JP2003523729A - Methods for modulating prokaryotic ribosome activity - Google Patents

Abstract

  (57) [Summary] The present invention relates to newly identified polynucleotides and the interaction of these polynucleotides and polypeptides and their production and use, as well as their variants, agonists and antagonists and their uses. In particular, in these and other respects, the invention relates to polynucleotides used in identifying compounds that modulate the activity of prokaryotic ribosomes.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to newly identified polynucleotides and the interaction of these polynucleotides with polypeptides, as well as their production and use, as well as their variants, agonists and antagonists and their uses. In particular, and in other respects, the invention relates to polynucleotides used to identify compounds that modulate the activity of prokaryotic ribosomes.

BACKGROUND OF THE INVENTION Pluromutilin is a type of protein synthesis inhibitor that has antimicrobial activity and selectively binds to prokaryotic ribosomes. The exact location of the pluromutilin ribosome binding site and the importance of various ribosomal protein and ribosomal RNA interactions is still poorly defined. A detailed knowledge of the structure, function and binding sites of any target is key to understanding the mechanism of drug action. Provided herein is data from a study of a series of pluromutilin derivatives that reveals a pluromutilin binding site on the 70S ribosome utilizing some mode of inhibition by pluromutilin and several in vitro translation and ribosome binding assays. These studies, in part, have led to the development of the present invention.

Emerging bacterial resistance to currently known classes of antibiotics is a major global health problem (1). Moreover, the most commonly used antibiotics (eg macrolides, beta-lactams and quinolones) were first marketed over 30 years ago. The increasing rate at which bacteria evolve drug resistance in combination with the lack of new classes of antibiotics has driven the need for new research approaches to discover new and develop chemotherapeutic agents in development. The choice of target for development confirms the functional importance of the target to the pathogen (essential) and guarantees that the host homologues in the infected host are significantly different from the pathogen target and that host processes are not inhibited by the drug (
Selectivity) and target are homologous in the selected pathogen and are based on a number of criteria including providing the desired antimicrobial spectrum. Interdisciplinary methods, including molecular biology, biochemistry, chemistry and more recently bioinformatics are needed to address these issues. This method, coupled with the pharmaceutical industry's interest in developing antibiotics,
This has led to the work disclosed herein on the pluromutilin class of molecules as potential antibiotics. Small molecules that inhibit prokaryotic ribosome function have long been known to be important antibacterial compounds as well as excellent tools for elucidating ribosome function (2-6
And references contained therein). The natural product pluromutilin was first isolated in 1951 from the basidiomycete Pleurotus mutilus (
Structure 1). Ploromutilin and its semi-synthetic analogues
It inhibits E. coli) protein synthesis and exhibits potent antimicrobial activity against Staphylococcus species, Streptococcus species, and Haemophilus influenzae (7). Semi-synthetic pluromutilin was introduced in the 1970s and 1
Although studied in the early 980s, no drug was successfully developed for human use.

[0004]

[Chemical 1] Chemical structure of pluromutilin Structure 1

Pluromutilin is a protein synthesis inhibitor that selectively binds to prokaryotic ribosomes. Studies on ribosome binding and in vitro protein synthesis performed with radiolabeled thiamulin and its derivatives in cell-free preparations from Escherichia coli have been reviewed by Hogenauer (8). . Competitive experiments with radiolabeled thiamulin show a good correlation between the ribosomal affinity of semi-synthetic pluromutilin and the minimum inhibitory concentrations (MICs) for E. coli, and the strong interaction of ribosomes in measuring activity. It was suggested that is basically important (9). Equilibrium dialysis and competition with other known antibiotics suggests that the drug binds to the 50S ribosomal subunit in close proximity to the peptidyl transferase center (9). In the functional translation assay, in the presence of pluromutilin, elongation of the existing peptide was followed, but no new peptide was initiated resulting in the rapid depletion of the active polysome pool of cells (10). There is also evidence to suggest that pluromutilin derivatives inhibit only the formation of the first peptide bond (11). Laboratory-developed E. coli mutants with tiamulin show modified ribosomal proteins S19, L3 or L4 when assessed by two-dimensional electrophoresis (12), and gene mapping experiments showed that It has been shown that these tiamulin resistance mutations coincide with the positions of ribosomal proteins L3 and L4 (13). The ability to functionally characterize targets is key to developing screens to identify inhibitory compounds. If the target can be analyzed on the basis of detailed studies, the desired properties of these compounds (eg efficacy, penetrance and stability) may be improved by rational design.

SUMMARY OF THE INVENTION The present invention provides (a) a polynucleotide having at least 70% identity to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1; or (b) complementary to the polynucleotide of (a). An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of certain polynucleotides is provided. The present invention further provides a method for producing a polynucleotide, which comprises expressing RNA from the host cell of the present invention. Further, there is provided a method for the treatment of an individual in need of inhibiting ribosomal polynucleotides, which comprises administering to the individual a therapeutically effective amount of a compound that binds to or interacts with a polynucleotide of the invention. Provided by the present invention.

Further, in order to assess the interaction of a compound, the composition comprising the polynucleotide is contacted with the compound to be screened under conditions that allow the interaction between the compound and the polynucleotide, such interaction comprising: Associated with a second component capable of providing a detectable signal in response to the interaction of the polynucleotide and the compound; and then detecting the presence or absence of the signal resulting from the interaction of the polynucleotide and the compound Thereby interacting with the polynucleotide of the invention, comprising the step of determining whether the compound interacts with the polynucleotide and activates or inhibits the activity of the polynucleotide, inhibiting or activating the activity of the polynucleotide. A method of identifying a compound to be converted is provided.

Another embodiment of the invention is to bind the compound to the bacterial 50S ribosomal subunit; to bind the compound to the bacterial 70S ribosomal; to bind the compound to the ribosome under tRNA binding conditions. Binding a compound to a ribosome under tRNA binding conditions using an activated ribosome programmed with a messenger RNA such as polyuridylic acid; binding a compound to ribosomal RNA and ribosomal proteins; binding a compound to Escherichia coli 23S rRNA Binding to a sequence; a compound at nucleotides 1971-2607 Escherichia coli 23S rR
Binding to NA; modification of RNA secondary structure formed by nucleotides 1971-2607 of Escherichia coli 23S rRNA; RNA formed by domain V of Escherichia coli 23S rRNA
Altering secondary structure; Modulating the binding of SB-328636 (Structure 2) or its derivatives to ribosomes; Modulating the binding of SB-352408 (Structure 3) or its derivatives to ribosomes; SB-328636 ( Ribosomal 23S RNA of structure 2) or derivative thereof
The binding to SB-352408 (Structure 3) or its derivatives ribosomal 23S RNA
Modulating the binding to;

Modulating the binding of SB-328636 (Structure 2) or its derivatives to domain V of Escherichia coli ribosomal 23S RNA; SB-352408 (Structure 3) Escherichia coli ribosomal 23S RN
Modulating binding of A to domain V; binding compounds to ribosomal RNA and ribosomal proteins; binding compounds to ribosomal proteins L4, L32, L33, L2 or L13; ribosomal proteins L4, L32, L33, Modulating the binding of L2 or L13 to the ribosome; modulating the binding of ribosomal proteins L4, L32, L33, L2 or L13 to ribosomal RNA; modulating the binding of a compound to G2061, A2062 or G2502; Modulating the binding of pluromutilin to G2061, A2062 or G2502; Modulating the binding of chloramphenicol to G2061, A2062 or G2502 ;; Altering the binding of p-azidopuromycin G2502 Modulating the binding of a compound to A2407 and U2408; modulating the binding of pluromutilin to A2407 and U2408; or by binding the compound to a nucleotide of 23S rRNA of SEQ ID NO: 1, 2 or 3 A polynucleotide comprising a nucleotide sequence that is at least 70% identical to the nucleotide sequence and SEQ ID NO: 1, 2 or 3
It is an antagonist that inhibits the activity or an agonist that activates the activity of a bacterial polynucleotide selected from the group consisting of polynucleotides containing the nucleotide sequence shown in.

Another method is provided by the present invention for the treatment of an individual suspected of being infected with a bacterium using an antagonist or agonist of the invention. Bacteria are members of the genus Staphylococcus, Staphylococcus aureus (
Staphylococcus aureus), a member of the genus Streptococcus and Streptococcus pneumoniae are preferred, and the methods and compositions of the present invention are preferred.

According to the present invention, also binding a compound to a bacterial 50S ribosomal subunit; binding a compound to a ribosome under A site tRNA binding conditions; using an activated ribosome programmed with polyuridylic acid , A site tRN
Binding the compound to ribosomes under A-binding conditions; binding the compound to ribosomal RNA and rivosome proteins; binding the compound to the Escherichia coli 23S rRNA sequence; binding the compound to nucleotides 1971-2607 Cori 23S rR
Binding to NA; modification of RNA secondary structure formed by nucleotides 1971-2607 of Escherichia coli 23S rRNA; RNA formed by domain V of Escherichia coli 23S rRNA
Altering secondary structure; Modulating SB-328636 (Structure 2) binding to ribosomes; Modulating SB-352408 (Structure 3) binding to ribosomes; SB-328636 (Structure 2) ribosome 23S Modulating binding to RNA; Modulating binding of SB-352408 (Structure 3) to ribosomal 23S RNA;

SB-328636 (Structure 2) Escherichia coli ribosome 23S RN
Modulating the binding of A to domain V; Escherichia coli ribosome 23S RN of SB-352408 (Structure 3)
Modulating binding of A to domain V; binding compounds to ribosomal RNA and ribosomal proteins; binding compounds to ribosomal proteins L4, L32, L33, L2 or L13; ribosomal proteins L4, L32, L33, Modulating the binding of L2 or L13 to the ribosome; modulating the binding of ribosomal proteins L4, L32, L33, L2 or L13 to ribosomal RNA; modulating the binding of a compound to G2061, A2062 or G2502; Modulating the binding of pluromutilin to G2061, A2062 or G2502; Modulating the binding of chloramphenicol to G2061, A2062 or G2502; Modulating the binding of p-azidopuromycin G2502 Modulating the binding of compounds to A2407 and U2408; modulating the binding of pluromutilin to A2407 and U2408; or a method of inhibiting the activity of bacterial ribosomes by binding the compound to nucleotides of 23S rRNA. Provided.

Various variations and modifications within the spirit and scope of the disclosed invention will be readily apparent to those skilled in the art after reading the following description and other parts of the disclosure.

Glossary of Terms The following explanations are provided to facilitate understanding of certain terms that are commonly used herein. “Disease (s)” includes upper respiratory tract infections (eg, otitis media, bacterial tracheitis, acute pharyngitis, thyroiditis), lower respiratory tract infections (eg, empyema, lung abscess), heart infections (
For example, infectious endocarditis), gastrointestinal infection (eg secretory diarrhea, splenic abscess, retroperitoneal abscess), CNS infection (eg cerebral abscess), eye infection (eg blepharitis, conjunctivitis,
Keratitis, endophthalmitis, anterior septal and orbital cellulitis, lacrimal cystitis), renal and ureteral infections (eg epididymitis, intrarenal and perirenal abscesses, toxic shock syndrome), skin infections (eg impetigo). Including diseases such as eruptions, folliculitis, skin abscesses, cellulitis, wound infections, bacterial myositis), and bone and joint infections (eg, septic arthritis, osteomyelitis),
It also means diseases caused by or associated with infection by bacteria. A "host cell" is a cell that has been introduced (eg, transformed or transfected) or can be introduced (eg, transformed or transfected) with an exogenous polynucleotide sequence.

“Identity” is known in the art and is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. Also, in the art, “identity” may, in some cases,
By the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the pairing between the chains of the sequences. "Identity" is Computational Mo
lecular Biology, Lesk, AM, Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, DW, Academic P
ress, New York, 1993; Computer Analysis of Sequence Data, ParpyrHrif
fin, AM and Griffin, HG, Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, Von Heinje, G. Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J. Ed., M Stockton.
Press, New York, 1991; and Carllio, H and Lipman, D., SIAM J. Applied Ma.
, 48: 1073 (1988), but not limited thereto, can be easily determined by known methods. Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to measure identity are codified in publicly available computer programs. Preferred computer program methods for determining the identity between two sequences include, for example:
GCG program package (Devereux, J. et al., Nucleic Acids Research (1984
) 12 (1): 387), BLASTP, BLASTN and FASTA (Atschul, SF et al., J. Molec. Bi
ol. (1990) 215: 403-410). The BLAST X program is available from NCBI and other sources (BLAST Manual, Altshul, S., et al., NCBI NLM NIH Bethesd
a, MD20894; Altschul, S .; Et al., J. Mol. Biol., 215: 403-410 (1990)). Identity can also be determined using the well known Smith Waterman algorithm.

Parameters for comparing polypeptide sequences include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970) Comparison matrix: Hentikoff and Hentikoff, Proc. Natl . Acad. Sci. USA.
89: 10915-10919 (1992) BLOSSUM62 Gap penalty: 12 Gap length penalty: 4 A useful program for these parameters is Genetics Computer Grou.
Publicly available as a "gap" program from p. Madison WI. The above parameters are the default parameters for polypeptide comparison (without penalty for end gaps). Preferred parameters for polynucleotide comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970) Comparison Matrix: Match = + 10, Mismatch = Gap Penalty: 50 Gap Long Penalties: 3 A useful program for these parameters is Genetics Computer Grou
Publicly available as a "gap" program from p. Madison WI. These parameters are the default parameters for polynucleotide comparisons.

Preferred meanings for “identity” for polynucleotides and polypeptides are given below in (1) and (2). (1) Further specific examples of polynucleotides include isolated polynucleotides comprising a polynucleotide sequence having at least 95, 97 or 100% identity to the control sequence of SEQ ID NO: 1, wherein said polynucleotide The polynucleotide sequence may be identical to the control sequence of SEQ ID NO: 1, or may have up to some number of nucleotide changes compared to the control sequence. Such changes are selected from the group consisting of deletions, substitutions (including transitions and transversions) or insertions of at least one nucleotide, wherein the changes are 5 of the control nucleotide sequence.
It may occur at the'or 3'terminal positions or at positions between those terminal positions, individually or interspersed in the nucleotides in the control sequence, or as one or more contiguous groups in the control sequence. The number of nucleotide changes is calculated by multiplying the total number of nucleotides in SEQ ID NO: 1 by the individual percent identity values (divided by 100) and subtracting the product from the total number of nucleotides in SEQ ID NO: 1. Determined by This is illustrated by the formula: n _n ≦ x _n − (x _n · y) where _{n n} is the number of nucleotide changes, x _n is the total number of nucleotides in SEQ ID NO: 1, and y is , 95% is 0.95, 97% is 0.97, and 100% is 1.00. · Is an operator of products, and a product which is not an integer of x _n and y is rounded down to the nearest integer. Then, subtract from x _n . Changes in the polynucleotide sequence encoding the polypeptide may result in nonsense, missense or frameshift mutations in the coding sequence, and thus the polypeptide encoded by the polynucleotide concomitant with such changes. Changes.

(2) Further embodiments of polypeptides include an isolated polypeptide comprising a polypeptide having at least 95, 97 or 100% identity to a polypeptide control sequence, wherein the polypeptide sequence is It may be identical to the control sequence, or it may have up to some number of amino acid changes compared to the control sequence.
Such changes are selected from the group consisting of deletions, substitutions (including conservative and non-conservative substitutions) or insertions of at least one amino acid, which changes are at the amino or carboxy terminal position of the control polypeptide sequence or at those positions. May occur individually or interspersed in amino acids in the control sequence, at positions between the terminal positions of, or as one or more contiguous groups in the control sequence. The number of amino acid changes is determined by multiplying the total number of amino acids by the integer showing the identity divided by 100 and subtracting the product from the total number of amino acids. This is explained by the following formula: n _a ≦ x _a − (x _a · y) where n _a is the number of amino acid changes, x _a is the total number of amino acids in the sequence, and y is 95%. 0.95, 97% is 0.97, 100% is 1.00,
Is a product operator, and a product that is not an integer of x _a and y is rounded down to the nearest integer, and then subtracted from x _a .

“Individual” includes, but is not limited to, metazoans, mammals, cows, monkeys,
Means multicellular eukaryotes, including primates, and humans. “Isolated” means altered “by the hand of man” from its natural state, ie, in the case of a natural product, altered and / or removed from its original environment. For example, a polynucleotide or polypeptide naturally present in a living organism is not “isolated”, but the same polynucleotide or polypeptide separated from its naturally occurring coexisting material is a term used herein. Has been "isolated". Furthermore, a polynucleotide or a polypeptide introduced into an organism (which may be alive or dead) by transformation, genetic engineering or any recombinant method, even if it is still present in the organism. However, it is "isolated".

“Organism (s)” means (i) Streptococcus, Staphylococcus, Bordete
lla, Corynebacterium, Mycobacterium, Neisseia, Haemophilus, Actinomyces
, Streptomyces, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella
, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus
, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clo
sterdium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Prote
us, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, L
egionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and
Members of (but not limited to) Mycoplasma genus, as well as St of Group A
reptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoni
ae, Streptococcus pyrogenes, Streptoxoxxus agalactiae, Streptococcus fae
calis, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae
, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermi
dis, Corynebacterium diptheriae, Garnella vaginalis, Mycobacterium tuber
culosis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium lepr
ae, Actinomyces israelli, Listeria monocytogenes, Bordetella pretusis, B
ordetella parapretusis, Bordetella bronchiseptica, Esherichia coli, Shig
ella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemoph
ilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi,
Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pest
is, Klebsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, V
ibrio cholera, Shigella dysenterii, Shigella flexneri, Pseudomonas aerug
inosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Ba
cillus cereus, Clostridium perfringens, Clostridium tetani, Clostridium
butulinum, Treponema pallidum, Rickettsia rickettsii and Chlamydia trac
Prokaryotes, including (but not limited to) members of certain species or groups of homitis, (ii) Archaebacteria, including (but not limited to) Archaebacter, and (
iii) Protozoa, fungi, members of Saccharomyces, Kluveromyces or Candida, but not limited to Saccharomyces cerevisiae, Kluveromyces
By unicellular or filamentous eukaryote, including but not limited to members of the lactis or Candida albicans species.

“Bacterium or bacteria” means (i) Streptococcus, Staphylococcus, Bordete
lla, Corynebacterium, Mycobacterium, Neisseia, Haemophilus, Actinomycete
s, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasture
lla, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacill
us, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, C
losterdium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Pro
teus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella
Members of the genera (but not limited to), Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and Group A.
Streptococcus, Group B Streptococcus, Group C Streptococcus,
Group D Streptococcus, Group G Streptococcus, Streptococcus pneu
moniae, Streptococcus pyrogenes, Streptoxoxxus agalactiae, Streptococcus
faecalis, Streptococcus faecium, Streptococcus durans, Neisseria gonorr
heae, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epid
ermidis, Corynebacterium diptheriae, Garnella vaginalis, Mycobacterium t
uberculosis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium
leprae, Actinomyces israelii, Listeria monocytogenes, Bordetella pretusi
s, Bordetella parapretusis, Bordetella bronchiseptica, Esherichia coli,
Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Hae
mophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typ
hi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia
pestis, Klebsiella pneumoniae, Serratia marcessens, Serratia liquefacien
s, Vibrio cholera, Shigella dysenterii, Shigella flexneri, Pseudomonas a
eruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis
, Bacillus cereus, Clostridium perfringens, Clostridium tetani, Clostrid
ium botulinum, Treponema pallidum, Rickettsia rickettsii and Chlamydia
By prokaryotes, including members of, but not limited to, certain species or groups of trachomitis, and (ii) archaebacteria, including, but not limited to, Archaebacters.

“Polynucleotide (s)” also generally refers to polyribonucleotides or polydeoxyribonucleotides, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotide” refers to DNA, single-stranded and double-stranded, among others, single- and double-stranded DNA, a mixture of single- and double-stranded regions or a mixture of single-, double- and triple-stranded regions. Double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, single-stranded or more typically double-stranded or triple-stranded, or a mixture of single-stranded and double-stranded regions, Includes, but is not limited to, hybrid molecules including DNA and RNA. In addition, "polynucleotide" as used herein refers to R
It means a triple-stranded region comprising NA or DNA or both RNA and DNA.
The chains in such regions may be from the same or different molecules. This region may include all of one or more of these molecules, but more typically includes only one region of some molecules. One of the molecules in the triple helix region is often an oligonucleotide. As used herein, the term "polynucleotide" includes a DNA or RNA as described above that contains one or more modified bases. Thus, DNA or RNA having a backbone that has been modified for stability or for other reasons is also referred to as "a" or "A" as intended herein.
"Polynucleotide". In addition, DNA or RNA containing unusual bases such as inosine, or modified bases such as tritylated bases (only two examples are shown),
The term polypeptide as used herein. It will be apparent that numerous modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide," as used herein, refers to such chemically, enzymatically, or metabolically modified forms of a polynucleotide and to cells, including viruses, especially simple and complex cells. Includes characteristic DNA and RNA chemical forms. "Polynucleotide" embraces short polynucleotides, often referred to as oligonucleotide (s).

As used herein, “polypeptide” refers to any peptide or protein that comprises two or more amino acids linked together by peptide bonds or modified peptide bonds. "Polypeptide" means both short chains, usually also referred to as peptides, oligopeptides and oligomers, and long chains, commonly referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptide" includes by natural processes such as processing and other post-translational modifications, as well as by chemical modifications. Such modifications are well described in the basic texts and more detailed papers as well as in the numerous research literature, which are well known to the person skilled in the art. It will be appreciated that the same type of modification may be present in the polypeptide at several sites in the same or varying degrees. Also, a given polypeptide may contain many types of modifications. Modifications can be anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxy termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphotidyl. Inositol covalent bond, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-linking formation, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation , Iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, etc.
There are A-mediated amino acid additions to proteins, and ubiquitination. For example, Pr
oteins-Structure and Molecular Properties, 2nd edition, TE Creighton, WHFre
eman and Company, New York (1993). For example, Posttran
slational Covalent Modification of Proteins, BC Johnson, Academic Press, New York (1983) Wold, F., Posttranslational Protein Modif
ications: Perspective and Prospects, pp. 1-12; Seifter et al., Meth. Enzymol. 1
82: 626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational M.
See odifications and Aging, Ann. NY Acad. Sci. 663: 48-62 (1992). The polypeptide may be branched or cyclic with or without branching. Ring,
Branched and branched and cyclic polypeptides may result from post-translational natural processes, as well as those produced by entirely synthetic methods.

“Recombinant expression system (s)” refers to an expression system or part thereof or that has been introduced or transformed into a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the invention. Refers to a polynucleotide of the invention. The term "variant" as used herein is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties. A typical polynucleotide variant differs in nucleotide sequence from another, reference polynucleotide. The alteration in the nucleotide sequence of the variant may or may not alter the amino acid sequence of the polypeptide encoded by the control polynucleotide. Nucleotide changes result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the control sequence, as discussed below. A typical polypeptide variant differs in amino acid sequence from another, reference polypeptide. Generally, the differences are limited to those sequences in which the control polypeptide and variant are largely similar in nature and are identical in many regions. Variant and control polypeptides may have their amino acid sequences altered by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. The invention also includes each variant of a polypeptide of the invention, ie, a polypeptide that is altered from the control sequence by a conservative amino acid substitution, whereby a residue is replaced by another residue having similar characteristics. Includes. Typical such substitutions are Ala, Val, Leu and Il.
between e; between Ser and Thr; between acidic residues Asp and Glu; between Asn and Gln; and between basic residues Lys and Arg; or between aromatic residues Phe and Tyr. Especially preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted or added in any combination. Variants of polynucleotides or polypeptides can be naturally occurring, eg, allelic variants, or can be variants not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be produced by mutagenesis techniques or direct synthesis and other recombinant methods known to those of skill in the art.

Description of the Invention The present invention relates to polynucleotides and polypeptides as described in more detail below. In particular, the invention relates to prokaryotic ribosomal polypeptides. The present invention particularly relates to ribosomal RNA transcribed from DNA having the nucleotide sequence shown in Table 1 [SEQ ID NO: 1, 2 or 3].

Table 1 Ribosomal Polynucleotide Sequences (A) SB-328636 and SB-352408 by RNase H digestion
E. coli 23S rRNA sequence (nucleotide 19
71-2607) (SEQ ID NO: 1) 5'-tggcgtaatgatggccaggctgtctccacccgagactcagtgaaattgaactcgctgtgaagatgcagt
gtacccgcggcaagacggaaagaccccgtgaacctttactatagcttgacactgaacattgagccttgatgt
gtaggataggtgggaggctttgaagtgtggacgccagtctgcatggagccgaccttgaaataccacccttta
atgtttgatgttctaacgttgacccgtaatccgggttgcggacagtgtctggtgggtagtttgactggggcg
gtctcctcctaaagagtaacggaggagcacgaaggttggctaatcctggtcggacatcaggaggttagtgca
atggcataagccagcttgactgcgagcgtgacggcgcgagcaggtgcgaaagcaggtcatagtgatccggtg
gttctgaatggaagggccatcgctcaacggataaaaggtactccggggataacaggctgataccgcccaaga
gttcatatcgacggcggtgtttggcacctcgatgtcggctcatcacatcctggggctgaagtaggtcccaag
ggtatggctgttcgccatttaaagtggtacgcgagctgggtttagaacgtcgtgagacagttcgg-3 '

(B) 23S Staphylococcus aureus ribosomal RNA-derived sequence (SEQ ID NO: 2) 5′-gattaagttattaagggcgcacggtggatgccttggcactagaagccgatgaaggacgttactaacgac
gatatgctttggggagctgtaagtaagctttgatccagagatttccgaatggggaaacccagcatgagttat
gtcatgttatcgatatgtgaatacatagcatatcagaaggcacacccggagaactgaaacatcttagtaccc
ggaggaagagaaagaaaattcgattcccttagtagcggcgagcgaaacgggaagagcccaaaccaacaagct
tgcttgttggggttgtaggacactctatacggagttacaaaggacgacattagacgaatcatctggaaagat
gaatcaaagaaggtaataatcctgtagtcgaaaatgttgtctctcttgagtggatcctgagtacgacggagc
acgtgaaattccgtcggaatctgggaggaccatctcctaaggctaaatactctctagtgaccgatagtgaac
cagtaccgtgagggaaaggtgaaaagcaccccggaaggggagtgaaatagaacctgaaaccgtgtgcttaca
agtagtcagagcccgttaatgggtgatggcgtgccttttgtagaatgaaccggcgagttacgatttgatgca
aggttaagcagtaaatgtggagccgtagcgaaagcgagtctgaatagggcgtttagtatttggtcgtagacc
cgaaaccaggtgatctacccttggtcaggttgaagttcaggtaacactgaatggaggaccgaaccgacttac
gttgaaaagtgagcggatgaactgagggtagcggagaaattccaatcgaacctggagatagctggttctctc
cgaaatagctttagggctagcctcaagtgatgattattggaggtagagcactgtttggacgaggggcccctc
tcgggttaccgaattcagacaaactccgaatgccaattaatttaacttgggagtcagaacatgggtgataag
gtccgtgttcgaaagggaaacagcccagaccaccagctaaggtcccaaaatatatgttaagtggaaaaggat
gtggcgttgcccagacaactaggatgttggcttagaagcagccatcatttaaagagtgcgtaatagctcact
agtcgagtgacactgcgccgaaaatgtaccggggctaaacatattaccgaagctgtggattgtcctttggac
aatggtaggagagcgttctaagggcgttgaagcatgatcgtaaggacatgtggagcgcttagaagtgagaat
gccggtgtgagtagcgaaagacgggtgagaatcccgtccaccgattgactaaggtttccagaggaaggctcg
tccgctctgggttagtcgggtcctaagctgaggccgacaggcgtaggcgatggataacaggttgatattcct
gtaccacctataatcgttttaatcgatggggggacgcagtaggataggcgaagcgtgcgattggattgcacg
tctaagcagtaaggctgagtattaggcaaatccggtactcgttaaggctgagctgtgatggggagaagacat
tgtgtcttcgagtcgttgatttcacactgccgagaaaagcctctagatagaaaataggtgcccgtaccgcaa
accgacacaggtagtcaagatgagaattctaaggtgagcgagcgaactctcgttaaggaactcggcaaaatg
accccgtaacttcgggagaaggggtgctctttagggttaacgcccagaagagccgcagtgaataggcccaag
cgactgtttatcaaaaacacaggtctctgctaaaccgtaaggtgatgtataggggctgacgcctgcccggtg
ctggaaggttaagaggagtggttagcttctgcgaagctacgaatcgaagccccagtaaacggcggccgtaac
tataacggtcctaaggtagcgaaattccttgtcgggtaagttccgacccgcacgaaaggcgtaacgatttgg
gcactgtctcaacgagagactcggtgaaatcatagtacctgtgaagatgcaggttacccgcgacaggacgga
aagaccccgtggagctttactgtagcctgatattgaaattcggcacagcttgtacaggataggtaggagcct
ttgaaacgtgagcgctagcttacgtggaggcgctggtgggatactaccctagctgtgttggctttctaaccc
gcaccacttatcgtggtgggagacagtgtcaggcgggcagtttgactggggcggtcgcctcctaaaaggtaa
cggaggcgctcaaaggttccctcagaatggttggaaatcattcatagagtgtaaaggcataagggagcttga
ctgcgagacctacaagtcgagcagggtcgaaagacggacttagtgatccggtggttccgcatggaagggcca
tcgctcaacggataaaagctaccccggggataacaggcttatctcccccaagagttcacatcgacggggagg
tttggcacctcgatgtcggctcatcgcatcctggggctgtagtcggtcccaagggttgggctgttcgcccat
taaagcggtacgcgagctgggttcagaacgtcgtgagacagttcggtccctatccgtcgtgggcgtaggaaa
tttgagaggagctgtccttagtacgagaggaccgggatggacatacctctggtgtaccagttgtcgtgccaa
cggcatagctgggtagctatgtgtggacacgggataagtgctgaaagcatctaagcatgaagcccccctcaaga
tgagatttcccaacttcggttataagatccctcaaagatgatgaggttaataggttcgaggtggaagcatgg
tgacatgtggagctgacgaatactaatcgatcgaagacttaatcaa-3 '

(C) 23S Escherichia coli ribosomal RNA-derived sequence (SEQ ID NO: 3)
) 5'-ggttaagcgactaagcgtacacggtggatgccctggcagtcagaggcgatgaaggacgtgctaatctgc
gataagcgtcggtaaggtgatatgaaccgttataaccggcgatttccgaatggggaaacccagtgtgtttttcg
acacactatcattaactgaatccataggttaatgaggcgaaccgggggaactgaaacatctaagtaccccga
ggaaaagaaatcaaccgagattcccccagtagcggcgagcgaacggggagcagcccagagcctgaatcagtg
tgtgtgttagtggaagcgtctggaaaggcgcgcgatacagggtgacagccccgtacacaaaaatgcacatgc
tgtgagctcgatgagtagggcgggacacgtggtatcctgtctgaatatggggggaccatcctccaaggctaa
atactcctgactgaccgatagtgaaccagtaccgtgagggaaaggcgaaaagaaccccggcgaggggagtga
aaaagaacctgaaaccgtgtacgtacaagcagtgggagcacgcttaggcgtgtgactgcgtaccttttgtat
aatgggtcagcgacttatattctgtagcaaggttaaccgaataggggagccgaagggaaaccgagtcttaac
tgggcgttaagttgcagggtatagacccgaaacccggtgatctagccatgggcaggttgaaggttgggtaac
actaactggaggaccgaaccgactaatgttgaaaaattagcggatgacttgtggctgggggtgaaaggccaa
tcaaaccgggagatagctggttctccccgaaagctatttaggtagcgcctcgtgaattcatctccgggggta
gagcactgtttcggcaagggggtcatcccgacttaccaacccgatgcaaactgcgaataccggagaatgtta
tcacgggagacacacggcgggtgctaacgtccgtcgtgaagagggaaacaacccagaccgccagctaaggtc
ccaaagtcatggttaagtgggaaacgatgtgggaaggcccagacagccaggatgttggcttagaagcagcca
tcatttaaagaaagcgtaatagctcactggtcgagtcggcctgcgcggaagatgtaacggggctaaaccatg
caccgaagctgcggcagcgacgcttatgcgttgttgggtaggggagcgttctgtaagcctgcgaaggtgtgc
tgtgaggcatgctggaggtatcagaagtgcgaatgctgacataagtaacgataaagcgggtgaaaagcccgc
tcgccggaagaccaagggttcctgtccaacgttaatcggggcagggtgagtcgacccctaaggcgaggccga
aaggcgtagtcgatgggaaacaggttaatattcctgtacttggtgttactgcgaaggggggacggagaaggc
tatgttggccgggcgacggttgtcccggtttaagcgtgtaggctggttttccaggcaaatccggaaaatcaa
ggctgaggcgtgatgacgaggcactacggtgctgaagcaacaaatgccctgcttccaggaaaagcctctaag
catcaggtaacatcaaatcgtaccccaaaccgacacaggtggtcaggtagagaataccaaggcgcttgagag
aactcgggtgaaggaactaggcaaaatggtgccgtaacttcgggagaaggcacgctgatatgtaggtgaggt
ccctcgcggatggagctgaaatcagtcgaagataccagctggctgcaactgtttattaaaaacacagcactg
tgcaaacacgaaagtggacgtatacggtgtgacgcctgcccggtgccggaaggttaattgatggggttagcg
caagcgaagctcttgatcgaagccccggtaaacggcggccgtaactataacggtcctaaggtagcgaaattc
cttgtcgggtaagttccgacctgcacgaatggcgtaatgatggccaggctgtctccacccgagactcagtga
aattgaactcgctgtgaagatgcagtgtacccgcggcaagacggaaagaccccgtgaacctttactatagct
tgacactgaacattgagccttgatgtgtaggataggtgggaggctttgaagtgtggacgccagtctgcatgg
agccgaccttgaaataccaccctttaatgtttgatgttctaacgttgacccgtaatccgggttgcggacagt
gtctggtgggtagtttgactggggcggtctcctcctaaagagtaacggaggagcacgaaggttggctaatcc
tggtcggacatcaggaggttagtgcaatggcataagccagcttgactgcgagcgtgacggcgcgagcaggtg
cgaaagcaggtcatagtgatccggtggttctgaatggaagggccatcgctcaacggataaaaggtactccgg
ggataacaggctgataccgcccaagagttcatatcgacggcggtgtttggcacctcgatgtcggctcatcac
atcctggggctgaagtaggtcccaagggtatggctgttcgccatttaaagtggtacgcgagctgggtttaga
acgtcgtgagacagttcggtccctatctgccgtgggcgctggagaactgaggggggctgctcctagtacgag
aggaccggagtggacgcatcactggtgttcgggttgtcatgccaatggcactgcccggtagctaaatgcgga
agagataagtgctgaaagcatctaagcacgaaacttgccccgagatgagttctccctgaccctttaagggtc
ctgaaggaacgttgaagacgacgacgttgataggccgggtgtgtaagcgcagcgatgcgttgagctaaccgg
tactaatgaaccgtgaggcttaacctt-3 '

The present invention provides a series of pluromutilins with different potentials for biochemical assays,
Disclosed are studies showing that they are associated with activity against antimicrobial agents, especially bacteria, such as Staphylococcus aureus. For example, tritium labeled derivatives have been synthesized and used in a Logan displacement assay to measure the binding and dissociation rates of various pluromutilin derivatives. Provided herein are methods of screening for novel antimicrobial compounds using this and other techniques and methods of treating patients infected with bacteria using such compounds. As provided herein, pluromutilin specifically binds to the 50S ribosomal subunit. After the 70S ribosome bound to the tritiated drug was subjected to a low magnesium sucrose gradient, separating the 70S ribosome into 30S and 50S particles, tritium was reduced to 50%.
It is detected only in the S fraction (Fig. 1). The rRNA of the present invention can be purified using any method known in the art. Purification can also be performed as described herein, such as by sucrose density gradient centrifugation (see Figure 1). Drug-derivative binding rates are usually in the order of minutes, with derivatives having a range of dissociation rates. The sucrose gradient of ribosomes bound with tritiated pluromutilin radioligand was analyzed. Radioligand bound to ribosomes under A site tRNA binding conditions using activated ribosomes programmed with polyuridylic acid (14). Ribosomal subunits were separated on a 10-30% sucrose gradient containing 0.8 mM Mg (OAc) ₂ (15). Black squares indicate absorbance units at 260 nm; open squares are D
³ shows ³ H-labeled drug in PM. The 50S and 30S ribosomal subunit peaks are shown for reference. In addition, a number of tritiated photoaffinity derivatives have been used to photolabel E. coli 70S ribosomes with the drug 50S.
It has been shown to crosslink only to subunits. Two-dimensional polyacrylamide gel electrophoresis and HPLC were used to identify ribosomal proteins that cross-link the agents of the invention and other compounds. Similarly, RNase H and reverse transcription experiments localized a nucleotide that bridges to the drug to the 23S rRNA. These observations lead, in part, to the development of the in vitro translation assay as well as the ribosome binding assay provided by the present invention.

Pluromutilin binding was tested under a variety of conditions, such as with P sites that were either filled or unfilled. Therefore, since there are several different types of tRNA binding sites, such as P, A, and E sites, pluromutilin binding is associated with tRN.
The conditions under which A is bound (herein, “tRNA binding conditions”) or substantially equivalent conditions are used.

The sequence of full length E. coli 23S rRNA is provided in Table 1 [SEQ ID NO: 2] and the sequence of Staphylococcus aureus 23S rRNA is provided in Table 1 [SEQ ID NO: 3]. A preferred method of the present invention is as a substrate for performing a binding assay,
E. coli 23S rRNA [SEQ ID NO: 2] or Staphylococcus aureus 23S rRNA [SEQ ID NO: 3] is used. E. coli 23S rRN
There is high homology between A [SEQ ID NO: 2] and Staphylococcus aureus 23S rRNA [SEQ ID NO: 3], especially in the region around the peptidyl transferase center.

The method of the invention that requires cross-linking, as this technique is provided herein,
Alternatively, it can be performed using a known method. In a preferred embodiment of the invention, the photoaffinity agent crosslinks 70S ribosomal subunits separated on a sucrose gradient. The label identified in the 50S peak indicates the presence of RNA and protein in the peak. This peak is extracted. A large subunit protein photocrosslinks to a photoaffinity compound, resulting in ³ H-SB
-304946 labeled L4, L32 and L33, ³ H-SB-3286
36 was determined to label L2, L13, L32 and L33. This is used in part as the basis of the present invention and indicates that L4, L32, L33, L2 and L13 are involved in pluromutilin binding. The activity is ribosomal protein L4
Purromutilin inhibits the activity of bacterial ribosomes, which is the modulation of the binding of L3, L32, L33, L2 or L13 to the ribosome or the modulation of the binding of ribosomal proteins L4, L32, L33, L2 or L13 to the RNA in the ribosome. A preferred method of modulating is provided.

[0033]

[Chemical 2]

The activity of the ribosome binds the compound to the bacterial 50S ribosomal subunit; binds the compound to the ribosome under A site tRNA binding conditions; A site tRNA using activated ribosomes programmed with polyuridylic acid. Binding the compound to ribosomes under binding conditions; binding the compound to Escherichia coli 23S
binding to the rRNA sequence; binding the compound to Escherichia coli 23S rRNA at nucleotides 1971-2607; Escherichia coli 2
Modification of RNA secondary structure formed by nucleotides 1971-2607 of 3S rRNA; modification of RNA secondary structure formed by domain V of Escherichia coli 23S rRNA; binding of SB-328636 (structure 2) to ribosome Modulating; binding of SB-352408 (Structure 3) to ribosomes; modulating SB-328636 (Structure 2) to ribosomal 23S rRNA; SB-352408 (Structure 3) to ribosomes 23S RN
Modulating binding to A; Modulating binding of SB-328636 (Structure 2) to domain V of Escherichia coli 23S RNA; SB-352408 (
Modulating binding of Escherichia coli 23S RNA of structure 3) to domain V; binding a compound to ribosomal RNA and ribosomal proteins;
Binding a compound to ribosomal proteins L4, L32, L33, L2 or L13; modulating the binding of ribosomal proteins L4, L32, L33, L2 or L13 to ribosomes; ribosomal proteins L4, L32, L33
, L2 or L13 binding to ribosomal RNA; 23S bound to pluromutilin (eg SB-328636 or SB-352408)
Modification of RNase H digestion pattern of rRNA; or compound as 23S rRNA
It is preferred in the method of the invention that the activity is selected from the group consisting of binding to a nucleotide of

FIG. 14 shows the secondary structure of E. coli 23S ribosomal rRNA (Gutell, RR,
Gray MW, and Schnare, MN (1993) Nucleic Acids Res. 21, 3055-3074). PA
The position of proteins photolabeled by L-1 and PAL-2 is highlighted (Brimacombe et al., (1990) In: Hill WE, Dahlberg A, Garrett RA, Moore.
PM, Schlessinger D and Warner JR (eds). The Ribosome. Structure Function
and Evolution. American Society for Microbioology. Washington, 93-106
). The region of the 23S rRNA shown to be cross-linked to PAL-1 by RNase H digestion and identified in the present invention is boxed in blue. L2 has been strongly implicated in the art as it is important for peptidyl transferase activity. L22 His22
Mutation of 9 to Gln results in loss of peptidyl transferase activity (Cooperman BS, Wooten T, Romero DP, and Traut RR, Biochem. Cell.
Biology, 1995, Nov-Dec., 73 (11-12) 1087-1094.). L13: 23S rRN
A secondary structure hairpin 35 is located near PTC in the tertiary structure (Xiong L, Sh
ah S, Mauvais P and Mankin A. Molecular Microbiology, 1999, 31 (2) 633-63
9.). L13 is located near hairpin 35 on the 23S rRNA secondary structure, consistent with the arrangement of other proteins and ribosomal RNA nucleotides that are cross-linked by photoaffinity labeled pluromutilin, indicating that this protein is PTC.
Means to be brought close to.

L32 and L33: Single-stranded sequence 2475 of 23S rRNA near PTC
A photolabile oligo-probe complementary to -2483 (2475 loop) site-specifically incorporates proteins L1, L13, L16, L32 and L33.
This is evidence that C2475 is within 21 ounces of L1, L13, L16, L32 and L33 (Muralikrishna P, Cooperman BS, Bio.
chemistry, 1995, Jan 10; 34 (1): 115-121). Pluromutilin PAL-1 and PAL-2 are antibacterial and specifically bind to the same binding pocket as other non-photoaffinity Pluromutilin derivatives, as disclosed herein. In the present invention, ribosomal proteins L2, L4,
L13, L32 and L33 were identified and eluted on a 2D gel (Solusol).
/ Combustion) at or near the pluromutilin binding site. In addition, E. coli 23S rRN by RNase H digestion
The identification of fragments of 23S rRNA containing domain V of A (nucleotides 2000-2500) is disclosed herein. Use of the photoaffinity derivative PAL1
Domain V (nucleotides 2000-2500) of E. coli 23S rRNA was identified by Nase H digestion at or near the pluromutilin binding site.

It is also taught by the present invention that pluromutilin specifically binds to the 50S subunit as shown by ligand binding and sucrose gradient analysis. Pluromutilin remains in the 50S subunit even after dissociation of the 70S complex. It was also shown that there is a strong correlation between binding capacity and functional inhibition and antibacterial capacity against some clinically important pathogens. Pluromutilin exhibits very rapid binding rates (kon) to E. coli 70S ribosomes in the order of minutes, with a range of slow dissociation rates (koff) t _1/2 values of 4 for various semisynthetic derivatives.
It varies from 0 minutes to 8 hours.

[0038] It is also taught by the present invention that pluromutilin inhibits initiation / restarting, as shown by the reduced signal of f- ^3H met incorporation into the peptide in the S30 natural translation initiation assay. While the preferred embodiments of the methods and compositions of the present invention reveal that poly U is the message used for programming, any messenger RNA capable of mediating translation can be used. The present invention also provides that pluromutilin is PTC, especially within the pluromutilin of the invention,
Provides direct binding to G2061, A2062 and G2502. In addition, chloramphenicol has been shown to imprint on A2062 and weakly compete with pluromutilin for binding. G2502 has also been shown to be the binding site for p-azidopuromycin. Purromutilin is also A240
It binds to 7 and U2408, but is not within the PTC on the secondary structure, but is found near the PTC and is associated with nucleotides 413-416 and L4 in long-range three-dimensional interactions. These data show that pluromutilin binds to L2, L4, L13, L32 and L33 as provided by the present invention.

The invention also relates to compositions of matter such as polynucleotide sequences that are identical throughout the sequence of SEQ ID NO: 1, as well as methods of using compositions of matter of SEQ ID NO: 2 or 3 or polynucleotides containing the same. I will provide a. The invention also includes a polynucleotide of the formula shown in Table 1 [SEQ ID NO: 1, 2 or 3]: wherein at the 5'end of the molecule X is hydrogen and at the 3'end of the molecule: Y is hydrogen or a metal, R ₁ and R ₂ are any nucleic acid residues, and n is an integer between 1 and 1000. Any R in which R is greater than 1
Nucleic acid residues of any length indicated by a group can be heteropolymers or homopolymers (preferably heteropolymers).

Further particularly preferred embodiments are some 2, 3, 5 to 10, 1
To 5, 1 to 3, 2, 1 or 0 nucleotidyl residues are substituted, deleted or added in any combination, Table 1 [SEQ ID NO: 1, 2 or 3] nucleotide sequence Is a polynucleotide having Particularly preferred among these are substitutions, additions and deletions that do not alter the properties and activities of the polynucleotides of the invention. Further preferred embodiments of the invention are polynucleotides that are at least 70% identical to the polynucleotides shown in Table 1 [SEQ ID NO: 1, 2 or 3] over their entire length and polynucleotides complementary to such polynucleotides. . Alternatively, a polynucleotide comprising a region that is at least 80%, 90% or 95% identical over its entire length to the polynucleotide of SEQ ID NO: 1, 2 or 3 and polynucleotides complementary thereto are most preferred.

A preferred embodiment is a polynucleotide that retains substantially the same biological function or activity as the DNA of Table 1 [SEQ ID NO: 1, 2 or 3]. The invention further relates to polynucleotides that hybridize to the sequences herein above. In this regard, the present invention particularly relates to polynucleotides that hybridize under stringent conditions to the herein above-described polynucleotides.
As used herein, the terms "stringent conditions" and "stringent hybridization conditions" refer to hybridizations that have at least 95% and preferably at least 97% identity between sequences. It only means that it will happen. Examples of stringent hybridization conditions are: 50% formic acid, 5xSSMC (150 mM NaCl, 15 mM
Trisodium citrate), 50 mM sodium phosphate, pH 7.6, 5x Denhardt's solution, 10% dextran sulfate and 20 microgram / ml denaturation, solution containing cleaved salmon semen DNA at 42 ° C. After overnight incubation, the hybridization support was washed with 0.1xSSMC at about 65 ° C. Hybridization and wash conditions are well known and may be found in Sambrook,
et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spr
ing Harbor, NY, (1989), especially chapter 11 thereof.

The present invention also consists essentially of SEQ ID NO: 1, 2 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence shown in SEQ ID NO: 1, 2 or 3 or a fragment thereof. Alternatively, a polynucleotide consisting of the polynucleotide sequence obtainable by screening an appropriate library containing the complete gene of the polynucleotide sequence shown in 3 and isolating the DNA sequence is provided. Fragments useful for obtaining such polynucleotides include, for example, the probes and primers described elsewhere herein.

As will be discussed further with respect to the polynucleotide assays of the present invention, for example, the polynucleotides of the present invention described herein above will isolate full-length cDNA and genomic clones encoding ribosomal proteins, and It may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate cDNA and genomic clones of other genes that have high sequence similarity to the gene. Such probes generally contain at least 15 bases. Preferably, such a probe has at least 30 bases and has at least 5 bases.
It may have 0 bases. A particularly preferred probe has at least 30 bases,
Have 50 or less bases.

The polynucleotides and polypeptides of the present invention are used, for example, as research reagents and materials for finding treatments and diagnoses of the diseases further discussed herein with respect to polynucleotide assays, particularly human diseases. It may be. The polynucleotide of the present invention, which is an oligonucleotide derived from the sequence of SEQ ID NO: 1, 2 or 3, may or may not be transcribed in the bacterium of infected tissue in whole or in part of the polynucleotide identified herein. May be used in the methods described herein, preferably PCR, to determine Such sequences are also recognized as having utility in diagnosing the stage of infection and type of infection reached by the pathogen.

Vectors, Host Cells, Expression The invention also includes the polynucleotides or polynucleotides of the invention, host cells genetically engineered with the vectors of the invention, recombinantly produced polypeptides of the invention. Regarding vectors. The cell-free translation system also includes the DN of the present invention.
RNA from the A construct can be used to produce such proteins. For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention. Introduction of polynucleotides into host cells includes calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection. Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1
986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd
Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989
) Is achieved by the methods described in many standard laboratory manuals.

Representative examples of suitable hosts are bacterial cells such as streptococci, staphylococci, enterococci E. coli, streptomyces and Bacillus subtilis; yeast cells and Aspergillus cells. And fungal cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells. A variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, among others, chromosomal, episomal and viral derived vectors,
For example, bacterial plasmid origin, bacteriophage origin, transposon origin,
Yeast episome origin, insertion element origin, yeast chromosome element origin, baculovirus,
Vectors derived from viruses such as papovaviruses such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabie virus and retrovirus, and vectors derived from plasmids and combinations thereof such as those derived from bacteriophage genetic elements such as cosmids and phagemids. including. Expression system constructs may contain control regions that regulate as well as engender expression. In this regard, in general, any system or vector suitable for maintaining, propagating or expressing a polynucleotide and / or expressing a polypeptide in a host is
It may be used for expression. Suitable DNA sequences are described, for example, in Sambrook et al., MO.
It may be inserted into the expression system by a variety of known and routine techniques such as those shown in LECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular milieu, suitable secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals. Polypeptides of the invention may be ammonium sulfate or ethanol precipitated, acid extracted,
Recoverable and purified from recombinant cell culture medium by known methods including anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. .. Most preferably high performance liquid chromatography is used for purification. If the polypeptide is denatured during isolation and / or purification, known techniques for refolding proteins may be used to regenerate the active conformation.

Mechanisms of Action and Methods of Use The polynucleotides of the present invention have also been used to assess the binding of small molecular substrates and ligands, eg, in cells, cell-free preparations, chemical libraries and natural product mixtures. Good. These substrates and ligands can be natural substrates and ligands, or can be structural or functional analogs. For example, see Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991).

The present invention also facilitates the action of ribosomal polypeptides or polynucleotides
(Agonist) or compound to identify those that inhibit (antagonist),
In particular, a method is provided for screening the compounds which are bacteriostatic and / or fungicidal agents. The method of screening may include high throughput methods. For example,
Synthetic reaction mixtures, membranes, cell compartments such as cell envelopes or cell walls or any preparation thereof containing ribosomal polypeptides or polynucleotides and labeled substrates or ligands for such polypeptides or polynucleotides for screening agonists or antagonists Are incubated in the absence or presence of candidate molecules, which may be ribosomal agonists or antagonists. The ability of the candidate molecule to agonize or antagonize ribosomal polypeptides or polynucleotides is reflected in decreased binding of labeled ligand or decreased production of product from such substrates. A molecule that binds unnecessarily, that is, without inducing the effect of a ribosomal polypeptide or polynucleotide,
It is likely to be a good antagonist. Molecules that bind well and increase the rate of product production from the substrate are agonists. Detection of the rate and level of product production from the substrate may be facilitated using a reporter system. Reporter systems useful in this regard include, but are not limited to, colorimetrically labeled substrates that are converted to products, reporter genes that respond to changes in ribosome polynucleotide or polypeptide activity, and binding assays known in the art. Not done.

Another example of a ribosome antagonist assay is that of a ribosome with a ribosome binding molecule, a recombinant ribosome binding molecule, a natural substrate or ligand, or a substrate or ligand analog under conditions suitable for competitive inhibition assays, and A competitive assay that binds potential antagonists. When the number of ribosomal molecules that bind to the binding molecule or are converted to the product can be accurately measured to assess the efficacy of potential antagonists, eg, by radioactive or colorimetric compounds, ribosomal polypeptides Alternatively, the polynucleotide can be labeled. Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polynucleotides or polypeptides of the invention, thereby inhibiting or extinguishing its activity. Potential antagonists are also
A closely related small molecule, peptide, that binds to the same site on a binding molecule, such as a binding molecule, without inducing ribosome-inducing activity, thus preventing the action of the ribosome by leaving no room for ribosome binding. It may be a polypeptide such as a protein or an antibody.

Small organic molecules of the present invention are preferably less than 2,000 daltons, more preferably between 300 and 1,000 daltons, most preferably 400 and 70.
It has a molecular weight between 0 daltons. Certain embodiments provide compounds that have not been published prior to the filing date of the present application, particularly pluromutilin compounds or structural or functional pluromutilin analogs. Potential antagonists include small molecules that bind to and occupy the binding site of a polypeptide, such that normal biological activity is prevented, thus preventing binding of cell binding molecules. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules. Other potential antagonists include antisense molecules (see Okano, J. Neuroc for a description of these molecules.
hem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GE
NE EXPRESSION, CRC Press, Boca Raton, FL (1988)). Potentially preferred antagonists include compounds associated with either variant of the ribosomal polynucleotide or the polypeptide.

Each of the DNA sequences provided herein may be used in the discovery and development of antibacterial compounds. Upon expression, the encoded protein can be used as a target for antibacterial agent screening. In addition, DNA sequences encoding the amino-terminal region of the encoded protein or Shine-Dalgarno or other mRNA-enhancing translational sequences can be used to construct antisense sequences to control expression of important coding sequences. . The antagonists and agonists of the present invention are useful for treating meningitis, for example otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis and especially, for example, infection of cerebrospinal fluid. It may also be used to inhibit and treat.

Compositions, Kits and Administration The present invention also relates to compositions comprising the polynucleotides or polypeptides described above, or their agonists or antagonists. The polypeptides of the present invention can be used in admixture with unsterilized or sterilized carriers used for cells, tissues or organisms, for example, a pharmaceutical carrier suitable for administration to a subject.
Such compositions include, for example, a solvent additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration. The formulation should suit the mode of administration. The present invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the composition components of the invention above.

The polypeptides of the invention and the other compounds may be used alone or in combination with other compounds such as therapeutic compounds. The pharmaceutical composition may be administered in any effective and convenient manner, for example by the topical, oral, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, or intradermal routes, among others. . In therapy, or as a prophylactic, the active substances may be administered to an individual as an injectable composition, for example as a preferably isotonic sterile aqueous dispersion. Alternatively, the composition may be formulated for topical application, such as in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouth washes, thread for impregnated bandages and sutures, and aerosols. They may also contain suitable conventional additives, such as preservatives, solvents to aid drug penetration, and ointments and creams containing emollients. Such topical formulations may contain compatible conventional carriers, such as cream or ointment bases and ethanol or oleyl alcohol for lotions. Such carriers may be from about 1% to about 98% by weight of the formulation, more usually up to about 80% by weight of the formulation. For administration to mammals, especially humans, the daily dose of the active substance is
It is from 0.01 mg / kg to 10 mg / kg, typically about 1 mg / kg. The physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and particular responsiveness of the individual. The above doses are typical of the average case. Of course, there are individual instances where high and low dose ranges are suitable and such examples are within the scope of this invention.

In-dwelling devices include surgical implants, prosthetic devices, catheters and the like, ie, those that are introduced into the body of an individual and remain in place for an extended period of time. Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis.
inuous ambulatory peritoneal dialysis (CAPD) catheter and the like. The compositions of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an indwelling device. After surgery, treatment may continue for the length of time the device is in the body. In addition, it can be used in covers that are spread during surgical procedures to protect against bacterial wound infections, especially Streptococcus pneumoniae wound infections.

Many orthopedic surgeons believe that for humans with prosthetic joints, antibiotic prophylaxis should be considered before dental procedures that can result in bacteremia. Delayed severe infection is a serious complication that sometimes leads to loss of prosthetic joints, with significant morbidity and mortality. It is therefore possible in this context to extend the use of active substances as an alternative to prophylactic antibiotics. In addition to the above-mentioned treatments, the compositions of the present invention are generally used as therapeutic agents for wounds,
Bacteria may be prevented from adhering to matrix proteins exposed to wound tissue, and may be used prophylactically in dental treatment instead of or in combination with antibiotic prophylaxis. Alternatively, the composition of the invention may be used to bathe an indwelling device immediately before insertion. To soak wounds or indwelling devices, 1 μl of active substance
A concentration of g / ml to 10 mg / ml is preferred.

The vaccine composition is conveniently in injectable form. Conventional adjuvants may be used to enhance the immune response. The unit dose suitable for vaccination is 0.5
It is 5 μg / kg, and such dose is preferably administered 1 to 3 times at intervals of 1 to 3 weeks. With the indicated dose range, no adverse toxicological effects are observed with the compounds of the invention which would preclude their administration to suitable individuals. Each document disclosed herein is considered to be incorporated herein by reference in its entirety. Any patent application to which this application claims priority is considered entirely incorporated herein by reference.

The present invention is further described by the following examples. The examples are provided merely to illustrate the invention by referring to specific embodiments. These exemplifications illustrate certain embodiments of the invention and are not intended to be limiting or to limit the scope of the disclosed invention. Certain terms used in this specification are explained in the above terminology. All examples were made using standard techniques, which are well known and routine to those of skill in the art, unless otherwise described in detail. Routine molecular biology techniques in the following examples are described in standard laboratory manuals, such as Sambrook et al. (1989) MOLECULAR CLONING.
: A LABORATORY MANUAL, 2nd Ed .; Cold Spring Harbor Laboratory Press, Col
d Spring Harbor, NY. Unless otherwise stated, all parts or amounts shown in the following examples are by weight.

Examples Example 1 RNase H Cleavage Experiment Photoaffinity labeled pluromutilin ³ H-SB-328636 and ³ H-SB-
RNA extracted from the 50S ribosomal subunit treated with 352408
Was subjected to ribonuclease H (RNase H) cleavage. The specific DNA oligomer was hybridized with 23S rRNA in rRNA and digested with RNase H, yielding a fragment 200-300 nucleotides long. To visualize the fragments, a polyacrylamide gel was run on Coomassie blue: each labeled 23S.
The rRNA showed the same cleavage pattern as the unlabeled rRNA. ^{The 3} H-labeled gel was run for several days on a molecular dynamics phosphoimizer screen (Mol
ecular Dynamics phosphorimager screen). A ³ H signal was observed in the RNase H digest corresponding to the boxed region in lanes 11 and 12. These fragments are 1971-2 in the E. coli 23S rRNA sequence.
Corresponding to 607. This region (see FIG. 3) contains part of domain IV and domain V (Gutell, RR, Gray MW, and Schnare, MN (1993) Nucleic Acids Res.
21, 3055-3074), containing a peptidyl transferase center. See Figure 2 showing a Coomassie blue stained gel of RNase H digested 23S rRNA.

Example 2 Crosslinking Experiment FIG. 4 shows photolabeled 23S by elution and scintillation counting.
3 shows the identification of labeled RNase H fragment of rRNA / drug complex. SB-3286
The total RNase H fragment of the crosslinked 23S rRNA for both 36 and SB-352408 was excised from a polyacrylamide gel, electroeluted, and confirmed the identification of bands visualized on a Molecular Dynamics Phosphoimizer screen. . The RNase H oligomer pair profile of each individual DNA and the percentage of DPM recovered relative to the total DPM initially added to the reaction are shown above. Note that RNase H does not cleave at 100% efficiency, so most of the ³ H-label added in each reaction remains in intact RNA (see Figure 4). 23S
The total radioactivity maps to the 1971-2607 containing fragment of rRNA as detected by scintillation counting.

Example 2 Protein Crosslinking Experiment Two-dimensional polyacrylamide gel electrophoresis and HPLC are used to identify proteins that crosslink to the drug. Specific rRNA nucleotides cross-linked to the photoaffinity derivative have a 23S rR at or near the peptidyl transferase center.
Localized to domain V of NA.

Example 3 In Vitro Translation Assays Examples of the invention provide a series of pluromutilin derivatives in several functional E. coli in vitro translation and ribosome binding assays tested. These analyzes showed that a series of pluromutilins with different potential in in vitro assays are associated with antimicrobial activity against S. aureus. Tritium labeled derivatives are synthesized and used for ligand displacement assays to determine various bonds Pururomuchirin derivative (k _on) and dissociation (k _off) rate. It has been determined that pluromutilin specifically binds to the 50S ribosomal subunit, has binding rates on the order of minutes, and a range of slow dissociation rates.

References 1. Heineman, JA. (1999) Drug Discovery Today, 4, 72-79. 2. Spahn CMT and Prescott CD (1996) Journal of Molecular Medicine, 7
4,423-439. 3. Kirillov S., Porse BT, Vester B, Woolley P., Garrett RA (1997) FEB
S Lett., 406, 223-233. 4. Rather PN (1998) Drug Resistance Updates, 1, 285-291. 5. Barriere JC, Berthaud N., Beyer D., Dutkamalen S., Paris JM, Desn
ottes JF (1998) Current Pharmaceutical Design, 4,155-180. 6. Weisblum B. (1995) Antimicrobial Agents & Chemotherapy, 39, 577-5852.
7. Dobler M. and Durr BG (1975) Cryst. Struct. Commun., 4, 259. 8. Hoge
nauer G. (1979) In Hahn FE (ed.), Antibiotics, Vol. V, (part 1), Sprin
ger-Verlag, pp. 344-360. 9. Hogenauer, G. and Ruf, C. (1981) Antimicrobial Agents & Chemotherapy, 1
9, 260-265. 10. Dornhelm P. and Hogenauer G. (1978) Eur. J. Biochem., 91, 465-473. 11. Hogenauer G, (1975) In Drews J. and Hahn F. (eds. ), Topics in Infecti
ous Diseases: Drug Receptor Interactions in Antimicrobial Chemotherapy, V
ol. I, Berlin-Heidelberg-New York: Springer, pp. 235-234. 12. Drews J., Georgopoulos A., Laber G., Schutze E. and Unger J. (1975) A
ntimicrob.Agents Chemother., 7, 507-516. 13. Bock A., Turnowsky F., and Hogenauer G (1982), J. Bacteriol., 151, 12
53-1260. 14. Cunningham, PR, Nurse, K., Bakin, A., Weitzmann, CJ, Pflumm, M.
, andOfengand, J. (1992) Biochemistry, 31, 12012-12022. 15. Nurse, K., Colgan, J., Denman, R., Wilhelm, J, and Ofengand, J. (198
7) Biochimie, 69,1105-1112.

[Brief description of drawings]

FIG. 1 is a graph showing that tritium is detected only in the 50S fraction after the 70S ribosome bound to a tritiated drug is subjected to a low magnesium sucrose gradient and the 70S ribosome is separated into 30S and 50S particles. Show.

FIG. 2 shows a Coomassie blue stained gel of 23S rRNA digested with RNase H.

FIG. 3. E. coli 23S identified in binding SB-328636 (Structure 2) and SB-352408 (Structure 3) by RNase H digestion.
The rRNA sequence (nucleotides 1971-2607) is shown and SB-32863
6 and E. coli 23S photolabeled and identified by SB-352408
The secondary structure of the part of rRNA is shown.

FIG. 4 shows identification of labeled RNase H fragment of 23S rRNA / drug complex photolabeled by elution and scintillation counting.

FIG. 5: Chemical structure of SB-328636 (Structure 2) and SB-352408 (Structure 2).

FIG. 6: Characterization of photoaffinity derivatives of pluromutilin. Four photoaffinity derivatives were prepared to help identify the binding site for pluromutilin on the E. coli 70S ribosome. Data for two of these photoaffinity agents is shown in this figure. As described anywhere herein, PAL-1 and PAL-2 are active against Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae, and have a radioligand binding assay (R
It was inhibitory in both LB) and a functional in vitro translation f-met assay.

FIG. 7 shows a sucrose gradient of PAL1 and PAL2 in the presence and absence of unlabeled pluromutilin competitor. Binding of PAL to the 50S subunit was detected only in the absence of unlabeled pluromutilin derivative,
Showed that they compete for the same site.

FIG. 8 shows analysis of 50S ribosomal protein by two-dimensional polyacrylamide gel electrophoresis. (Method Madjer JJ, Michel S, Cozzone AJ, Reboud
JP. (1979) Analytical Biochemistry 92, 174-182). Crosslinking efficiencies were 1.4% and 0.2% for PAL-1 and PAL-2, respectively, so protein band detection could not be detected by phosphorimaging and fluorography. It was Therefore, each band was excised and quantified by ³ H counting.

FIG. 9 shows the identification of photolabeled proteins from 2D gels by independent methods (Solusol and combustion). Each band was excised from the 2D gel and quantified by ³ H counting. Gel slices from two different gels
olusol) or Packard ( ³ H is captured as ³ H ₂ O)
Burned in oxidizer and quantified. Packard
The oxidizer results are described above.

FIG. 10 shows that ³ H-PAL-1 specifically labels ribosomal proteins. ³ H-PAL-1 labels L2, L13, L32 and L33. Preincubation with unlabeled pluromutilin derivative inhibited photolabeling, indicating that PAL-1 specifically binds to the pluromutilin binding site. Note that addition of unlabeled pluromutilin does not completely inhibit L2 labeling.

FIG. 11 shows that ³ H-PAL-2 specifically labels ribosomal proteins. ³ H-PAL-2 labels L4, L32 and L33. Preincubation with unlabeled pluromutilin derivative inhibits photolabeling and
It shows that AL-2 is specifically bound at the pluromutilin binding site.

FIG. 12 H of ³ H-PAL-1 cross-links to 50S ribosomal protein.
PLC is shown. RP-HPLC started as an independent method to confirm the identity of proteins labeled with photoaffinity derivatives. The protein identities shown here are based on a reference chromatogram of the 50S protein performed under similar conditions (Kerlavage AR, Weitzmann CJ and Cooperman BS. (1984) J Chromatogr.
317, 201-212).

FIG. 13 shows the ³ H profile of the HPLC fraction. Fractions from the 5 ³ H peaks were collected to confirm the presence of protein on the silver stained gel,
It will be identified by mass spectrometry. Free ³ H-PAL-1 elutes at a retention time of 65 minutes close to ³ H peak 5. Samples preincubated with the unlabeled competitor, pluromutilin, inhibit the photolabeling of 50S protein, confirming the 2D gel burning results.

FIG. 14 shows the secondary structure of E. coli 23S ribosomal RNA.

FIG. 15 shows radioligands synthesized for binding experiments ((A) Chemical structure of pluromutilin, (B) Tritiated semisynthetic radiolabeled derivative with double bond cleavage at position 12). , (C) under the conditions of A-site tRNA binding using activated ribosome programmed with polyuridylic acid, the radioligand is 70S.
Bound to the ribosome). To separate the subunits, ribosomal subunits were separated on a 10-30% sucrose gradient containing low magnesium. Total radioactivity binds to the 50S subunit.

FIG. 16 shows a pluromutilin radioligand binding assay. The assay was developed to monitor changes in binding affinity of pluromutilin derivatives in response to structural modifications. The 70S ribosomes are incubated with the ligand, filtered and bound ³ H radioligand is measured by scintillation counting.

FIG. 17 shows a functional translation assay. The S30 natural translation initiation assay
Designed to monitor protein synthesis initiation in the presence of pluromutilin derivatives. S30 lysates in the appropriate buffer are incubated with pluromutilin in the presence of [ ³ H] fmet-tRNA ^met . The synthesized polypeptide is precipitated with hot trichloroacetic acid, filtered, and counted.

FIG. 18 shows a functional translation assay. Transcription linked to S30 natural translation /
Translation assay. In S30 lysate and appropriate buffer, the luciferase gene (Pro
The plasmid with mega) is incubated to transcribe and translate the luciferase gene. Addition of luciferase substrate produces luminescence. In the presence of pluromutilin, luciferase translation is inhibited, resulting in decreased luminescence.

FIG. 19 shows selected radioligand binding displacement curves for a set of pluromutilin derivatives. Low for S. aureus (0.25 microgram /
ml), intermediate range (2 microgram / ml) and high MICs (8 and> 6)
4 microgram / ml) indicated drug concentration versus percent displacement.

FIG. 20: Low for S. aureus (0.25 microgram / m
l), intermediate range (2 microgram / ml) or high MICs (8 and> 6)
4 microgram / ml) of radioligand binding and reaction of pluromutilin derivatives in a functional assay. There is a good inter-assay correlation between functional and binding assays that correlates with antimicrobial activity; less potent binders are less potent (MIC) in whole cell antimicrobial assays.

FIG. 21 shows inhibition of translation initiation / reinitiation by a lead-pluromutilin derivative. ^(A) S30 3 in H-fmet assay, _{IC 50} measurement of varying the S30 lysate concentrations. The IC ₅₀ depends on the lysate concentration (ie ribosome concentration). (B) The linear relationship of lysate concentration vs. IC ₅₀ indicates that pluromutilin can titrate ribosomes.

FIG. 22 shows the binding rate of ³ H pluromutilin radioligand. Binding rates were measured by running a radioligand binding assay at various ribosome concentrations with aliquots removed at a given time. Binding of ³ H-pluromutilin radioligand is rapid (t _{1 /} 2-20 sec).

FIG. 23 shows a pluromutilin dissociation measurement. (A) Moderately strong derivative S
BM dissociation rate. Over 1000-fold unlabeled drug was added to 70S ribosomes under radioligand binding conditions. Ribosomes were then separated on a sucrose gradient to isolate the ribosome-drug complex. Excess ³ H pluromutilin radioligand was then added and the amount of radioligand bound at time intervals was measured by a filtration binding assay. The dissociation rate of unlabeled drug is reflected in the amount of labeled drug that can bind to ribosomes. (B) Dissociation rate of other pluromutilin derivatives. The antimicrobially active strong binder did not dissociate to a detectable rate.

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 45/00 A61K 48/00 4C084 48/00 A61P 1/12 4C086 A61P 1/12 1/18 4C206 1 / 18 9/00 9/00 11/00 11/00 13/02 105 13/02 105 13/12 13/12 17/00 101 17/00 101 19/02 19/02 25/28 25/28 27/02 27/02 27/16 27/16 31/04 31/04 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 C12P 19/34 A 5/10 C12Q 1/68 A C12P 19 / 34 G01N 33/15 Z C12Q 1/68 33/50 Z G01N 33/15 C12R 1:19 33/50 1: 445 // (C12Q 1/68 1:46 C12R 1:19) C12N 15/00 ZNAA ( C12Q 1/68 5/00 A C12R 1: 445) (C12Q 1/68 C12R 1:46) (31) Priority claim number 60 / 139,09 (32) Priority date June 14, 1999 (June 14, 1999) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES , FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), JP, US (71) Applicant SmithKline Beecham Public Limited Company SmithKline Beecham p. l. c. United Kingdom Tida Brew 89 Jesus, Middlesex, Brentford, Great West Road 980 (72) Inventor Lisa Ann Heg United States 19333 Tanglewood Lane 554 (72) Devon, PA United States Therese Ann Sterner United States 19333 Sugartown Road 340 (72) Devon, Pennsylvania Inventor Kelvin Sea Nurse United States 19067 Nox Drive 1366 (72) Jadry, Pennsylvania Inventor Dona Em Ferguson United States 19382 Pennsylvania West Chester, Hunt Drive No. 916 F term (reference) 2G045 AA40 DA12 DA13 DA14 4B024 AA01 AA13 CA01 CA04 CA11 4B063 QA07 QQ06 QQ43 QR39 QR75 4B064 AF27 CA19 CC24 DA01 4B065 AB01 BA02 CA23 CA44 4C0814 CA44A02 AA13 A02 ZA33 ZA34 ZA59 ZA73 ZA82 ZA90 ZA96 ZB35 4C086 AA01 AA02 EA16 EA18 MA02 NA14 ZA02 ZA33 ZA34 ZA59 ZA73 ZA75 ZA82 ZA90 ZB35 4C206 AA01 AA02 DB03 GA02 MA02 NA14 ZA02 ZA33 ZA34 ZA59 ZA73 ZA75 ZA82 ZA90 ZA59

Claims (15)

[Claims] 1. A polynucleotide comprising at least 70% identity to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, or (b) consisting of a polynucleotide which is complementary to the polynucleotide of (a). An isolated polynucleotide comprising a polynucleotide sequence selected from the group. 2. The polynucleotide according to claim 1, wherein the polynucleotide is DNA. 3. The polynucleotide according to claim 1, wherein the polynucleotide is RNA. 4. The polynucleotide according to claim 2, which comprises the nucleic acid sequence shown in SEQ ID NO: 1. 5. A vector containing the polynucleotide according to claim 1. 6. A host cell containing the vector according to claim 5. 7. A method for producing a polynucleotide, which comprises expressing RNA from the host cell according to claim 6. 8. A method for treating an individual in need of inhibiting ribosomal polynucleotide, which comprises administering to the individual a therapeutically effective amount of a compound that binds to or interacts with the polynucleotide of claim 1. 9. To assess the interaction of a compound, contacting a composition comprising a polynucleotide with a compound to be screened under conditions that allow the compound and the polynucleotide to interact, such interaction comprising: Is associated with a second component capable of providing a detectable signal in response to the interaction of the polynucleotide and the compound; and then detecting the presence or absence of the signal resulting from the interaction of the polynucleotide and the compound Thereby interacting with and inhibiting the activity of a polynucleotide, the method comprising the step of determining whether the compound interacts with the polynucleotide and activates or inhibits the activity of the polynucleotide. Alternatively, a method for identifying a compound to be activated. 10. Binding a compound to a bacterial 50S ribosomal subunit; binding a compound to a bacterial 70S ribosomal; binding a compound to a ribosome under tRNA binding conditions; with a messenger RNA such as polyuridylic acid. Binding the compound to the ribosome under tRNA binding conditions using a programmed activated ribosome; binding the compound to the Escherichia coli 23S rRNA sequence; binding the compound to nucleotides 1971-2607 at Escherichia coli 23S rR
Binding to NA; modification of RNA secondary structure formed by nucleotides 1971-2607 of Escherichia coli 23S rRNA; RNA formed by domain V of Escherichia coli 23S rRNA
Altering secondary structure; Modulating SB-328636 (Structure 2) binding to ribosomes; Modulating SB-352408 (Structure 3) binding to ribosomes; SB-328636 (Structure 2) ribosome 23S Modulating binding to RNA; Modulating SB-352408 (Structure 3) binding to ribosomal 23S RNA; SB-328636 (Structure 2) Escherichia coli ribosomal 23S RN
Modulating the binding of A to domain V; Escherichia coli ribosome 23S RN of SB-352408 (Structure 3)
Modulating binding of A to domain V; binding compounds to ribosomal RNA and ribosomal proteins; binding compounds to ribosomal proteins L4, L32, L33, L2 or L13; ribosomal proteins L4, L32, L33, Modulating the binding of L2 or L13 to the ribosome; modulating the binding of ribosomal proteins L4, L32, L33, L2 or L13 to ribosomal RNA; modulating the binding of a compound to G2061, A2062 or G2502; Modulating the binding of pluromutilin to G2061, A2062 or G2502; Modulating the binding of chloramphenicol to G2061, A2062 or G2502 ;; Altering the binding of p-azidopuromycin G2502 Modulating the binding of a compound to A2407 and U2408; modulating the binding of pluromutilin to A2407 and U2408; or by binding the compound to a nucleotide of 23S rRNA of SEQ ID NO: 1, 2 or 3 A polynucleotide comprising a nucleotide sequence that is at least 70% identical to the nucleotide sequence and SEQ ID NO: 1, 2 or 3
An antagonist or an agonist that inhibits the activity of a bacterial polynucleotide selected from the group consisting of polynucleotides containing the nucleotide sequence shown in. 11. A method for treating an individual suspected of being infected with a bacterium, which comprises using the antagonist or agonist according to claim 10. 12. The method of claim 10, wherein the bacterium is selected from the group consisting of Staphylococcus spp., Staphylococcus aureus spp., Streptococcus spp., And Streptococcus pneumoniae. 13. Binding a compound to a bacterial 50S ribosomal subunit; Binding a compound to a ribosome under A site tRNA binding conditions; Using an activated ribosome programmed with polyuridylic acid, an A site tRN.
Binding the compound to ribosomes under A-binding conditions; binding the compound to the Escherichia coli 23S rRNA sequence; binding the compound to nucleotides 1971-2607 at Escherichia coli 23S rR
Binding to NA; modification of RNA secondary structure formed by nucleotides 1971-2607 of Escherichia coli 23S rRNA; RNA formed by domain V of Escherichia coli 23S rRNA
Altering secondary structure; Modulating SB-328636 (Structure 2) binding to ribosomes; Modulating SB-352408 (Structure 3) binding to ribosomes; SB-328636 (Structure 2) ribosome 23S Modulating binding to RNA; Modulating SB-352408 (Structure 3) binding to ribosomal 23S RNA; SB-328636 (Structure 2) Escherichia coli ribosomal 23S RN
Modulating the binding of A to domain V; Escherichia coli ribosome 23S RN of SB-352408 (Structure 3)
Modulating binding of A to domain V; binding compounds to ribosomal RNA and ribosomal proteins; binding compounds to ribosomal proteins L4, L32, L33, L2 or L13; ribosomal proteins L4, L32, L33, Modulating the binding of L2 or L13 to the ribosome; modulating the binding of ribosomal proteins L4, L32, L33, L2 or L13 to ribosomal RNA; modulating the binding of a compound to G2061, A2062 or G2502; Modulating the binding of pluromutilin to G2061, A2062 or G2502; Modulating the binding of chloramphenicol to G2061, A2062 or G2502; Modulating the binding of p-azidopuromycin G2502 Modulating the binding of compounds to A2407 and U2408; modulating the binding of pluromutilin to A2407 and U2408; or a method of inhibiting the activity of bacterial ribosomes by binding the compound to nucleotides of 23S rRNA. 14. The method of claim 13, wherein the bacterium is selected from the group consisting of members of the genus Staphylococcus, Staphylococcus aureus, members of the genus Streptococcus, and Streptococcus pneumoniae. 15. A method of treating an individual infected with a bacterium comprising the step of contacting an individual suspected of being infected with the bacterium with an antimicrobially active amount of a composition comprising a pluromutilin compound, wherein said contacting is: Binding the compound to the bacterial 50S ribosomal subunit; binding the compound to the ribosome under A site tRNA binding conditions; using an activated ribosome programmed with polyuridylic acid, the A site tRN
Binding the compound to the ribosome under A-binding conditions; binding the compound to the Escherichia coli 23S rRNA sequence; binding the compound at nucleotides 1971-2607 to Escherichia coli 23S rR
Binding to NA; modification of RNA secondary structure formed by nucleotides 1971-2607 of Escherichia coli 23S rRNA; RNA formed by domain V of Escherichia coli 23S rRNA
Altering secondary structure; Modulating SB-328636 (Structure 2) binding to ribosomes; Modulating SB-352408 (Structure 3) binding to ribosomes; SB-328636 (Structure 2) ribosome 23S Modulating binding to RNA; Modulating SB-352408 (Structure 3) binding to ribosomal 23S RNA; SB-328636 (Structure 2) Escherichia coli ribosomal 23S RN
Modulating the binding of A to domain V; Escherichia coli ribosome 23S RN of SB-352408 (Structure 3)
Modulating the binding of A to domain V; binding the compound to ribosomal RNA and ribosomal proteins; binding the compound to ribosomal proteins L4, L32, L33, L2 or L13; ribosomal proteins L4, L32, L33, Modulating the binding of L2 or L13 to the ribosome; modulating the binding of ribosomal proteins L4, L32, L33, L2 or L13 to ribosomal RNA; modulating the binding of a compound to G2061, A2062 or G2502; Modulating the binding of pluromutilin to G2061, A2062 or G2502; Modulating the binding of chloramphenicol to G2061, A2062 or G2502; Modulating the binding of p-azidopuromycin G2502 Rukoto; method results in inhibition of bacterial ribosomal activity by binding or compound nucleotide 23S rRNA; A2407 and that modulates the binding of U2408 compound; it modulates binding to A2407 and U2408 of Pururomuchirin.