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-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.
Hori
Y
Y
School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, UK.
Bichenkova
E V
EV
Wilton
A N
AN
El-Attug
M N
MN
Sadat-Ebrahimi
S
S
Tanaka
T
T
Kikuchi
Y
Y
Araki
M
M
Sugiura
Y
Y
Douglas
K T
KT
D001665:Binding Sites; D006690:Bisbenzimidazole / Q000378:metabolism; D018983:DNA Footprinting; D004722:Endoribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D008024:Ligands; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D012322:RNA Precursors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012329:RNA, Bacterial / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016337:RNA, Catalytic / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012343:RNA, Transfer / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012344:RNA, Transfer, Ala / Q000037:antagonists & inhibitors / Q000378:metabolism; D012354:RNA, Transfer, His / Q000037:antagonists & inhibitors / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000037:antagonists & inhibitors / Q000378:metabolism; D012366:RNA, Transfer, Val / Q000037:antagonists & inhibitors / Q000378:metabolism; D043262:Ribonuclease P; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D008024:Ligands; D012322:RNA Precursors; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012344:RNA, Transfer, Ala; D012354:RNA, Transfer, His; D012360:RNA, Transfer, Phe; D012366:RNA, Transfer, Val; D012343:RNA, Transfer; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D006690:Bisbenzimidazole