SUNY at Albany
June 19-23, 2001
Processing of pre-transfer RNA by ribonuclease P : design of synthetic inhibitors of a ribozyme.
In all organisms studied to date the transfer RNA genes express tRNA as precursors requiring processing to give the functional tRNA molecules. Such maturation of the tRNA 5¢-terminus is carried out by ribonuclease P (RNase P). This endoribonuclease in eubacteria consists of an RNA subunit (~400 nucleotides) and a 14kDa protein subunit. The RNA provides the catalytic activity and can even cleave its substrate correctly in the absence of protein under suitable conditions. The protein subunit appears to aid product release. Some aminoglycosides inhibit RNase P and, as RNase P is essential and shows differences between species, has these antibiotics may be lead structures for drug design. We show here the design of strong RNaseP inhibitors.
We have found that heterocyclic derivatives, e.g. 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 can be strong ligands for tRNA. Binding isotherms show apparent dissociation constants in the mM range. Hydroxyl radical footprinting was carried out of their binding to tRNA and indicates that they may bind in the D and T loops. Based on this tRNA-recognition as a rationale, they were tested as inhibitors of the processing of precursor tRNAs by the RNA subunit of E. 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 of the particular tRNA precursor substrate for tRNAAla, tRNAVal and tRNAHis. I50 values (concentrations for 50% inhibition) were between 5.3 and 20.8 the mM, making these compounds amongst 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 inhibitor. They provide novel, small-molecule, inhibitor frameworks for this endoribonuclease ribozyme.
Acknowledgements. We are grateful to the British Council for a Collaborative Research Project Grant (KTD), the Libyan General Secretariat of Education and Vocational Formation (MNEA) and the "Research for the Future" programme of the Japan Society for the Promotion of Science, grant JSPS-RFTF97I00301 (YK).
Yoshiaki Hori (2), Elena V. Bichenkova (1), Amanda N. Wilton (1), Mohamed N. El-Attug (1),
Sch'l of Pharmacy & Pharmaceutical Sci. (1),
Univ. of Manchester, M13 9PL, U.K.