Recognition in the Minor Groove of DNA at 5’-(A,T)GCGC(A,T)-3’ by a Four Ring Tripeptide Dimer. Reversal of the Specificity of the Natural Product Distamycin

The tripeptide ImPImP containing alternating imidazole and pyrrole carboxamides specifically binds the designated six base pair site 5’-d(A,T)GCGC(A,T)-3’ in the minor groove of DNA. Quantitative footprint titration experiments demonstrate that ImPImP binds the sites 5’-AGCGCT-3’ and 5’-TGCGCA-3’ with apparent first order binding affinities of 3.8 x lo5 M-I and 3.6 x lo5 M-I, respectively (25 mM tris acetate, 10 mM NaC1, pH 7.0 and 22 “C). Affinity cleaving experiments with ImPImP-EDTA-Fe reveals equal cleavage on both sides of the 5’-(A,T)GCGC(A,T)-3’ site, consistent with a side-by-side antiparallel arrangement of the four ring peptides in the minor groove. This reversal of specificity of the natural product distamycin which prefers to bind pure A,T sequences underscores the utility of 2: 1 peptide-DNA models for the design of ligands for sequence-specific recognition in the minor groove of DNA. By extending these peptides to four ring systems, a new lower limit of six base pair binding by 2: 1 peptide-DNA complexes-has been defined. The natural products netropsin (N) and distamycin A (D) are crescent shaped diand tripeptides, respectively, that bind in the minor groove of DNA at sites of four or five successive A,T base pairs (bp).1-3 The structures of a number of peptideDNA complexes have been determined by X-ray diffraction4 and NMR ~pectroscopy,~ and the thermodynamic profiles have been studied for these complexes.6 This work suggests that favorable electrostatic interactions and extensive van der Waals contacts between the peptide and the floor and walls of the minor groove contribute to complex stability. The carboxamide NH’s of the peptides participate in bifurcated hydrogen bonds with adenine N3 and thymidine 0 2 atoms on the floor of the minor groove. The aromatic hydrogens of the N-methylpyrrole rings are set too deeply in the minor groove to allow room for the guanine 2-amino group of a G,C base pair, affording binding specificity for A,T-rich sequences. Although this model has @ Abstract published in Advance ACS Abstracts, March 15, 1995. (1) For a review, see: Zimmer, C.: Wihnert, U. Prog. Biophys. Molec. Bid. 1986, 47, 31-112. (2) (a) Van Dyke, M. W.: Hertzberg, R. P.: Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 1982, 79,5470-5474. (b) Van Dyke, M. W.: Dervan, P. B. Cold Spring Harbor Symposium on Quantitative Biology 1982,47,347353. (c) Van Dyke, M. W.; Dervan, P. B. Biochemistry 1983, 22, 23732377. (d) Harshman, K. D.; Dervan, P. B. Nucl. Acids Res. 1985,13,48254835. (e) Fox, K. R.: Waring, M. J. Nucl. Acids Res. 1984, 12, 92719285. (0 Lane, M. J.; Dabrowiak, J. C.; Vournakis, J. Proc. Narl. Acad. Sci. U.S.A. 1983, 80, 3260-3264. (3) (a) Schultz, P. G.; Taylor, J. S.: Dervan, P. B. J. Am. Chem. Soc. 1982, 104, 6861-6863. (b) Taylor, J. S.: Schultz, P. G.: Dervan, P. B. Tetrahedron 1984,40,457-465. (c) Schultz, P. G.: Dervan, P. B. J. Biomol. Strucr. Dyn. 1984, 1, 1133-1147. (4) (a) Kopka, M. L.: Yoon, C.; Goodsell, D.: Pjura, P.: Dickerson, R. E. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 1376-1380. (b) Kopka, M. L.; Yoon, C.: Goodsell, D.; Pjura, P.: Dickerson, R. E. J. Mol. Biol. 1985, 183, 553-563. (c) Coll, M.: Frederick, C. A,; Wang, A. H.-J.; Rich, A. Proc. Narl. Acad. Sci. U.S.A. 1987, 84, 8385-8389. (5) (a) Patel, D. J.; Shapiro, L. J. B id . Chem. 1986, 261, 1230-1240. (b) Klevitt, R. E.; Wemmer, D. E.: Reid, B. R. Biochemistry 1986, 25, 3296-3303. (c) Pelton, J. G., Wemmer, D. E. Biochemistry 1988,27,80888096. (6) (a) Markey, L. A.: Breslauer, K. J. Proc. Natl. Acad. Sci. U.S.A. 1987, 84,4359-4363. (b) Breslauer, K. J.; Remeta, D. P.: Chou, W.-Y.; Ferrante, R.: Curry, J.; Zaunczkowski, D.; Snyder, J. G.: Marky, L. A. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 8922-8926. aided in the design of oligopeptides for recognition of longer tracts of A,T-rich DNA,’ efforts based on a 1:l peptide-DNA model to design peptides capable of binding sequences containing both A,T and G,C base pairs have met with limited S U C C ~ S S . ~ ~ 2:l Peptide-DNA Complexes. The observation that distamycin (at 2-4 mM) is capable of binding in the minor groove of 5’-AAA1T-3’ as a side-by-side dimer required consideration of 2: 1 complexes for recognition in the minor groove of DNA.’O This explained why the nonnatural peptides pyridine-2-carboxamidenetropsin (2-PyN) and 1-methylimidazole-2-carboxamidenetropsin (2-1“) bind the unanticipated sequence 5’(A,T)G(A,T)C(A,T)-3’ (Figure l).Il This example of specific recognition of sequences containing both A.T and GC base pairs suggests that the 2:l motif may serve as a new model for the design of peptide analogs for specific recognition of other sequences (Figure l).‘] The 2: 1 models differ significantly from that of 1: 1 models in that each ligand of the dimer interacts with one of the DNA strands in the minor groove. The imidazole nitrogen of each ligand hydrogen bonds specifically with one guanine amino group. The side-by-side combination of one imidazole ring on one ligand and a pyrrole ring on the second ligand recognizes _ (7) Dervan, P. B. Science 1986, 232, 464-471. (b) Youngquist, R. S.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 1985. 82. 2565-2569. (c) Youngquist, R. S.: Dervan, P. B. J. Am. Chem. SOC. 1987, 109, 75647566. (8) For early examples of hybrid molecules for the recognition of mixed sequences, see: (a) Dervan, P. B.; Sluka, J. P. New Synthetic Methodology and Functionally Interesting Compounds: Elsevier: New York, 1986; pp 307-322. (b) Griffin, J. H.; Dervan, P. B. J. Am. Chem. SOC. 1987, 109, 6840-6842. (9) Kissinger, K.; Drowicki, K.: Dabrowiak, J. C.; Lown, J. W. Biochemistry 1987, 26, 5590. (10) (a) Pelton, J. G.; Wemmer, D. E. Proc. Natl. Acad. Sci. U.S.A. 1989, 86,5723-5727. (b) Pelton, J. G.: Wemmer, D. E. J. Am. Chem. SOC. 1990, (11) (a) Wade, W. S., Dervan, P. B. J. Am. Chem. SOC. 1987,109, 15741575. (b) Wade, W. S.; Mrksich, M.; Dervan, P. B. J. Am. Chem. SOC. 1992, 114, 8783-8794. (c) Mrksich, M.; Wade, W. S.: Dwyer, T. J.; Geierstanger, B. H.; Wemmer, D. E.: Dervan, P. B. Proc. Nail. Acad. Sci. U.S.A. 1992, 89, 7586-7590. (d) Wade, W. S.: Mrksich, M.: Dervan, P. B. Biochemistry 1993, 32, 11385-11389. 112, 1393-1399. 0002-7863/95/1517-3325$09.00/0