al.,20 an analogous intercalation site is dened by the same six bases as in the present structures, but the dierent crystal packing makes available two more cytosines which determine an overall 8 bases intercalation site. In both Berberine and Sanguinarine/DNA complex crystal structures, the binding mode is determined by pp interactions mainly involving the C5 and G6 residues of chain A and the G8 residue of chain D, the interaction being reinforced by the presence of a positively charged nitrogen on each drug skeleton. No H-bonds were found between the DNA atoms and the ethereal oxygens. It is to be underlined that in both structures, pairs of DNA columns, bridged by a couple of symmetry related Ca2+ ions, grow along the (100), (010) or (110) direction, and that the 5 0 -cytosine groups, ipped out from the helix by the drugs, establish contacts with adjacent not parallel DNA columns. As a result, couples of parallel DNA columns, bridged by the metal centres, develop along the cell sides and one face diagonal directions (Fig. S3, ESIw). Large voids remain in the crystal packing, that should be lled by the not detected cytosine groups, as well as additional disordered water molecules. In Fig. 3 are shown the OMIT electron density map of Sanguinarine alone (a) and the lateral and top views of the skeletons of the two drugs overlapped superimposing DNA coordinates from the two structures (b). Interestingly, the electron density map of the Sanguinarine/DNA adduct corresponds to the iminium form (Scheme 1), thus conrming the ndings that the planar and charged iminium form, which in aqueous solution exists only in the pH range 1.06.0, is actually the DNA-binding species also in physiological conditions.1 Both Berberine and Sanguinarine have a tetracyclic skeleton bearing, respectively, a quaternary protoberberine moiety, which is 5,6-dihydro[a,g]quinolizinium, and a benzo[c]phenanthridine group. Noteworthy, in spite of their dierent skeletons, the two alkaloids occupy almost the same volume of space in the intercalative binding site. The overall dimensions of the fused ring systems (ve or six rings for Berberine or Sanguinarine, respectively), however,

Downloaded by Ajou University Central Library on 02 July 2011 Published on 22 March 2011 on http://pubs.rsc.org | doi:10.1039/C1CC10971E

Fig. 2 Sanguinarine molecule intercalated at the interface of two two-molecules DNA units. DNA chains color scheme: A = orange, B = yellow, C = green, D = blue.

Fig. 3 (a) Skeleton and OMIT electron density map for Sanguinarine contoured at 1.5s level and (b) top and lateral view of overlapped Berberine (pink) and Sanguinarine (cyan) molecules.

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seem too bulky to give rise to the classic parallel intercalation19 usually found in the crystal structures of less hindered compounds such as acridine or anthraquinone derivatives. On the other hand, the p-stacking overlapping obtained with this non-classic binding site is signicantly higher than in the perpendicular intercalation shown by anthracycline derivates. Remarkably, the CGTACG sequence was found to adopt the present packing only in adducts with highly bulky functionalized acridine or anthracycline derivatives,20 while with smaller compounds it is able to give the classic parallel intercalation.21 In conclusion, the ligand interaction data presented here evidence a non-classic intercalation of the studied ligands with the CGTACG DNA sequence, thus conrming the already reported ndings for these alkaloids concerning their poor ability in interacting according to a pure intercalative binding mode.1418 In our opinion, even if the studied sequence is too short to be biologically relevant in itself, nevertheless our results should be regarded with interest considering that intercalation, similar to that observed in the present structures, could be attained in vivo, especially in the case of particular DNA sequences able to assume foldings dierent from the canonical double helix, such as hairpins or Holliday junctions.22 We thank the ESRF Synchrotron (Grenoble, France) for beam-time.

Notes and references

z Berberine d(CGTACG)2 complex: a = 29.99 A, c = 119.06 A, trigonal, space group P3221, Resolution = 2.3 A, Rsym = 0.076, Completeness = 100%. Sanguinarine d(CGTACG)2 complex: a = 30.37 A, c = 118.26 A, trigonal, space group P3221, Resolution = 2.3 A, Rsym = 0.052, Completeness = 96.3%. The DNA-drug complexes were crystallized at 296 K using the sitting drop vapor diusion method from a solution containing 10% MPD, 40 mM Na Cacodylate pH = 6.5, 12 mM spermine tetra-HCl, 80 mM sodium chloride and 20 mM magnesium chloride. Drops were equilibrated against a 30% MPD reservoir. Details of the crystallization process are reported within ESI.w Data collection on crystals of the DNA-drug complexes were performed at 100 K, using as a cryoprotectant the mother liquor solution with an increased concentration of MPD (30%). Data for the Berberine and the Sanguinarine complexes were collected on an Oxford Diraction instrument and using synchrotron radiation at the ID29 Beamline, ESRF Grenoble, respectively. Data were integrated and scaled using the program XDS.23 Data of the two complexes are hemihedrally twinned. The possibility of twinning of the data was suggested by the analysis of the intensity distribution performed using the phenix.xtriage module of the PHENIX program24 (see also ESIw). The structure of the Berberine complex was solved by the Molecular Replacement technique using the program Molrep25 and the coordinates of the hexamer duplex d(CGTACG) (PDB code 1XCS), without all the heteroatoms, as a search model. The structure of the Sanguinarine complex was solved by the same technique using the coordinates of the two molecules unit of the Berberine complex. The two models were rened with the program Refmac526 from the CCP4 program suite27 using the twin option. Manual rebuilding of the model was performed using the program Coot.28 (Data Collection and Renement statistics are reported in ESI.w) Final coordinates and structure factors have been deposited with the Protein Data Bank (PDB accession number 3NP6, 3NX5). 1 K. Bhadra and G. S. Kumar, Mini-Rev. Med. Chem., 2010, 10(13), 12351247 and references therein. 2 M. Maiti and G. S. Kumar, Med. Res. Rev., 2007, 27(5), 649695.

3 M. Maiti and G. S. Kumar, Bioactive Heterocycles IV, From Topics in Heterocyclic Chemistry, 2007, vol. 10, pp. 155209. 4 V. Simanek, in The Alkaloids, ed. A. Brossi, Academic Press, New York, 1985, vol. 26, pp. 185234. 5 K. C. Singhal, Indian J. Exp. Biol., 1976, 14, 345347. 6 T. Satou, N. Akao, R. Matsuhashi, K. Koike, K. Fujita and T. Nikaido, Biol. Pharm. Bull., 2002, 25, 16511654. 7 C. L. Kuo, C. W. Chi and T. Y. Liu, Cancer Lett., 2004, 203, 127137. 8 T. N. Graf, K. E. Levine, M. E. Andrews, J. M. Perlmutter, S. J. Nielsen, J. M. Davis, M. C. Wani and N. H. Oberlies, J. Agric. Food Chem., 2007, 55, 12051211. 9 J. Ziegler and P. J. Facchini, Annu. Rev. Plant Biol., 2008, 59, 735769. 10 Y. Sun, K. Xun, Y. Wang and X. Chen, Anti-Cancer Drugs, 2009, 20, 757769. 11 Some recent literature for Berberine: (a) Z. Liu, Q. Liu, B. Xu, J. Wu, C. Guo, F. Zhu, Q. Yang, G. Gao, Y. Gong and C. Shao, Mutat. Res., 2009, 662, 7583; (b) M. S. Choi, J. H. Oh, S. M. Kim, H. Y. Jung, H. S. Yoo, Y. M. Lee, D. C. Moon, S. B. Han and J. T. Hong, Int. J. Oncol., 2009, 34, 12211230. 12 Some recent literature for Sanguinarine: (a) K. M. Ansari, A. Dhawan, S. K. Khanna and M. Das, Food Chem. Toxicol., 2005, 43(1), 147153; (b) M.-C. Chang, C.-P. Chan, Y.-J. Wang, P.-H. Lee, L.-I. Chen, Y.-L. Tsai, B.-R. Lin, Y.-L. Wang and J.-H. Jeng, Toxicol. Appl. Pharmacol., 2007, 218(2), 143151. 13 Most cited works published from 2008 to present: (a) X. Tian, Y. Song, H. Dong and B. Ye, Bioelectrochemistry, 2008, 73(1), 1822; (b) P. Giri and G. S. Kumar, J. Photochem. Photobiol., A, 2008, 194(23), 111121; (c) M. Hossain and G. S. Kumar, J. Chem. Thermodyn., 2009, 41(6), 764774; (d) J. Urbanova, P. Lubal, I. Slaninova, E. Taborska and P. Taborsky, Anal. Bioanal. Chem., 2009, 394(4), 9971002; (e) A. Adhikari, M. Hossain, M. Maiti and G. S. Kumar, J. Mol. Struct., 2008, 889(13), 5463; (f) L.-P. Bai, Z. Cai, Z.-Z. Zhao, K. Nakatani and Z.-H. Jiang, Anal. Bioanal. Chem., 2008, 392(4), 709716. 14 D. S. Pilch, C. Yu, D. Makhey, E. J. LaVoie, A. R. Srinivasan, W. K. Olson, R. R. Sauers, K. J. Breslauer, N. E. Geacintov and L. F. Liu, Biochemistry, 1997, 36, 1254212553. 15 S. A. Kim, Y. Kwon, J. H. Kim, M. T. Muller and I. K. Chung, Biochemistry, 1998, 37, 1631616324. 16 J. J. Zhu, J. J. Zhang and H. Y. Chen, Spectrosc. Lett., 1998, 31, 17051718. 17 W. Y. Li, H. Lu, C. X. Xu, J. B. Zhang and Z. H. Lu, Spectrosc. Lett., 1998, 31, 12871298. 18 S. Mazzini, M. C. Bellucci and R. Mondelli, Bioorg. Med. Chem., 2003, 11, 505514. 19 J. L. Lisgarten, M. Coll, J. Portugal, C. W. Wright and J. Aymami, Nat. Struct. Biol., 2002, 9(1), 5760. 20 N. Valls, R. A. Steiner, G. Wright, G. N. Murshudov and J. A. Subirana, JBIC, J. Biol. Inorg. Chem., 2005, 10, 476482 and references therein. 21 A. Adams, J. M. Guss, W. A. Denny and L. P. G. Wakelin, Nucleic Acids Res., 2002, 30, 719725 and references therein. 22 (a) A. L. Brodgen, N. H. Hopcroft, M. Searcey and C. J. Cardin, Angew. Chem., Int. Ed., 2007, 46, 38503854; (b) D. M. J. Lilley, Q. Rev. Biophys., 2000, 33(2), 109159. 23 W. Kabsch, J. Appl. Crystallogr., 1993, 26, 795800. 24 P. D. Adams, R. W. Grosse-Kunstleve, L.-W. Hung, T. R. Ioerger, A. J. McCoy, N. W. Moriarty, R. J. Read, J. C. Sacchettini, N. K. Sauter and T. C. Terwilliger, Acta Crystallogr., Sect. D, 2002, 58, 19481954. 25 A. Vagin and A. Teplyakov, J. Appl. Crystallogr., 1997, 30, 10221025. 26 G. N. Murshudov, A. A. Vagin and E. J. Dodson, Acta Crystallogr., Sect. D, 1997, 53, 240255. 27 Collaborative Computational Project, Number 4. Acta Crystallogr., Sect. D 1994, 50, 760763. 28 P. Emsley, B. Lohkamp, W. Scott and K. Cowtan, Acta Crystallogr., Sect. D, 2010, 66, 486501.

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