Synthetic inhibitors of the processing of pretransfer RNA by the ribonuclease P ribozyme: Enzyme inhibitors which act by binding to substrate

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-hydroxyyhenyl)-6,6 '-bis benzimidazole are shown by UV-visible and fluorescence spectrophotome try to be strong ligands for tRNA, giving simple, hyperbolic binding isothe rms 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 inhi bitors of the processing of precursor tRNAs by the RNA subunit of Escherich ia coli RNase P (M1 RNA). Preliminary studies show that inhibition of the p rocessing of Drosophila tRNA precursor molecules by phosphodiester bond cle avage, releasing the extraneous 5'-portion of RNA and the mature tRNA molec ule, was dependent on both the structure of the inhibitor and the structure of the particular tRNA precursor substrate for tRNA(Ala), tRNA(Val), and t RNA(His). In more detailed followup using the tRNA(His) precursor as the su bstrate, experiments to determine the concentration dependence of the react ion showed that inhibition took time to reach its maximum extent. I-50 valu es (concentrations for 50% inhibition) were between 5.3 and 20.8 muM, makin g these compounds among the strongest known inhibitors of this ribozyme, an d 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 provi de novel, small-molecule, inhibitor frameworks for this endoribonuclease ri bozyme.