Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-30
2002-03-12
Zitomer, Stephanie W. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S173100, C435S454000, C435S488000, C435S489000, C435S173600, C435S320100, C536S023100, C536S024200, C536S063000, C514S002600, C424S009100
Reexamination Certificate
active
06355426
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of pharmaceutical screening methods for novel drugs and for the ribonucleotide targets to which they bind.
BACKGROUND OF THE INVENTION
A ribonucleoprotein (RNP) complex is formed between ribonucleic acid (RNA) and protein. Such RNPs are shown to participate in almost all macromolecular processes, including RNA processing, protein synthesis RNA editing, and the signal recognition of proteins targeted for export. Knowledge of the functional importance of RNA is ever increasing, as exemplified by the indication that, for example, 23S rRNA plays a key role in peptidyl transferase [H. Noller et al.,
Science,
256:1416-1419 (1992)]. The translation apparatus (i.e., that which decodes RNA for protein synthesis) is essential to all living cells and represents one of the major targets for antibiotics and other pharmaceutically useful compositions.
An understanding of the precise mechanism of drug action is dependent on detailed knowledge at the molecular level, of the structure and function of the drug-RNP complex, e.g., the ribosome and its associating factors. However, such understanding at the molecular level of RNA structure and RNA-ligand interactions has been hampered by the size and complexity of, for example, the ribosome and other RNP particles.
As one example of RNP particles, the ribosome is likely to have evolved from autonomously assembled structural sub-domains. Domain organization occurs within the ribosome. Partial ribonuclease digestion of the 30S subunit releases a RNP complex containing ribosomal proteins (r-proteins) S7, S9, S19, S13, or S14 and fragments derived from the 3′ half of 16S rRNA [J. Morgan et al.,
Eur. J. Biochem.,
29:542-552 (1972); A. Yuki et al,
Eur. J. Biochem.,
56:23-34 (1975)]. Specific RNP particles encompassing the 5′ and central domains have also been isolated [R. A. Zimmerman,
Ribosomes,
(Nomura, M. et al. eds.) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1974), p. 225-269]. The small subunit ribosomal RNA (rRNA) from
E. coli
(16S rRNA) is organized into three major domains: the 5′, central and 3′ domains. Ribosomal RNA fragments representing each of these subdomains can reassemble with specific subsets of ribosomal proteins [Noller, H. F. et al,
Science,
212: 402-411 (1981)].
Several lines of evidence support the notion that the ribosome can be fragmented into smaller, functional subdomains that retain organizational and ligand-binding properties characteristic of the intact particle. For example, in vitro assembly of intact 30S subunits have further demonstrated two to three independent nucleation events for various domains within the 30S subunit. This provides a further indication for the existence of independent assembly domains [Nomura, M. et al,
J. Cell Physiol.,
74: 241-252 (1974); W. A. Held et al.,
J. Biol Chem.,
249:3103-3111 (1974)].
More recently, a fragment corresponding to the 5′ domain (nucleotides 1-526) assembled with r-proteins S4, S16, S17, and S20 [C. J. Weitzmann et al.,
FASEB J.,
7:177 (1993)], and a fragment of the 3′ domain of 16S rRNA (nucleotides 923-1542) reconstituted together with eight r-proteins formed a structure that resembled the head of the 30S subunit [Samaha, R. R. et al,
Proc. Natl. Acad. Sci.,
91:7884-7888 (1994)]. This particle retains the property of being able to bind the antibiotic, spectinomycin, which specifically protects the N-7 position of G1064 from attack by dimethylsulphate in both 30S subunits and the sub-particle [Samaha et al, cited above; Moazed, D. et al, Nature (London), 327: 389-394 (1987)].
The degree of protection to both particles shows the same dependence on drug concentration, indicating that spectinomycin binds with similar affinity to each particle. Resistance mutations have been mapped to structural changes in either ribosomal protein S5 or within helix 34 of 16S rRNA formed by base pairing between nucleotides 1046-1065 and 1191-1211 [Brink, M. F. et al,
Nucleic Acids Res.,
22:325-331 (1994) and Johanson, U. et al,
Nucleic Acids Res.,
23:464-466 (1995)]. See FIG.
1
.
Further dissection of the ribosome has been achieved and expanded to include interactions with ligands other than ribosomal proteins. An oligoribonucleotide analogue of the decoding region located near to the 3′ end of 16S rRNA interacts with both antibiotic (neomycin) and RNA ligands (tRNA and mRNA) of the 30S subunit in a manner that resembles normal subunit function {Purohit, P. et al,
Nature
(London), 370: 659-662 (1994)}. Accordingly, fragmentation of RNP complexes and indeed large RNAs can be considered as a potent strategy in the analysis of such molecules [Schroeder, R.,
Nature
(London), 370:597-598 (1994)].
The binding of the antibiotic, spectinomycin, is independent of S5 indicating that the rRNA is the major determinant of the binding site [Samaha et al, cited above]. The G1064-C1192 base pair is likely directly involved in the binding of spectinomycin as revealed by the chemical footprinting data and the existence of resistance mutations that reflect either a disruption of the base pair or replacement of the base pair [Brink et al, cited above].
Mutations in
E. coli
16S rRNA that confer spectinomycin resistance include C1192U,G,A, G1064U,C,A, G1064U-C1192A, G1064-C1192G, G1064A-C1192U and C1066U. The major effect of spectinomycin in vitro is proposed to inhibit the translocation of peptidyl-tRNAs from the A-site to the P-site by preventing the binding of elongation factor G (EF-G) to the ribosome [Bilgin, N. et al.,
EMBO J.,
9: 735-739 (1990)]. Helix 34 has been proposed to melt during the elongation cycle and spectinomycin exerts its inhibitory effect by stabilizing the helix [Brink et al, cited above]. Helix 34 has the potential to exhibit two structural conformers similar to the phylogenetic model, without disruption of the overall base pairing arrangement [Prescott, C. D. et al., Biochimie 1991, 73, 1121-1129] (See FIG.
2
). The conformers reflect the alternate availability of either an “upper” (1199-1201) or “lower”(1202-1204) 5′-UCA-3′ triplet.
To date, characterization of the interaction between drug and rRNA has been based on the above-described in vitro approaches, for example, ligand binding to an oligoribonucleotide analogue of the decoding region located near to the 3′ end of 16S rRNA. This RNA fragment interacts with both antibiotic (neomycin) and RNA ligands (tRNA and mRNA) of the 30S subunit in a manner that resembles normal subunit function.
Despite the wealth of research in this area, there remains a need in the art for methods and compositions useful for identifying pharmaceutically useful compounds, e.g., antibiotics, which bind cellular RNA targets.
SUMMARY OF THE INVENTION
As one aspect, the present invention provides a method for identifying an RNA fragment that mimics the structure of a binding site of a target RNA molecule (hereafter referred to as a “mimicking RNA fragment”). In this method, the target molecule is a defined, known RNA molecule. The method includes the steps of providing a defined DNA fragment, by either fragmenting DNA encoding the target RNA molecule with one or more restriction enzymes or chemically synthesizing a DNA fragment encoding a portion of the RNA target molecule. The defined fragment is cloned into a plasmid which, under suitable conditions, permits synthesis of the RNA fragment encoded by the DNA fragment. The plasmid is transfected into a host cell which contains the target RNA molecule. Untransfected host cells are cultured in the presence of a compound which inhibits cell growth or kills the cells. The transfected cells are similarly cultured in the presence of the compound. If the transfected cells permit the synthesis of an RNA fragment that mimics the target molecule, the RNA fragment imparts drug resistance to the t
Fedon Jason C.
Gimmi Edward R.
Kinzig Charles M.
Smithkline Beecham Corporation
Wilder Cynthia B.
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