Molecular Anchoring of Duplex and Triplex DNA by Disubstituted Anthracene-9,10-diones: Calorimetric, UV Melting, and Competition Dialysis Studies Ihtshamul Haq, † John E. Ladbury, ‡ Babur Z. Chowdhry, † and Terence C. Jenkins* ,§ Contribution from the School of Chemical and Life Sciences, The UniVersity of Greenwich, Wellington Street, London SE18 6PF, UK, Department of Biochemistry, UniVersity College London, Riding House Street, London W1P 8BT, UK, and CRC Biomolecular Structure Unit, The Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG, UK ReceiVed June 6, 1996 X Abstract: Isothermal titration calorimetry, UV melting, and competition dialysis techniques have been used to examine the binding of isomeric 1,4- and 2,6-bis(ω-aminopropionamido)-substituted anthracene-9,10-diones (anthraquinones) with dA n ‚dT n duplexes and dT n -dA n ‚dT n triplexes. Recent footprinting studies [Fox, K. R.; Polucci, P.; Jenkins, T. C.; Neidle, S. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7887-7891] indicate that 2,6 derivatives, but not their 1,4 counterparts, differentially stabilize triple-stranded DNA and may have application in antigene chemotherapy. Thermodynamic investigations are here reported for interaction with dA 18 ‚dT 18 and dT 18 -dA 18 ‚dT 18 . The 2,6 compound shows preferential triplex binding, with K b values of 1.8 × 10 4 M (duplex) -1 and 2.2 × 10 5 M (triplex) -1 at 25 °C in aqueous solution, pH 6.0, whereas the 1,4 isomer favors duplex binding, with K b values of 1.1 × 10 5 M (duplex) -1 and 3.5 × 10 4 M (triplex) -1 . Binding to the preferred DNA is enthalpically driVen for each ligand, whereas binding to the disfavored DNA is either entropically driVen or enthalpy/entropy compensated. Further, the binding site sizes (3.6 base pairs/base triplets) suggest DNA intercalation. Competition dialysis studies with poly- (dA)‚poly(dT) and poly(dA)‚poly(dT) 2 confirm these binding preferences, and qualitative support is provided from UV melting experiments. Such studies reveal triplex disruption by the 1,4 isomer at low drug concentrations while the 2,6 compound effects stabilization toward thermal triplex denaturation. Spectrophotometric studies of each free ligand indicate self-association in aqueous solution, with dimerization constants at 25 °C of (2.9 ( 0.2) × 10 3 and (3.2 ( 0.1) × 10 3 M -1 respectively for the 1,4 and 2,6 isomers. Taken together, these data provide a firm thermodynamic basis for the contrasting duplex/triplex binding preferences of this isomeric family of ligands. Introduction The ability of DNA to form triple-helical structures has been known for almost 40 years. 1,2 Current interest in triplex DNA has largely been stimulated by potential applications of triplex- forming oligonucleotides (TFOs) as therapeutic agents, particu- larly as part of a DNA duplex-targeted antigene strategy. 3,4 Thus, for example, a TFO may be used to artificially control the expression of regulatory genes by inhibiting either transcription or regulatory protein binding after sequence-selective recognition and hybridization to a target double-stranded DNA site (for reviews, see refs 2-4). Several recent studies have highlighted the biological potential and viability of the oligonucleotide- directed antigene approach. 5,6 Intermolecular DNA triplexes can form when an oligopyrim- idine strand binds in the major groove of a host homopurine‚ homopyrimidine duplex sequence, with the formation of T-A‚T and C + -G‚C (i.e., Py-Pu‚Py) base triplets, such that the introduced strand adopts a parallel orientation relative to the host purine strand. In the latter case this only occurs at low pH (e5.5) since the third-strand cytosines must be ring protonated to facilitate interstrand hydrogen bonding. An alternative triplex can be produced when an oligopurine strand binds to the DNA duplex in an antiparallel fashion, leading to A-T‚A and G-C‚G (i.e., Pu-Py‚Pu) triplets. 2,7 Hence, subject to certain conditions, 4,8 site-specific triplex binding can lead to a recognition of target DNA duplex sequences. Triplex instability under physiological conditions represents a major limiting difficulty to the therapeutic use of TFOs, since the C + -G‚C triplet requires a low pH and the T-A‚T triplet is only stable under conditions of high ionic strength. Several * Author to whom correspondence should be addressed at the follow- ing: The Institute of Cancer Researchstelephone (+44) 181 643-8901, FAX (+44) 181 770-7893, E-mail t.jenkins@icr.ac.uk. † University of Greenwich. ‡ University College London. § The Institute of Cancer Research. X Abstract published in AdVance ACS Abstracts, October 1, 1996. (1) Felsenfeld, G.; Davies, D. R.; Rich, A. J. Am. Chem. Soc. 1957, 79, 2023-2024. (2) Soyfer, V. N.; Potaman, V. N. Triple-Helical Nucleic Acids; Springer-Verlag: New York, 1996. (3) He ´le `ne, C. Anti-Cancer Drug Des. 1991, 6, 569-584. (4) Thuong, N. T.; He ´le `ne, C. Angew. Chem., Int. Ed. Engl. 1993, 32, 666-690. (5) (a) McShan, W. M.; Rossen, R. D.; Laughter, A. H.; Trial, J.; Kessler, D. J.; Zendegui, J. G.; Hogan, M. E.; Orsan, F. M. J. Biol. Chem. 1992, 267, 5712-5721. (b) Ing, N. H.; Beekman, J. M.; Kessler, D. J.; Murphy, M.; Jayaraman, K.; Zendegui, J. G.; Hogan, M. E.; O’Malley, B. W.; Tsai, M. J. Nucleic Acids Res. 1993, 21, 2789-2796. (c) Grigoriev, M.; Praseuth, D.; Guieysse, A. L.; Robin, P.; Thuong, N. T.; He ´le `ne, C.; Harrel-Bellan, A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3501-3505. (6) (a) Helm, C. W.; Shrestha, K.; Thomas, S.; Shingleton, H. M.; Miller, D. M. Gynacol. Oncol. 1993, 49, 339-343. (b) Mayfield, C.; Ebbinghaus, S.; Gee, J.; Jones, D.; Rodu, B.; Squibb, M.; Miller, D. J. Biol. Chem. 1994, 269, 18232-18238. (c) Vasquez, K. M.; Wensel, T. G.; Hogan, M. E.; Wilson, J. H. Biochemistry 1995, 34, 7243-7251. (d) Macaulay, V. M.; Bates, P. J.; McLean, M. J.; Rowlands, M. G.; Jenkins, T. C.; Ashworth, A.; Neidle, S. FEBS Lett. 1995, 372, 222-228. (7) Howard, F. B.; Miles, H. T.; Ross, P. D. Biochemistry 1995, 34, 7135-7144. (8) (a) Roberts, R. W.; Crothers, D. M. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 9397-9401. (b) Mergny, J-L.; Sun, J-S.; Rougee ´, M.; Montenay- Garestier, T.; Barcelo, F.; Chomilier, J.; He ´le `ne, C. Biochemistry 1991, 30, 9791-9798. 10693 J. Am. Chem. Soc. 1996, 118, 10693-10701 S0002-7863(96)01907-5 CCC: $12.00 © 1996 American Chemical Society