Electrostatic Interactions in RNA Aminoglycosides Binding

Aminoglycoside antibiotics interact with seemingly unrelated families of RNA molecules. They bind to 16S ribosomal RNA and interfere with the decoding mechanism by distorting the codon-anticodon recognition.1,2 Similar aminoglycosides specifically inhibit self-splicing group I introns3 and the hammerhead ribozyme.4 Recently, these antibiotics have been found to competitively block the binding of the HIV Rev protein to its viral RNA recognition site (RRE), thereby inhibiting virus production.5 While the molecular details of these important RNA-drug interactions are yet to be elucidated,6 aminoglycosides provide an entry into the largely unexplored area of RNAsmall-molecules recognition.7 As highly functionalized polycationic oligosaccharides, interactions between polar residues of the aminoglycosides (i.e., amino and hydroxyl groups) and the RNA backbone and/or heterocyclic bases are likely to occur.8 The reported structureactivity relationships for the natural aminoglycosides suggest that electrostatic interactions are important for RNA binding.3-5 The most active derivatives contain at least 5 or 6 amino groups that are predominantly charged at pH 7.0.9 The role played by the hydroxyl groups is much less clear. Kanamycin B is 20fold less active than tobramycin (its 3′-deoxy derivative) in inhibiting self-splicing of group I introns,3 and a similar trend has been found in the inhibition of Rev-RRE binding.5 Will the removal of additional hydroxyl groups enhance RNA binding? Comparing the basicity of ethylamine (pKa ) 10.7) to ethanolamine (pKa ) 9.50)10 indicates that the presence of a vicinal hydroxyl lowers the basicity of the amine by more than one pKa unit. We therefore hypothesized that deoxygenated aminoglycoside antibiotics may be stronger RNA binders due to an increased basicity of the neighboring amino groups. The aminoglycosides studied are shown in Figure 1 and include the following: kanamycin B (1), tobramycin (2), dibekacin (4′deoxytobramycin, 3), 6′′-deoxytobramycin (4), 4′′-deoxytobramycin (5), and 2′′-deoxytobramycin (6). This series represents a complete set of aminoglycoside derivatives in which the hydroxyl groups are removed one at a time while the remaining functional groups are kept intact.11 The natural and modified aminoglycosides have been tested for their ability to inhibit the hammerhead ribozyme (Figure 2).12,13 At pH 7.3, the ribozyme E16 cleaves its substrate S16 with a pseudo-first-order rate constant of 0.075 min-1 (Table 1 and Figure 3).11 At a 100 μM aminoglycoside concentration, tobramycin (2) decreases the cleavage rate 6-fold. Dibekacin (3) and 4′′-deoxytobramycin (5), lacking a single secondary hydroxyl each, decrease the cleavage rate by a factor of 20. 2′′-Deoxytobramycin (6), lacking the other secondary hydroxyl on the 3′′-amino-3′′-deoxy-R-D-glucopyranosyl ring is found to be the most potent inhibitor among the deoxy derivatives, resulting in an almost 40-fold decrease in the cleavage rate. These deoxygenated derivatives are therefore far better inhibitors of the hammerhead ribozyme when compared to tobramycin, the parent aminoglycoside, and reach the level of neomycin B (8), the strongest inhibitor hitherto known.4 In contrast, 6′′deoxytobramycin (4), lacking the only primary hydroxyl in the tobramycin skeleton, is less effective than tobramycin (2), slowing down the ribozyme only 4-fold, similar to kanamycin B (1), the least active aminoglycoside tested.14 The most potent ribozyme inhibitors are the deoxygenated aminoglycoside derivatives lacking the secondary hydroxyl groups (Table 1). The 4′-OH is part of a 3-aminopropanol fragment and is likely to interact with the primary 6′-amino group. The 4′′and 2′′-hydroxyls are vicinal to the 3′′-amino group. Additionally, the 2′′-OH is within a hydrogen-bonding (1) Moazed, D.; Noller, H. F. Nature 1987, 327, 389-394. (2) Purohit, P.; Stern, S. Nature 1994, 370, 659-662. (3) von Ahsen, U.; Davies, J.; Schroeder, R. Nature 1991, 353, 368370. von Ahsen, U.; Davies, J.; Schroeder, R. J. Mol. Biol. 1992, 226, 935941. (4) Stage, T. K.; Hertel, K. J.; Uhlenbeck, O. C. RNA 1995, 1, 95-101. (5) Zapp, M. L.; Stern, S.; Green, M. R. Cell 1993, 74, 969-978. Werstuck, G.; Zapp, M. L.; Green, M. R. Chem. Biol. 1996, 3, 129-137. (6) Jiang, L.; Suri, A. K.; Fiala, R.; Patel, D. J. Chem. Biol. 1997, 4, 35-50. Hendrix, M.; Priestly, E. S.; Joyce, J. F.; Wong, C.-H. J. Am. Chem. Soc. 1997, 119, 3641-3648. (7) Wilson, W. D.; Ratmeyer, L.; Zhao, M.; Strekowski, L.; Boykin, D. Biochemistry 1993, 32, 4089-4104. Ratmeyer, L.; Zapp, M. L.; Green, M. R.; Vinayak, R.; Kumar, A.; Boykin, D. W.; Wilson, W. D. Biochemistry 1996, 35, 13689-13696. Park, W. K. C.; Auer, M.; Jaksche, H.; Wong, C.-H. J. Am. Chem. Soc. 1996, 118, 10150-10155. Wang, Y.; Killian, J.; Hamasaki, K.; Rando, R. R. Biochemistry 1996, 35, 12338-12346. Lato, S. M.; Roles, A. R.; Ellington, A. D. Chem Biol. 1995, 2, 291-303. (8) Hendrix, M.; Alper, P. B.; Priestley, E. S.; Wong, C.-H. Angew. Chem., Int. Ed. Engl. 1997, 36, 95-98. (9) Dorman, D. E.; Paschal, J. W.; Merkel, K. E. J. Am. Chem. Soc. 1976, 98, 6885-6888. Botto, R. E.; Coxon, B. Ibid. 1983, 105, 10211028. Szilágyi, L.; Sz. Pusztahelyi, Z.; Jakab, S.; Kovács, I. Carbohydr. Res. 1993, 247, 99-109. (10) For the pKa for the conjugate acid in H2O at 25 °C, see; Lide, D. R., Ed.; CRC Handbook of Chemistry and Physics, 75th ed.; CRC Press: Boca Raton, FL, 1994; pp 8-45. (11) See the Supporting Information for experimental details. (12) Uhlenbeck, O. C. Nature 1987, 328, 596-600. Fedor, M. J.; Uhlenbeck, O. C. Biochemistry 1992, 31, 12042-12054. (13) Aminoglycosides have been shown to interact preferentially with the enzyme-substrate complex and inhibit the cleavage step by displacing critical Mg2+ ions. See ref 4 and: Clouet-d’Orval, B.; Stage, T. K.; Uhlenbeck O. C. Biochemistry 1995, 34, 11186-11190. (14) Lowering the concentration of the aminoglycosides to 10 μM results in a similar trend (Table 1). Figure 1. Natural and synthetic aminoglycosides studied.