Study on Specific Binding of Chelerythrine to Single-Base Bulged DNA by Using ESI-MS

1. Study on Specific Binding of Chelerythrine to Single-Base Bulged DNA by Using ESI-MS 應用 ESI-MS 方法研究白屈菜紅醎與單醎基Bulged DNA 的特異性結合 Zhi-Hong Jiang, PhD 姜 志 宏

2. J. Drews, Science, 287, 1960 (2000).

3. A Brief Introduction to Interaction of Small Organic Molecules with DNA and RNA • The specific interactions of small organic molecules with DNA and RNA are a topic of increasing interests in the fields of bioorganic chemistry, medicinal chemistry and chemical biology. Investigations on such interactions can not only provide better elucidation of their bioactivity mechanism, but also guide the rational design of anti-tumor, antiviral and antibiotic drugs. • The interactions of drug molecules with DNA comprise covalent bonding and noncovalent binding. It has been evidenced that noncovalent binding with DNA is the molecular basis of many antitumor, antiviral and antibiotic drugs.

4. Chemical Structures of Quarternary Benzophenanthridine Alkaloids

5. Plants Containing These Three Alkaloids • Papaveraceae:Chelidonium ; Eomecon; Papaver; Macleaya • Rutaceae:Zanthoxylum • Fumariaceae:Corydalis; Fumaria

6. Chelidonium majus（白屈菜） Zanthoxylum nitidum (两面针) Radix Zanthoxyli Nitidi (入地金牛) Herba Chelidonii （白屈菜） Chinese Herbal Medicines

7. Pharmacological Activities • Anti-tumor：apoptosis; cytotoxic; anti-angiogenesis; topoisomerases I and II inhibition • Anti-microbial • Anti-inflammatory • Anti-plaque

8. Sequence Selectivity of ds-DNA-Binding of Three Benzophenanthridine Alkaloids 1. The binding capacity of three alkaloids toward CT-DNA are in the order of sanguinarine > nitidine ≥ chelerythrine. Sanguinarine showed much higher selectivity toward poly[dG-dC]·poly[dG-dC] than chelerythrine and nitidine. 2. For the sequence selectivity, both sanguinarine and nitidine bind preferentially to d(TGCGCA)2 (alternating GC base pairs). However, chelerythrine shows a high specificity for 5′-TGGGGA-3′/3′-ACCCCT-5′ (contiguous GC base pairs), exhibiting quite distinct sequence selectivity from sanguinarine. 3. Molecular planarity plays an important role for DNA-binding activities of sanguinarine and chelerythrine. 4. Three alkaloids interact even with 8-mer double-stranded oligodeoxynucleotide d(ATGCGCAT)2 at submicromolar concentration, showing that they are extremely strong DNA-binding agents. L.-P. Bai, Z. Zhao, Z. Cai, Z.-H. Jiang. Bioorg. & Med. Chem., 14, 5439-5445 (2006).

9. DNA Bulge Binder Bioorg. Med. Chem. Lett.2005, 15, 2673

10. Nucleic acid bulge is an important biological motif Bulge structures in nucleic acids are of general biological significance. They have been proposed as intermediates in a multitude of processes including RNA splicing, frame-shift mutagenesis, intercalator-induced mutagenesis, imperfect homologous recombination. Bulges have also been suggested as binding motifs for regulatory proteins involved with viral replication, including the TAR region of HIV-1. Additionally, the etiology of at least 12 human neurodegenerative genetic diseases has been attributed to genetic variations in the lengths of triplet repeats in genomic DNA (e.g., myotonic dystrophy, Huntington’s diseases, Friederich’s ataxia, and fragile X syndrome). The unstable expansion of triplet repeats has been attributed to reiterative synthesis due to slippage and bulge formation in the newly formed DNA strand. Compounds capable of binding to bulges could have significant therapeutic potential. Z. Xi, G.-S. Hwang, I. H. Goldberg. Chem. & Biol. 2002,9, 925-931

11. DNA Duplex Bulged DNA Bulged Structure of Nucleic Acids

12. T C T A T T A T C T G A T A A T A G T T C T A T T A T C T G A T A A T A G T N DNA Hairpins Used in This Research Regular hairpin (N=C, T, G, A) Single-base bulged hairpin

13. Fluorescence spectra DNA Fluorescence Intensity Wavelength (nm) Nonlinear curve fitting Software: KaleidaGraph (Version 3.6) Fluorometric Titration Method I/I0=1+ ((I∞-I0)/2I0)×{([DNA]0+[alkaloid]0+1/Ka)-(([DNA]0+[alkaloid]0+1/Ka)2-4[DNA]0[alkaloid]0)1/2} H. J. Schneider, A. K. Yatsimirski. In Principles and methods in supramolecular chemistry, New York: J. Wiley, 2000, pp 137-143

14. Bulge Base Slectivity of Chelerythrine and Sanguinarine Table 1. Association constants (Ka) of chelerythrine and sanguinarine with DNA hairpins obtainedfrom fluorometric analysis

15. Both chelerythrine and sanguinarine bind more tightly to bulged hairpins than to the regular hairpin, they show a preference for the binding to the single pyrimidine (C, T) bulge over the purine (A, G) bulge. • Additionally, the binding selectivity of chelerythrine to bulged hairpin is much stronger than that of sanguinarine.

16. Selectivity of Chelerythrine to T-bulged Hairpins Table 2. Association constants (Ka) of chelerythrine with four T-bulged hairpins containing different loop obtained from fluorometric analysis

17. Chelerythrine shows approximately equivalent RA values (20.8~28.0) to all T-bulged hairpins with different loops. This clearly reveals that chelerythrine selectively binds to the bulge site in DNA hairpins independent of the nature of hairpin loop.

18. ESI-MS Investigation on the Complexes of Chelerythrine and Bulged Oligodeoxynucleotides • As a sensitive and effective analytical technique, electrospray ionization mass spectrometry (ESI-MS) has played an active role in the investigations of noncovalent ligand-DNA complexes, owing to the gentle nature of the electrospray ionization process, which allows a wide range of noncovalent complexes to be introduced intact into the gas phase. • Four advantages: specificity, sensitivity, speed and stoichiometry • ESI-MS method in studying noncovalent ligand-DNA complexes has been validated by comparison with other well-established techniques, and successfully employed for analysis of noncovalent complexes of small organic molecules with oligodeoxynucleotides. • J. M. Danial, S. D. Friess, S. Rajagopalan, S. Wenda, R. Zenobi. Int. J. Mass Spectrom.2002, 216,1-27. • K. X. Wan, T. Shibue, M. Gross. J. Am. Chem. Soc.2000, 122, 300-307. • W.-H. Chen, C.-L. Chan, Z. Cai, G.-A. Luo, Z.-H. Jiang. Bioorg. & Med. Chem. Lett.14, 4955-4959 (2004).

19. Free DNA Drug-DNA complex Regular hairpin:Che=1:2 [H-4H]4- [H-5H]5- [H+Che-5H]4- [H+che-6H]5- [H+2Che-6H]4- [H+2Che-7H]5- C-bulge:Che=1:2 [C+Che-6H]5- [C+Che-5H]4- [C-5H]5- [C-4H]4- [C+2Che-6H]4- [C+2Che-7H]5- RA=1.06 Relative Affinity (RA)= [Complex] / [Free DNA] RA=5.15 Fig. 1 Negative ESI-TOF-MS spectra of complexes between chelerythrine and five hairpins

20. Table 3. Relative binding affinities of chelerythrine toward bulged and regular hairpins derived from ESI-MS a RA denotes relative affinity, the ratio of peak area of [DNA-drug complex] / [free DNA]. Referred to K. X. Wan, T. Shibue, M. Gross. J. Am. Chem. Soc.2000, 122, 300-307.

21. C-bulge T-bulge G-bulge A-bulge Regular hairpin Relative Affinity Fig. 2 Relative affinities of chelerythrine to five hairpins The relative affinities of all bulged hairpins are larger than that of the regular hairpin

22. [H-4H]4- [H+Che-6H]5- [H+Che-5H]4- [C+Che-5H]4- [C+Che-5H]5- [H-5H]5- C-bulge & Hairpin [C-4H]4- [C-5H]5- [H-4H]4- [T+Che-5H]5- T-bulge & Hairpin [H-5H]5- Fig. 3 Competitive binding of chelerythrine between each bulged hairpin and regular hairpin [T-4H]4- [T+Che-5H]4- [T-5H]5- [H-4H]4- [G+Che-5H]5- G-bulge & Hairpin [H-5H]5- [G+Che-5H]4- [G-5H]5- [G-4H]4- [H-4H]4- [A+Che-5H]5- [H-5H]5- A-bulge & Hairpin [A-4H]4- [A+Che-5H]4- [A-5H]5-

23. Table 4. Relative binding affinities of chelerythrine with four pairwise bulged and regular hairpins obtained from ESI-MS Chelerythrine does much prefer bulged hairpin to the regular hairpin

24. C-bulge & T-bulge [T-5H]5- [C+Che-6H]5- [T-4H]4- [C+Che-5H]4- [T+Che-6H]5- [C-4H]4- [C-5H]5- [T+Che-5H]4- Fig. 4 Competitive binding of chelerythrine between three pairwise bulged hairpins T-bulge & G-bulge [G-4H]4- [G-5H]5- [T+Che-5H]4- [T+Che-6H]5- [T-4H]4- [T-5H]5- [G+Che-6H]5- [G+Che-5H]4- G-bulge & A-bulge [G-4H]4- [G-5H]5- [A+Che-5H]4- [A-4H]4- [A-5H]5- [A+Che-6H]5- [G+Che-5H]4- [G+Che-6H]5-

25. Table 5. Relative binding affinities of chelerythrine with three pairwise bulged hairpins obtained from ESI-MS Bulge base selectivity of chelerythrine: C-bulge > T-bulge >> G-bulge ≥ A-bulge

26. -0.2℃ 6.4℃ 3.8℃ 5.0℃ 3.0℃ Normalized DNA Thermal Denaturation Profiles in Presence and Absence of Chelerythrine

27. Table 6. Melting temperature of five hairpins in presence(+) and absence(-) of chelerythrine

28. UV melting study indicated that chelerythrine can provide thermodynamic stabilization to a single nucleotide bulge following the order of: C-bulge > T-bulge > A-bulge ≥ G-bulge

29. Conclusions • ESI-MS study showed that chelerythrine binds more tightly to bulge sites than to the duplex region of regular hairpin DNA. It exhibits a significant preference for the binding site of single pyrimidine bulge (C, T) over the purine bulge (A, G). These results were fully supported by the findings obtained from fluorimetric analysis and UV-melting study. • Fluorimetric titration results also showed that the binding selectivity of chelerythrine to bulges is much stronger than that of sanguinarine • A combination of ESI-MS study, fluorimetric titration and UV-melting investigations has unambiguously demonstrated that chelerythrine specifically binds to single pyrimidine bulge site in DNA. • The preferential binding of chelerythrine to bulge site in DNA suggested that it may have therapeutic effects on human neurodegenerative geneticdiseases. • The specific recognition of chelerythrine to pyrimidine bulges could be applicable to the detection of the single pyrimidine bulge in duplex DNA.

30. Our Papers on DNA-Binding of Alkaloids from Chinese Medicinal Herbs • L.-P. Bai, Z. Zhao, Z. Cai, Z.-H. Jiang. Nucleic Acids Symposium Series No. 50, 197~198, 2006. • L.-P. Bai, Z. Zhao, Z. Cai, Z.-H. Jiang. Bioorg. & Med. Chem., 14, 5439-5445 (2006). • Y. Qin, W.-H. Chen, J. Pang, Z. Zhao, L. Liu, Z.-H. Jiang. Chem. & Biodiv., in press, 2006. • J. Pang, W.-H. Chen, Y. Long, Z.-H. Jiang. Bioorg. & Med. Chem Lett., in press, 2006. • J.-F. Wu, L.-X. Chen, G.-A. Luo, Y.-S. Liu, Y.-M. Wang, Z.-H. Jiang. J. Chromatogr. B, 833, 158-164 (2006). • Y. Long, L.-P. Bai, Y. Qin, J.-Y. Pang, W.-H. Chen, Z.-H. Jiang. Bioorg. & Med. Chem., 14, 4670-4676 (2006). • Y. Qin, J.-Y. Pang, W.-H. Chen, Z. Cai, Z.-H. Jiang. Bioorg. & Med. Chem., 14, 25-32 (2006). • J. Pang, Y. Qin, W.-H. Chen, G. A Luo, Z.-H. Jiang. Bioorg. & Med. Chem., 13, 5835-5840 (2005). • W.-H. Chen, J.-Y. Pang, Y. Qin, Q. Peng, Z. Cai, Z.-H Jiang. Bioorg. & Med. Chem. Lett., 15, 2689-2692 (2005). • W.-H. Chen, Y. Qin, Z. Cai, C.-L. Chan, G.-A. Luo, Z.-H. Jiang. Bioorg. & Med. Chem., 13, 1859-1866 (2005). • W.-H. Chen, C.-L. Chan, Z. Cai, G.-A. Luo, Z.-H. Jiang. Bioorg. & Med. Chem. Lett.14, 4955-4959 (2004).

31. Ongoing Studies • DNA G-Quadruplex Binding • RNA, particulary pre-microRNA (Bulged-Hairpins) and micro-RNA (22-nucleotides) • DNA Covalent Adduct Formation

32. Financial Supports 1. Research Grant Committee, University Grant Council, Hong Kong (HKBU 2455/06M; HKBU 2130/04M) 2. Department of Health, Hong Kong 3. Faculty Research Grant, Hong Kong Baptist University

33. Acknowledgement Postgraduate Students: Li-Ping Bai Yong Qin Collaborators: Dr. Wen-Hua Chen Dr. Jiyan Pang Prof. Zongwei Cai Prof. Zhongzhen Zhao

34. Thank You !