Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-11-02
2003-01-28
Cochrane Carlson, Karen (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C530S350000, C530S300000, C536S023100, C514S002600
Reexamination Certificate
active
06511813
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed, in part, to methods of using elongation factor p (efp) and related constituents of ribosomal complexes which comprise efp, the 50S ribosomal subunit, the 30S ribosomal subunit, the 70S ribosome, and related proteins, cofactors and enzymes to identify compounds that modulate prokaryotic cell function. Antibiotic compounds affecting such cell functions and methods of using those compounds to treat microbial infections in mammals are also described.
BACKGROUND OF THE INVENTION
An important catalytic function of ribosomes is the synthesis of peptide bonds. Various studies have suggested that the 70S ribosome, which is comprised of the 30S and 50S ribosomal subunits, is important for protein synthesis.
Models of protein synthesis assume that once the aminoacyl-tRNA is bound to the ribosomal A-site of the 70S ribosome complex, peptidyltransferase, an integral part of the 50S ribosomal subunit, can condense all twenty-one aminoacyl-tRNAs with equal efficiency, without intervention of exogenous proteins and GTP. However, several studies indicate that in vitro, the peptidyltransferase condenses predominantly hydrophobic amino acids. Peptide bond synthesis in vitro is also dependent upon aminoacyl moieties. In particular, prokaryotic 70S ribosomes cannot efficiently incorporate certain amino acids into polypeptides from cytidyl aminoacyl-adenosine (CA) (analogues of the 3′-terminal end of aminoacyl-tRNAs). As well, several antibiotics, such as anisomycin and chloramphenicol, inhibit peptide bond synthesis with some aminoacyl-tRNAs and not with others.
A prokaryotic gene encoding one of several known types of elongation factor proteins, elongation factor p (efp) was cloned and sequenced. Aoki et al.,
Nucleic Acids Research
, 1991 (19), pp. 6215-6220, the disclosure of which is incorporated herein by reference in its entirety. Efp has been found to be essential for cell viability. Efp stimulates the efficiency of the peptidyltransferase activity of procaryotic ribosomes between fMet-tRNA
f
Met
and analogues of various aminoacyl-tRNAs. For example, the K′ for the cytidyl(3′-5′)-[2′(3′)-O-L-CA-Gly is enhanced 50-fold, whereas that for CA-Phe is essentially unaltered by efp. Efp may modulate the efficiency of protein synthesis by controlling the rate of synthesis of certain peptide bonds. There are 800-900 molecules of efp per
E. coli
, or about 0.1 to 0.2 copy per ribosome, suggesting that efp may function catalytically in the cell. The preparation and isolation of efp can be found in M. C. Ganoza et al.,
Eur. J. Biochem
., 1985, vol. 146, pp. 287-294, and/or D-G. Chung et al. Chapter 4, pp. 69-80 of Ribosomes and Protein Synthesis, A Practical Approach, edited by G. Spedding, 1990, IRL Press at Oxford University Press, Oxford, N.Y. and Tokyo, the disclosures of which are incorporated herein by reference in their entirety.
The requirements of peptide-bond and ester-bond formation stimulated by efp have been studied with fMet-tRNA
f
Met
bound to 30S subunits and native or reconstituted 50S subunits. Efp functions in both peptide- and ester-bond synthesis promoted by the peptidyltransferase. The L16 protein (N-terminal fragment) of the 50S subunit is required for the efp-mediated synthesis of peptide bonds, whereas the L11, L15, and L7/L12 are not required in this reaction, suggesting that efp may function at a different ribosomal site than most other translation factors.
Of interest is the fact that efp differentially stimulates peptide bond synthesis when various amino acids are covalently linked to aminoacyl-adenosine (CA). It is possible that efp preferentially acts on weak acceptors for the peptidyltransferase. The specific mechanism whereby efp stimulates bond synthesis is not entirely clear. Efp may help accommodate fMet-tRNA
f
Met
or peptidyl-tRNAs, or both, within the active center of the peptidyltransferase or it could affect peptidyltransferase directly.
The position occupied by each species of aminoacyl-tRNA on the ribosomes has been studied using antibiotics that are known to inhibit specific sites on the ribosome. Two types of A sites can be distinguished by their different reactivities towards specific antibiotics. The first site (of the i type) occur after fMet-tRNA
f
Met
has directly entered the ribosomal P-site, where the E-site is free. The second A-site (of the e type) is the one normally used to bind aminoacyl-tRNAs to 70S ribosomes during the course of chain elongation.
The antibiotics neomycin, thiostrepton, and hygromycin appear to inhibit translocation and occupation of the A site, but they inhibit only about 20% or have no effect on efp reaction. These antibiotics also have no effect on formation of the fMet-tRNA
f
Met
/ribosome translation complex nor on the peptide-bond synthesis which occurs in the absence of efp.
Streptomycin at 2×10
−5
M, which causes misreading and also inhibits A-site occupation of the e type, is a potent inhibitor of the efp-mediated reaction. The efp-mediated reaction is one in which purified efp is added to a translation complex of fMet-tRNA
f
Met
: 70S ribosome:mRNA; and then puromycin or an appropriate amino acid-charged tRNA is added. Efp mediates the formation of a peptide bond between the fmet and the second amino acid. Streptomycin is known to interact with two sites on the 16S rRNA of the 30S subunit. It is unknown, however, whether streptomycin acts to directly inhibit efp.
Lincomycin inhibits peptidyltransferase and occupation of the A-site of the e type. Lincomycin has marginal effects on the synthesis of polyphenylalanine, but inhibits the puromycin reaction and nullifies the ability of efp to stimulate synthesis of peptide bonds. Erythromycin, also inhibits peptidyltransferase and it destabilizes the peptidyl-tRNA/ribosome/mRNA complex but it has no apparent effect on the efp reaction at 5×10
−5
.
The present invention involves the surprising discovery of the critical role that efp may have in the procaryotic cell, and its role as a key component in the search for novel antimicrobial agents. These and other aspects of the invention are described below.
SUMMARY OF THE INVENTION
There is a need for more rapid and direct methods to screen compounds which may modulate ribosome mediated peptide bond formation. Such screening assays may discover new and useful antibiotics. New screens to detect and characterize compounds that affect efp and its functioning in the 70S ribosome, the 50S and 30S ribosomal subunits, and related proteins are disclosed herein. Newly discovered compounds or agents may promote cell death. The new understanding of the mechanism of action of known antimicrobials disclosed here may extend the usefulness of those antimicrobial agents.
Because of the surprising discovery disclosed here for the critical role that efp plays in the procaryotic cell, we can now disclose several aspects of this invention. Described herein are new methods or procedures to screen for, detect and/or characterize new compounds that modulate the function of efp in the prokaryotic cell. These methods or procedures include new in vitro methods as well as new in vivo methods.
In some embodiments of the invention, methods for identifying a compound which modulates activity of a prokaryotic elongation factor p in an in vitro assay, a cell based assay to determine the affect of the compound on cell function, a cell free extract assay to determine the affect of the compound on cell function are provided. The in vitro assay preferably comprises the steps of exposing elongation factor p with a compound, determining whether the compound modifies activity of the elongation factor p, and the cell-based assay preferably comprises determining whether the compound modifies activity of cell function. In some embodiments of the invention, the in vitro assay comprises determining whether the activity of the elongation factor p is decreased, determining whether the elongation factor p binds to the compound, determining whether th
Marotti Keith R.
Poorman Roger A.
Shinabarger Dean L.
Wells Peter A.
Carlson Karen Cochrane
O'Connor P.C. Cozen
Pharmacia & Upjohn Company
Robinson Hope A.
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