Synthetic Inhibitors of the Processing of Pretransfer RNA by the Ribonuclease P Ribozyme:  Enzyme Inhibitors Which Act by Binding to Substrate

Abstract

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.

A real-time fluorescence polarization activity assay to screen for inhibitors of bacterial ribonuclease P

“…We have demonstrated that the FP/FA assay can detect small ligand binding to pre-tRNA if the interactions alter the solution structure (Figure 4). This assay is more sensitive for measuring small ligands binding to tRNA than previous methods relying on changes in the intrinsic fluorescence of modified bases on native tRNA (49) or of the compounds (24,54,55), and other spectroscopic methods such as UV-VIS and circular dichroism (24,54–56). The FP/FA assay reported here using end-labeled pre-tRNA provides a general and convenient way of measuring binding and stabilization of in vitro transcribed tRNA by non-fluorescent molecules.…”

“…Aminoglycosides are also weak non-competitive inhibitors of eukaryal RNase P ( K i = 143 μM for neomycin B to Dictyostelium discoideum RNase P) (23). A number of synthetic compounds, including bis-benzimidazoles ( IC 50 = 5–21 μM) (24), porphines and porphyrins ( K i = 1–4 μM) (25), inhibit E. coli RNase P activity mainly through binding to pre-tRNA. Spiramycin, a macrolide antibiotic, was reported to activate the steady-state turnover catalyzed by E. coli RNase P (26).…”

RNA-targeted therapeutics: prospects and promise

“…Footprinting experiments indicate that they bind in the D and T loop regions. A few of these compounds, in particular the bis(4hydroxyphenyl) derivative, were found to inhibit tRNA processing by RNase P, also at the low micromolar levels [37]. Although no patenting activity on these compounds for an RNA-binding utility has yet appeared, the compounds are [101,104,120,121].…”

Kinetics of Hyperprocessing Reaction of Human Tyrosine tRNA by Ribonuclease P Ribozyme fromEscherichia coli

“…Recently, however, certain tRNAs have been found to be disrupted, some without base modiˆcation and some under in vitro conditions. [3][4][5][6][7][8][9][10][11] In other wors, not all tRNAs are rigid, and their cloverleaf shape is not always stable. The study of such destabilized or unstable tRNAs should give clues about the ways in which tRNA molecules are stabilized and also about the origin of tRNA molecules.…”

“…[3][4][5][6][7][8][9][10] The change from a cloverleaf to a double-hairpin can be monitored by the reaction of ribonuclease P from Escherichia coli. [3][4][5][6][7][8][9][10] Ribonuclease P also has been found in all species examined. The enzyme catalyzes the 5?-processing reaction of tRNA precursors to produce a mature 5?-end in tRNA molecules.…”