The increase in fluorescence on binding of m-phenyl substituted hydroxy derivatives of Hoechst 33258 with poly-[d(A-T)], d(CGCGAATTCGCG)2, and with the corresponding T4-looped 28-mer AATT hairpin was used to monitor binding by equilibrium titrations and stopped-flow kinetics. Replacing the p-OH substituent of Hoechst 33258 (association constant Ka ) 5.2 × 10 8 M -1 for 28-mer hairpin) by m-OH increases the AATT site binding energy by 1.1 kcal mol -1 , Ka ) 3.8 × 10 9 M -1 . Addition of a second m-hydroxy group (bis-m-OH Hoechst) further strengthens binding, giving Ka ) 1.9 × 10 10 M -1 , and the binding energy increases by about 2.1 kcal mol -1 compared to p-OH Hoechst. The value of Ka determined at equilibrium equaled that determined from the ratio of association and dissociation rate constants from stopped-flow studies. The increase in affinity of the monohydroxy Hoechst analogue (m-OH) may originate from water-mediated hydrogen bonding with the minor groove. The further increase in affinity of the bis-m-OH derivative (whose second m-OH group must be directed away from the DNA minor groove floor) may arise from a hydrogen-bonded network of water molecules. The potential to increase binding strength through relayed water molecules is proposed as an additional possible input for lead drug design at DNA targets.
2,2'-p-Phenylene bis[6-(4-methyl-1-piperazinyl)]benzimidazole, 2,2'-bis(3,5-dihydroxyphenyl)-6,6'-bis benzimidazole, and 2,2'-bis(4-hydroxyphenyl)-6,6'-bis benzimidazole are shown by UV-visible and fluorescence spectrophotometry to be strong ligands for tRNA, giving simple, hyperbolic binding isotherms with apparent dissociation constants in the micromolar range. Hydroxyl radical footprinting indicates that they may bind in the D and T loops. On the basis of this tRNA recognition as a rationale, they were tested as inhibitors of the processing of precursor tRNAs by the RNA subunit of Escherichia coli RNase P (M1 RNA). Preliminary studies show that inhibition of the processing of Drosophila tRNA precursor molecules by phosphodiester bond cleavage, releasing the extraneous 5'-portion of RNA and the mature tRNA molecule, was dependent on both the structure of the inhibitor and the structure of the particular tRNA precursor substrate for tRNA(Ala), tRNA(Val), and tRNA(His). In more detailed followup using the tRNA(His) precursor as the substrate, experiments to determine the concentration dependence of the reaction showed that inhibition took time to reach its maximum extent. I(50) values (concentrations for 50% inhibition) were between 5.3 and 20.8 microM, making these compounds among the strongest known inhibitors of this ribozyme, and the first inhibitors of it not based on natural products. These compounds effect their inhibition by binding to the substrate of the enzyme reaction, making them examples of an unusual class of enzyme inhibitors. They provide novel, small-molecule, inhibitor frameworks for this endoribonuclease ribozyme.
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