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Número de publicaciónWO1999052357 A1
Tipo de publicaciónSolicitud
Número de solicitudPCT/US1999/008134
Fecha de publicación21 Oct 1999
Fecha de presentación14 Abr 1999
Fecha de prioridad14 Abr 1998
Número de publicaciónPCT/1999/8134, PCT/US/1999/008134, PCT/US/1999/08134, PCT/US/99/008134, PCT/US/99/08134, PCT/US1999/008134, PCT/US1999/08134, PCT/US1999008134, PCT/US199908134, PCT/US99/008134, PCT/US99/08134, PCT/US99008134, PCT/US9908134, WO 1999/052357 A1, WO 1999052357 A1, WO 1999052357A1, WO 9952357 A1, WO 9952357A1, WO-A1-1999052357, WO-A1-9952357, WO1999/052357A1, WO1999052357 A1, WO1999052357A1, WO9952357 A1, WO9952357A1
InventoresCarol S. Dammel, Julie C. Watson, Victor J. Hernandez, Charles Gluchowski
SolicitanteRibogene, Inc.
Exportar citaBiBTeX, EndNote, RefMan
Enlaces externos:  Patentscope, Espacenet
Assays for inhibitors of bacterial translation initiation factor 3
WO 1999052357 A1
Resumen
The invention is directed to a high-throughput screening system for identifying new antibacterial agents specifically targeted against an essential component of the bacterial translation apparatus, IF3. The assay uses a reporter gene system in whole cells, and is based on the ability of IF3 to discriminate against translation initiation at the atypical start codon of the reporter gene. The figure is a schematic diagram of the pAUU-CAT reporter gene construct in which the CAT gene has its ATG initiator codon replaced with ATT. Inhibitors of IF3 are expected to result in increased growth of specially-designed test cells due to higher levels of expression from the reporter gene. The invention further provides secondary assays which are used to further study the test compounds which produced positive results in the screening assays of the invention. The invention also encompasses reporter gene constructs, test cells, and compounds identified by the methods of the invention.
Reclamaciones  (El texto procesado por OCR puede contener errores)
WHAT IS CLAIMED;
1. A method for screening for test compounds that inhibit the activity of bacterial translation initiation factor 3 (IF3) comprising:
(a) contacting a test cell with a test compound for a time sufficient to allow the test compound to inhibit IF3 activity in the test cell, wherein the test cell contains a reporter gene operably linked to a promoter which is expressible in the test cell, and wherein the reporter gene comprises an atypical start codon; and
(b) detecting the translation of the reporter gene mRNA, wherein a specific increase in the translation of the reporter gene mRNA in the test cell contacted with the test compound relative to the translation of the reporter gene mRNA in a test cell not contacted with the test compound, indicates that the test compound inhibits IF3 in the test cell.
2. A method for screening for test compounds that inhibit the activity of bacterial translation initiation factor 3 (IF3) comprising:
(a) contacting a test cell with a test compound for a time sufficient to allow the test compound to inhibit IF3 activity in the test cell, wherein the test cell contains a reporter gene operably linked to a promoter which is expressible in the test cell, and wherein the reporter gene comprises an atypical start codon; and
50 - (b) detecting the signal generated by the reporter gene product, wherein a specific increase in the signal in the test cell contacted with the test compound relative to the signal generated by the reporter gene product in a test cell not contacted with the test compound, indicates that the test compound inhibits IF3 in the test cell.
3. A method for screening for test compounds that inhibit the activity of bacterial translation initiation factor 3 (IF3) comprising:
(a) culturing a test cell under selection;
(b) contacting the test cell with a test compound for a time sufficient to allow the test compound to inhibit IF3 activity in the test cell, wherein the test cell contains a reporter gene operably linked to a promoter which is expressible in the test cell such that the test cell can overcome the selection, and wherein the reporter gene comprises an atypical start codon; and
(c) detecting growth of the test cell under selection, wherein an increase in growth of the test cell contacted with the test compound relative to the growth of the test cell not contacted with the test compound, indicates that the test compound inhibits IF3 in the test cell.
4. The method of claim 1, 2, or 3 wherein the test cell is Escherichia coli.
51 -
5. The method of claim 1, 2, or 3 wherein the test cell is selected from the group consisting of Salmonella typhimurium, Klebsiella pneumoniae, Serratia marcescens, Proteus vulgaris, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, Bacillus subtilis, and Bacillus stearothermophilus.
6. The method of claim 1, 2, or 3 wherein the atypical start codon is ATT.
7. The method of claim 1, 2, or 3 wherein the reporter gene is an antibiotic resistance gene.
8. The method of claim 1, 2, or 3 wherein the reporter gene is an auxotrophic gene.
9. The method of claim 3 wherein the selection is caused by culturing the test cell in media containing antibiotics selected from the group consisting of chlormaphenicol , tetracycline, and kanamycin.
10. The method of claim 3 wherein the reporter gene is the E. coli chloramphenicol acetyltransferase gene.
11. A method for screening for test compounds that cause IF3 inhibition in bacteria comprising: (a) inoculating a plate containing a solid medium comprising about 2 mg/1 to 40 mg /l chloramphenicol with E. coli test cells containing a E. coli chloramphenicol acetyltransferase gene operably linked to the lac promoter in the test cell, and wherein the chloramphenicol acetyl
- 52 - transferase gene comprises the atypical ATT start codon;
(b) adding a test compound to a point of application in the plate and incubating
,. the plate for a time sufficient to allow the test compound to contact the test cell and inhibit IF3 activity; and
(c) detecting growth of the test cells contacted with the test compound, wherein an increase in growth of the test cell contacted with 0 the test compound relative to the growth of test cells not contacted with the test compound indicates that the test compound inhibits IF3 activity in the test cell.
12. The method of claim 11 wherein the solid medium 5 in the plate comprises about 6 mg/1 chloramphenicol.
13. The method of claim 1 further comprising comparing the translation of the reporter gene mRNA to the translation of the reporter gene mRNA in a test cell contacted with lincomycin, wherein the test cell contacted with 0 lincomycin serves as a positive control.
14. The method of claim 2 further comprising comparing the signal generated by the reporter gene product to the signal generated by the reporter gene product in a test cell contacted with lincomycin, wherein the test cell contacted with lincomycin serves as a positive control.
15. The method of claim 3 further comprising comparing the growth of the test cell under selection to the 0 growth of the test cell under selection contacted with lincomycin, wherein the test cell contacted with lincomycin serves as a positive control .
53 -
16. A method for screening for test compounds that inhibit the activity of bacterial translation initiation factor 3 (IF3) comprising:
(a) incubating in a reaction comprising a
-. reporter gene mRNA comprising an atypical start codon, a cell extract comprising IF3 a test compound for a time sufficient to allow the test compound to inhibit IF3 activity in the cell extract; and
(b) detecting the translation of the reporter gene mRNA in the reaction, wherein a specific increase in the translation of the reporter gene mRNA in the reaction containing the test compound relative to the translation of the reporter gene mRNA 5 in a reaction not containing the test compound, indicates that the test compound inhibits IF3 in the reaction.
17. The method of claim 16 wherein the atypical start codon is ATT. 0
18. The method of claim 16 wherein the reporter gene is the firefly luciferase gene.
19. A method for screening for test compounds that 5 inhibit the activity of bacterial translation initiation factor 3 (IF3) comprising:
(a) incubating in a reaction comprising bacterial 16S ribosomal RNA, IF3, and a test compound for a time sufficient to 0 allow the test compound to interfere with the binding of IF3 to 16S ribosomal RNA; and
54 (b) determining the binding characteristics of IF3 to the bacterial 16S ribosomal RNA, wherein a change in the binding characteristics of IF3 to 16S ribosomal RNA in the presence of the test compound relative to the binding characteristics of IF3 to 16S ribosomal RNA in the absence of the test compound indicates that the test compound inhibits the activity of IF3.
20. A method for screening for test compounds that bind bacterial translation initiation factor 3 (IF3) comprising :
(a) incubating in a reaction comprising IF3 and a test compound for a time sufficient to allow the test compound to bind IF3 ; and
(b) detecting in the reaction the presence of a complex comprising IF3 and the test compound .
21. The method of claim 20 wherein the test compound is detectably labeled by a dye, an enzyme, an isotope, a fluorescent group or a luminsecent group.
22. The method of claim 20 wherein the IF3 is present on a solid phase.
23. A compound that inhibits IF3 activity in bacteria that is identified by the method of claim 1, 2, 3, or 16.
24. A pharmaceutical composition comprising a compound that inhibits IF3 activity in bacteria that is
- 55 identified by the method of claim 1, 2, 3, or 16 and a pharamceutically acceptable carrier.
25. A method for inhibiting the growth of bacteria comprising contacting the bacteria with a compound that inhibits IF3 activity in bacteria as identified by the method of claim 1, 2, 3, or 16.
26. A method for treating a subject with an infectious disease caused by bacteria comprising administering to the subject a therapeutically effective amount of a compound that inhibits IF3 activity in bacteria as identified by the method of claim 1, 2, 3, or 16.
27. The method of claim 26 further comprising adjunctively administering to the subject a second antibacterial compound.
28. The method of claim 26 wherein the second antibacterial compound is a member of an antibiotic group selected from the group consisting of aminoglycosides, amphenicols, ansamycins, ╬▓-lactams, cephalosporins, cephamycins, monobactams, oxacephems, penicillins, lincosamides, macrolides, polypeptide antibiotics, tetracyclines, 2 , 4-diaminopyrimidines, nitrofurans, quinolones, sulfonamides, sulfones, oxazolidinones and glycylcycines .
29. A pharmaceutical composition comprising a compound that inhibits IF3 activity in bacteria that is identified by the method of claim 1, 2, 3, or 16, a second antibacterial compound, and a pharamceutically acceptable carrier.
56
30. The method of claim 26 wherein the subject is human .
31. The method of claim 26 wherein the subject is a companion animal or a livestock animal.
32. A method for sterilizing bacteria-contaminated object or material comprising contacting the bacteria- contaminated object or material with a compound that inhibits IF3 activity in bacteria as identified by the method of claim 1, 2, 3, or 16.
57
Descripción  (El texto procesado por OCR puede contener errores)

ASSAYS FOR INHIBITORS OF BACTERIAL TRANSLATION INITIATION FACTOR 3

This application claims priority under 35 U.S.C. §119 (e) to provisional patent application no. 60/081,736 filed April 14, 1998, the entire contents of which is incorporated herein by reference in its entirety.

1. INTRODUCTION

The present invention relates to methods for high- throughput screening for compounds with antibiotic activity. Specifically, the invention relates to high-throughput screens which target translation initiation factor 3 (IF3) in bacteria. The invention further relates to novel compounds identified using such screening methods.

2. BACKGROUND

New antibiotics are desperately needed as clinically significant bacterial pathogens have acquired resistance to nearly all existing antibiotics (Chopra et al . , 1997, Antimicrob. Agents Chemother, 37:1563-1571; Cohen,

1992, Science, 257:1050-1055; Kunin, 1993, Ann. Intern. Med. , 118:557-561; Neu, 1992, Science, 257:1064-1073; Tenover and Hughes, 1996, JAMA, 275:300-304).

The variety of mechanisms by which bacteria acquire resistance to antimicrobial drugs is remarkable (Arthur and

Courvalin, 1993, Antimicrob. Agents Chemother., 37:1563-1571;

Chopra, 1992, J. Antimicrob. Chemother, 30:737-739; Kunin,

1993, Ann. Intern. Med., 118:557-561; Russell and Chopra, 1996, Understanding antibacterial action and resistance, 2nd ed. Ellis Horwood, New York, NY; Jacobs, 1994, Clin. Infect. Dis., 19:1-10; Hooper and Wolfson, 1993, Am. Soc . Microbiol . , 1993:97-118). This robust fitness is likely a reflection of the high degree of evolutionary, metabolic, molecular and ecological diversity seen in the microbial world. The emergence and spread of resistant bacterial organisms are primarily caused by acquisition of drug resistance genes, resulting in a broad spectrum of antibiotic resistance (e.g., extended-spectrum cephalosporin-resistant mutant β-lactamases found in several bacterial organisms) . Genetic exchange of multiple-resistance genes, by transformation, transduction and conjugation, combined with selective pressures in settings such as hospitals where there is heavy use of antibiotic therapies, enhance the survival and proliferation of antimicrobial agent -resistant bacterial strains occurring by, e.g., spontaneous mutants. Id . Although the extent to which bacteria develop resistance to antimicrobial drugs and the speed with which they do so vary with different types of drugs, resistance has inevitably developed to all antimicrobial agents (Gold and Moellering, Jr., 1996, New Eng. J. Med., 335 (19) : 1445-1453) . Prevention of life threatening microbial infections coupled with medical practice aimed at minimizing the development of drug resistance are certainly important (Moellering, 1990, Scand. J- Infect. Dis., Suppl . 70:18-24; McCaig and Hughes, 1995,

JAMA 273:214-219; Guillemot, 1998, JAMA, 279:365-370).

To prevent or delay the buildup of a resistant pathogen population, different chemicals that are effective against a particular disease-causing bacterium must be available. Thus, there is a need to develop methods for identifying compounds which can penetrate and specifically kill the pathogenic bacterial cell, or arrest its growth without also adversely affecting its human, animal, or plant host .

Much research is needed to discover more effective ways of using existing drugs, such as chemical modification of existing antibiotics, with the goals of circumventing bacterial resistance and finding more potent activities (Russell and Chopra, 1996, Understanding antibacterial action and resistance, 2nd ed. Ellis Horwood, New York, NY; Kuckers and Bennett, 1987, The use of antibiotics, a comprehensive review with clinical emphasis, 4th ed. Heinemann, Oxford, c United Kingdom; Moellering et al . , 1989, J. Antimicrob.

Chemother., 24 (Suppl. A):l-7). Direct testing of compounds for their ability to inhibit bacterial growth using standard microbiological methods has been the approach traditionally used to discover antibiotics. However, the task of identifying the mechanism of action of compounds identified 0 this way would be daunting. Without such knowledge it is difficult to predict selectivity, efficacy, or the likelihood that resistance will develop.

An alternative powerful new approach, called "targeted screening" , has recently become possible with the 5 advent of the field of genomics and our increasing understanding of bacterial molecular genetics. Using this approach, one can select a molecule or cellular process that might be useful as a drug target, and design screens to identify compounds that interfere with such molecule or 0 process. Targeted screening can be fast and inexpensive, and can allow rapid high-throughput screening of a large number of compounds. In contrast to simple growth inhibition, the targeting of specific molecular targets seeks to restrict drug discovery to compounds that act by a specific mechanism of action. One of the advantage is that the drug's target is 5 known in advance, reducing the necessity of lengthy drug development. If the assay is carefully designed to target an essential bacterial process that is not found in mammalian cells, then efficacy and selectivity can be maximized. The remaining challenge, and a more serious one, is finding the 0 right molecule or process to target in the assay that will identify an antibiotic that is effective against a broad spectrum of pathogens, while remaining harmless to the cells of the host organism.

The genomes of several pathogenic microorganisms, such as Escherichia coli , Helicobacter pylori , and Chlamydia

5 trachomatis, have recently been sequenced (Blattner et al . ,

1997, Science 277: 1453; Tomb et al . , 1997, Nature, 388: 539-

547) . The availability of gene sequences encoding all proteins of these bacteria provides an unprecedented opportunity for understanding and manipulating bacterial genomes at the molecular level . A number of genes are known 10 or are suspected to be essential to growth, survival or virulence. Such genes could be ideal targets in screening for novel antibiotics.

Translation is the process used by cells to make proteins and is an essential cellular process for all living

-1-5 organisms, including infectious pathogens. Translation requires the coordinated interplay of mRNA, ribosomes, tRNAs, and a number of specialized proteins (Hill et al . , 1990, The Ribosome . Structure, Function and Evolution. Washington, DC: Am. Soc . Microbiol . ; Nierhaus et al . , 1992, The Translational

20 Apparatus. Structure, Function, Regulation, Evolution. New

York: Plenum.; Matheson et al . , 1995, Biochem. Cell Biol .

73:739-1227; Zimmermann et al . , 1996, Ribosomal RNA.

Structure, Evolution, Processing, and Function in Protein

Biosynthesis. Boca Raton, FL : CRC) . Ribosomes are highly conserved macromolecular structures responsible for the 25 translation of mRNA. The E. coli ribosome is composed of two large particles, the 30S and the 50S subunits. The 30S subunit consists of a 16S rRNA molecule and 21 small r- proteins. The 50S subunit is composed of two RNA molecules,

23 S and 5S rRNA, and 31 large r-proteins . It is convenient

3 to divide the process of translation into three broad phases: initiation, elongation and termination. Initiation requires the ribosome to begin peptide chain formation at the correct initiation codon. In addition to ribosomes, initiation requires mRNA, initiator tRNA and initiation factors. Elongation is the complicated process by which a peptide chain is extended through the coordinated action of ribosomes, mRNA, charged elongator tRNAs and two elongation factors EF-Tu and EF-G. Termination is the process used by the ribosome to correctly terminate peptide chain formation, employing a termination codon UAA, UAG, or UGA and the termination factors RF-1, RF-2 and RF-3.

One can consider initiation of translation to be a natural starting point for the ribosome cycle. Initiation in bacteria begins with the formation of a ternary complex containing initiator fMet-tRNA, mRNA and the 30S subunit. Three distinct essential initiation factors (IF1, IF2, and IF3) , ribosomal subunits, fMet-tRNA, mRNA and GTP are required for in vivo initiation of translation in E. coli . Models for the function of initiation factors have been reviewed most recently in Gualerzi and Pon (1990, Biochem. , 29:5881-5889) .

IF2 directs binding of initiator tRNA to the 3 OS subunit (Grunberg-Manago, 1980, Ribosomes: Structure,

Function and Genetics (Chambliss, G. et al . , eds) . University

Park Press, Baltimore, NY pp.445 -477; Canonaco et al . , 1986,

Fed. Eur. Biochem. Soc . 207: 198-204; Hartz et al . , 1989,

Genes Dev. 3: 1899-1912). GTP is bound to the 30S initiation complex by IF2 and its hydrolysis is required to release IF2 from the 70S complex. Failure to release IF2 results in a blockade of peptide bond formation. IF1 appears to stimulate the activities of IF2 and IF3 (Grunberg-Manago and Gros,

1977, Progr. Nucl . Acid Res. Mol . Biol . , 20:209-284; Hartz et al., 1989, Genes Dev., 3:1899-1912). IF1 will bind 30S ribosomal subunits only in the presence of IF2, GTP, fmet- tRNA and mRNA. Like IF2, it is released from the complex following union with the 50S subunit, but unlike IF2, release of IF1 does not require GTP hydrolysis; union alone suffices.

IF3 binds the 3 OS subunit, preventing the association of 50S and 3OS subunits and appears to promote the proper binding of mRNA and initiator tRNA to the 3OS subunit (Subramanian and

5 Davis, 1970, Nature (London), 228:1273-1275; Hartz et al . ,

1989, Genes Dev., 3:1899-1912).

Protein initiation factor IF3 is essential and encoded by the infC gene in E. coli (Sacerdot et al . 1982,

EMBO J., 1:311-315). IF3 favors dissociation of 70S complexes, and is thought to function at the level of ternary 10 complex stability, increasing the level of fidelity by inspecting both the tRNA and the start codon in the P site.

IF3 protects specific nucleotides in the 30S subunit from base-specific chemical probes (Moazed et al . , 1995, J. Mol .

Biol., 248:207-210). These protected sites overlap with

15 those protected by 50S subunits and P-site-bound tRNA. This observation is consistent with the role of IF3 in subunit dissociation and initiator tRNA selection (Chapman and Noller, 1977, J. Mol. Biol., 109:131-149; Herr et al . , 1979, J. Mol. Biol., 130:433-449; Moazed and Noller, 1990, J. Mol.

20 Biol. , 211:135-145) .

3. SUMMARY OF THE INVENTION

This present invention provides a method of screening for therapeutic compounds useful in the treatment of bacterial infections. The methods are designed to 25 identify compounds that specifically target the bacterial initiation factor IF3, a factor integral to protein translation and believed to be essential to bacterial growth.

In various embodiments, the invention provides a primary screening assay to identify inhibitors of bacterial IF3,

Q 0 secondary in vi tro IF3 assays, and lead compounds that scored positive in these assays. The assays of the invention are adaptable to high-throughput screening. In one embodiment, the screening assays of the invention targets initiation factor IF3 which is unique to bacteria and essential for growth. The assay can identify compounds that bind to bacterial IF3 and affect its function

5 in translation. Any bacterial IF3 can be used in the screening assays. The IF3 of E. coli is preferably used as a model system. In a preferred embodiment, the primary screening assay uses a reporter gene system in whole cells, and is based on the ability of IF3 to discriminate against translation initiation at the atypical start codon of the 10 reporter gene. Specifically, inhibitors of IF3 are expected to result in increased growth of specially-designed test cells due to higher levels of expression from the reporter gene .

In other embodiments, secondary assays are provided

15 to evaluate hits identified in the primary assay. Reporter constructs analogous to these used in the primary assay will be used in in vi tro translation reactions to confirm that inhibitors identified in the primary assay target IF3 function. Further follow up assays are provided which are

2o designed to characterize the mode of action of inhibitors on

IF3 function. Compounds identified in the primary assay based on growth inhibition of a test species, such as E. coli , and Staphylococcus aureus, will be tested for efficacy against other bacterial pathogens to confirm that the positive compounds are effective against a broad range of microorganisms. Furthermore, these compounds will be tested for toxicity in mammalian cells to confirm the bacterial selectivity of the target.

The primary screening assay is designed to screen chemical compound libraries at high-throughput . Such •a Q libraries contain single compounds, mixtures of compounds with known structures, and natural product extracts.

Mixtures of compounds can be deconvoluted to identify the active compound. The active component (s) in natural product extracts that test positive in the assay will be separated and identified. The high-throughput screening assay is an effective method for identifying compounds that interfere π with IF3 function. The follow-up assays will confirm the mechanism of action of compounds that inhibit IF3 function in the screening assay. Knowledge of their mechanism of action will facilitate the chemical optimization process and further structure-activity relationship studies to identify drug candidate compounds . 0

In a preferred embodiment, the assay uses a strain of E. coli test cell that produces wild-type IF3, and carries a plasmid encoding a reporter gene, such as chloramphenicol acetyltransferase (CAT) , which has had its start codon changed from the typical ATG to the atypical ATT. The test 5 cells grow normally on LB, but grows very poorly on LB plates containing low doses of chloramphenicol (Cam) . This is due to the low expression of the CAT gene, the translation of which initiates with the AUU codon. For example, a lawn of test cells is plated onto LB-Cam plates. Test compounds are o spotted to the plates which are incubated for an appropriate period at 37°C. Compounds which interfere with IF3 function in the test cell are expected to result in zones of increased growth against a background of relatively poor growth. This increase in growth is due to, an increased expression of the

CAT gene containing the modified start codon, which occurs 5 when IF3 function is inhibited.

In another embodiment, an in vi tro translation assay is provided to confirm the mechanism of the action of positive compounds identified in the primary assay. The in vi tro assay uses cell extract comprising IF3 which can be prepared from E. coli , or other target bacterial cells of interest. For example, the in vi tro assay uses messenger RNA

(mRNA) derived from a construct containing a reporter gene, such as the firefly luciferase gene, which has had its native

AUG start codon changed to AUU. Because this assay uses mRNA, the assay can distinguish compounds which might result in false positives by causing increases in transcription or c translation of the AUU construct by a non-IF3 mediated mechanism. Compounds which truly affect IF3 function will be expected to result in an increase in firefly luciferase expression in vi tro analogous to the increase of CAT expression seen in vivo .

The methods of the invention have a high 0 probability of identifying useful drugs for several reasons.

First, the screening system targets an essential factor that is ubiquitous and conserved throughout the bacterial kingdom.

The target is highly selective as no homologous mammalian counterpart exists. The high-throughput primary screen of 5 the invention allows for the easy visible identification of positive hits. Compounds testing positive in this whole cell growth assay have penetrated the cell permeability barrier and are functional in the intracellular environment.

0 4. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Multiple sequence alignment of IF3 amino acid sequences. Multiple sequence alignments were performed by the method of F. Corpet, 1988. Consensus amino acid symbols: ! is any one of I or V; $ is any one of L or M; % is any one of F or Y; # is any one of N, D, Q, E or B . 5

Organism abbreviations: E. coli , Escherichia coli ; S . typh, Salmonella typhimurium; K. pnew, Klebsiella pneumoniae; P. vulg, Proteus vulgar is; S . marc, Serratia marcescens ; B . aphi , Buchnera aphidicola; H. infl , Haemophilus influenzae; P. syri , Pseudomonas syringae; R . spha, 0

.ftodoJbacter sphaeroides ; M. xant, Myxococcus xanthus ;

B . stea, Bacillus stearothermophilus; B . subt, Bacillus subtilis; Synech, Synechocystis sp . ; P.purp, Porphyra

- 9 - purpurea; M. tube, Mycobacterium tuberculosis; B . burg,

Borrelia burgdorferi ; H. pylo, Heli cobacter pylori ; M. ferm,

Mycoplasma fermentans; E. grac, Euglena gracilis; M. pnew,

Mycoplasma pneumoniae; M. geni , Mycoplasma geni talium. Figure 2. Schematic diagram of pAUU-CAT.

Transcription terminators are derived from the ribosomal rrnB operon; plac indicate the lac promoter minus operator sequences; infC RBS refers to the ribosome binding site of the E. coli infC gene. CAT refers to the chloramphenicol acetyltransferase gene which has had its ATG initiator codon replaced with ATT. The parent plasmid is pSE420.

Figure 3. Primary paradoxical growth assay. DR599 containing pAUU-CAT grown in LB-Amp media to saturation was diluted 1:1000 and 1ml was plated to LB agar containing 6mg/l chloramphenicol . The plate was dried and compounds were added to the agar by an automated pipetting machine (Microlab

2200 Automated Pipetting system, Hamilton Co.). Six 96-well plates of test compounds are tested per agar plate for a total of 480 compounds. Plates were incubated at 37°C for 30 hours before scoring results.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for screening for compounds with antibiotic activity. Specifically, the invention provides assays in which bacterial translation initiation factor (IF3) serves as the drug target. The invention also encompasses compounds that inhibit IF3 activity as identified by the screening methods.

Although some of the fundamental mechanisms and components of translation have been highly conserved throughout evolution (01sen and Woese, 1997, Cell, 89:991-

994; Kyrpides and Woese, 1998, Proc . Natl. Acad. Sci.,

95:224-228), significant adaptive changes in the process have occurred independently in bacteria and eukaryotes . The

- 10 - protein factors required for initiation of translation are examples of this divergent evolution. For example, eukaryotic initiation is complex involving a larger number of proteins, many of which comprise multiple subunits (Pain 5 1996, Eur. J. Biochem., 236:747-771; Clark et al . , 1996, Biochimie, 78:1119-1122). In contrast, initiation of translation in bacteria requires only three monomeric factors, initiation factor 1 (IF1) , initiation factor 2 (IF2) , and initiation factor 3 (IF3) .

IF3 is an essential gene, acting to prevent 0 association of ribosomal subunits and recognize the correct initiation codon. Many of the genes coding for the different initiation factors have been isolated and sequenced from diverse bacterial species (see Figure 1) . The cloning and sequencing of the infC gene encoding IF3 from four 5 enterobacterial species, Salmonella typhimurium, Klebsiella pneumoniae, Serratia marcescens, and Proteus vulgaris, have been reported (1993, Liveris et al . , FEMS Microbiol . Letters, 112:211-216) . The amino acid sequence of IF3 and infC gene sequence of Bacillus stearothermophilus has also been reported 0 (1983, Kimura et al . FEBS Lett. 160:78-81; 1989, Pons et al . , Mol. Gen. Genet. 218:355-357). Apparently, IF3 is ubiquitous and conserved throughout the bacterial kingdom. Thus, it is likely that any compounds which are effective at inhibiting IF3 of one bacterial species may have an inhibitory effect on the IF3 or its functional equivalents in a wide range of bacteria. Importantly, IF3 has no functional homolog in mammalian cells, which means that the target for the compounds identified by screening assays of the invention, is absent in a subject's cells. As a result, the potential toxicity of the desired compounds is likely to be low. With 0 this in mind, the inventors of the present invention recognize that the differences between bacterial and eukaryotic initiation combined with the ubiquitous and

- 11 - conserved nature of bacterial initiation factor IF3 make it an attractive target for antibiotic development. The compounds identified by the methods of the invention are likely to be highly selective against bacteria and effective c against a broad range of such pathogens .

In general, the invention provides bacterial translation initiation factor 3 (IF3) as a specific target for screening antibiotic compounds. Any methods of detecting interactions between IF3 and a test compound for the purpose of finding an antibiotic is within the scope of the 0 invention. Test compounds that inhibit IF3 activity are likely to be useful as an antibiotic for treatment of infectious diseases caused by bacteria in animals, and sterilization of bacteria-contaminated objects and materials. In particular, the invention provides methods for 5 the identification of compounds that inhibit the activity of IF3 comprising contacting test cells with a test compound, determining the effect of the compound has on IF3 activity, and comparing the effect obtained in test cells which have not been contacted with the test compound, such that if the 0 activity of IF3 in the cells contacted with the test compound is lower from those cells not contacted, the test compound is identified as a potential drug candidate.

In one embodiment of the invention, instead of measuring directly the interaction of a test compound and IF3 in test cells, the inventors devised a strategy for detecting 5 the functional activity of IF3 efficiently which is adaptable to high-throughput screening. The strategy is based on the ability of IF3 to discriminate against atypical start codons during initiation of translation.

Initiation of translation in bacteria begins with 0 the triplet ATG (or AUG in RNA) in the vast majority of cases

(Schneider et al . , 1986, J. Mol. Biol., 188:415-431). Other atypical 3' or 5 ' wobbling codons GUG, UUG, and AUU are

- 12 occasionally found at translation start sites on messenger

RNA.

The structural gene for IF3, infC, is unusual in that its expression initiates at a rare AUU start codon. This AUU codon in infC appears to be conserved in bacteria and plays a key role in translational regulation of infC gene expression. It was observed that the cellular level of IF3 negatively regulates or represss the expression of infC at the translational level in vivo and in vi tro (Butler et al . ,

1986, J. Mol. Biol., 192:767-780; 1987, Proc . Natl . Acad.

Sci. USA, 84:4022-4025; Brombach and Pon, 1987, Mol. Gen.

Genet. 208:94-100; LaTeana et al . , 1993, Proc. Natl. Acad.

Sci. USA., 90:4161-4165). Although several models pertaining to the mechanism of IF3 -mediated start codon discrimination have been proposed, the available biochemical and genetic evidence indicate that IF3 itself discriminates against atypical start codons. This discrimination manifests itself in some bacterial mutants which are apparently deficient in

IF3 function, where translation from atypical start codons is increased 3-5 fold. Normally translation initiating from a messenger RNA containing the rare AUU start codon is approximately 2% of that seen for an identical mRNA containing an AUG start codon. The increase in expression as a result of IF3 inhibition does not occur when these codons are changed to AUG, and translation from an AUG codon is unaffected by these bacterial mutants (Sussman et al . , 1996,

Mol. Micro., 21:347-360). A reduction in wild-type IF3 levels in vivo also results in increased translation from a gene with a AUU start codon (Olsson et al . , 1996, Mol. Gen.

Genet. 250:705-714) .

While not limited to any theory on how the activity of IF3 discriminates against initiation of translation from atypical start codons, the methods of the invention are based in part on the inventors' recognition that when IF3 activity

- 13 - is low in a cell, such as when it is due to inhibition by a chemical compound, there is a corresponding increase in expression of genes initiating with atypical start codons.

This aspect has been advantageously exploited by the c inventors in the development of a primary screening assay for identifying compounds that inhibit IF3 activity.

The term "translation initiation factor 3" or "IF3" as used in this invention encompasses E. coli translation initiation factor 3 and translation initiation factor 3 or the equivalents in other bacteria, and also functional 0 equivalents of bacterial translation initiation factor 3.

Non-limiting examples of IF3 and its functional equivalents are provided in Figure 1.

The term "test cell" as used herein, refers to bacterial cells that are genetically manipulated for use in 5 the screening assays of the invention. The term also encompasses any progeny of the subject test cell.

The term "atypical start codon" as used herein, refers to start codons other than ATG (in DNA) , or AUG (in

RNA) . Atypical start codons useful in the invention using E. 0 coli as test cells include CTG (CUG) , ATC (AUC) , ATA (AUA) , and ACG, and preferably ATT or (AUU in RNA) .

The term "reporter gene" as used herein, refers to a gene sequence encoding a protein the expression of which facilitates detection and measurement of translation, and wherein the native start codon which is ATG (or AUG) has been 5 changed to an atypical start codon. The term "control reporter gene" as used herein, refers to the gene sequence encoding the reporter molecule wherein the first codon is ATG or AUG.

The term "test compound" refers to a compound to be 0 tested by one or more screening assays of the invention as a putative agent that inhibits IF3 activity. The test compounds of the invention encompass numerous classes of

14 chemical molecules, though typically they are organic molecules, and preferentially of low molecular weight.

The primary assay is based on measuring translation of a reporter gene which is initiated with an atypical start c codon in a test cell containing wild type functional IF3. The translation product of the reporter gene, the reporter molecule, can by design be easily detected and quantitated by techniques known in the art. By comparison with test cells which have not been contacted with the test compound, an increase in the amount or activity of reporter molecule 0 produced in the test cell indicates that the test compound is a potential drug candidate.

In a preferred embodiment, a test cell in the screening assay of the invention is placed under selection while it is contacted with a test compound, wherein the 5 expression of the reporter gene allows the survival of or confers a growth advantage to the test cells in the screen. The sensitivity of the assay can be adjusted by applying the appropriate level of selection such that even a slight increase in the level of reporter gene expression as a result 0 of IF3 inhibition can be detected. Many combinations of reporter genes and selection conditions are known in the art, such as but not limited to, the use of antibiotic selection and gene(s) encoding factors conferring resistance to the respective antibiotics, and the use of specialized growth media and the appropriate auxotrophic gene(s). Essentially, 5 the primary assay is a specialized whole cell growth assay which is amenable to scale-up and automation.

Other methods known in the art for detecting and measuring the activity of IF3 can also be used to screen compounds for potential drug candidate. For example, the use 0 of a direct binding assay for detecting the binding of a test compound to IF3 can be used. Some of these techniques are adopted and modified for use as secondary assays for further

15 analysis of positive compounds identified in the primary assay as discussed in later sections.

In other embodiments, the invention provides a reporter gene in which the native start codon which is ATG (or AUG in the transcript) has been changed to an atypical start codon. The invention also provides a reporter gene construct in which the reporter gene is operably linked to a promoter which facilitates transcription of the reporter gene in a test cell. The transcript (or mRNA) of the reporter gene comprises the atypical start codon which will be used for initiation of translation in the test cell. The invention also provides a reporter gene construct as described above further comprising a broad host range origin of replication such that the reporter gene construct can be transferred and stably maintained in a plurality of different bacteria.

In yet another embodiment, the invention provides a genetically manipulated test cell which contains a reporter gene wherein the native start codon of the reporter gene which is ATG (or AUG) has been changed to an atypical start codon, and wherein the reporter gene is operably linked to a promoter which facilitates transcription of the reporter gene in the test cell. The atypical start codon in the transcript

(or mRNA) of the reporter gene is used in the initiation of translation in the test cell. The test cells are used in the assays of the invention.

Test compounds that score positive in the screening assays of the invention are putative agents that inhibit IF3 activity, and, as such, may be useful as lead compounds for the development of therapeutic agents useful for the treatment of infectious diseases. Drugs based on such lead compounds are likely to be effective in controlling growth in a broad spectrum of bacteria while causing minimal side effects to the treated subject.

16 The invention further provides secondary assays which are preferably used to further characterize the test compounds which produced positive results in the primary screening assays of the invention. The invention further provides antibiotic compounds which can be used as positive controls in assays for determining the functional activity of IF3 such as the assays of the invention.

During the evaluation of positive compounds of the invention and selection of leads for further development, the two main considerations will be antibacterial activity and chemical structure. To be considered for further development, a compound should inhibit preferably the growth of both Gram positive and Gram negative bacteria. Highest priority will be given to compounds which inhibit Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis . The chemical structure should preferebly be amenable to further chemical modification, so that analogs and derivatives can be synthesized and analyzed for structure-activity relationship and other preclinical studies, such as toxicology, pharmacokinetics, and drug metabolism.

5.1. THE PRIMARY SCREENING ASSAY

The methods of the invention are designed to identify compounds that inhibit IF3 activity in test cells. IF3 is essential to bacterial growth and serves as the target of the assay. Generally, the screening assays comprise contacting a test compound with test cells for a time sufficient to allow the test compound to cause IF3 inhibition, and determining the effect of IF3 inhibition.

In one embodiment, the invention provides an assay that uses a strain of test cells which comprises a reporter gene which is expressible in the test cell, and wherein the

17 first codon of the reporter gene is an atypical start codon.

The reporter gene is operably linked to a promoter which enables the transcription of the reporter gene in the test cell resulting in the production of messenger RNA (mRNA) encoding the reporter molecule. The reporter mRNA comprises the atypical start codon which is used for initiation of translation. In the test cells, a fully functional IF3 discriminates against the translation of any mRNA which comprises an atypical start codon, including the reporter mRNA. By discrimination it is meant that, in the presence of a functional IF3 , the ternary complex comprising an atypical codon is unstable relative to ternary complex comprising an

AUG codon, and thus, overall translation is preferentially initiated at an AUG start codon. Without being bound by any theory, under normal conditions, in the test cells of the invention, the translation of the reporter mRNA comprising an atypical codon is very low relative to the translation of other mRNA comprising the AUG start codon. Although any atypical start codon may be used to make the reporter gene, the atypical start codon ATT in DNA (or AUU in RNA) is preferred.

In the presence of a test compound which inhibits

IF3 activity, the ability of IF3 to discriminate against translation initiation at atypical codons is impaired. As a result, initiation of translation at atypical start codons is enhanced and the level of reporter molecule in the test cells is detectably increased. Since a decrease of IF3 activity has little effect on the translation of mRNA comprising the typical start codon (ATG or AUG) , the increase in translation is specific to mRNA comprising an atypical codon, including the reporter mRNA, while the level of translation remains constant for the vast majority of mRNAs in the cell which comprise the AUG start codon.

18 Accordingly, the invention provides a method for screening for test compounds that inhibit IF3 activity in bacteria comprising:

(a) contacting a test cell with a test compound for a c time sufficient to allow the test compound to inhibit IF3 activity in the test cell, wherein the test cell comprises a reporter gene operably linked to a promoter which is expressible in the test cell, and wherein the reporter gene comprises an atypical start codon; and 0

(b) detecting the translation of the reporter mRNA, wherein a specific increase in the translation of the reporter gene mRNA in the test cell contacted with the test compound relative to the translation of reporter gene mRNA in a test cell not contacted 5 with the test compound, indicates that the test compound inhibits IF3 activity in the test cell.

Any method known in the art for detecting or measuring translation of a species of mRNA are applicable in this invention. Since translation of the reporter mRNA leads to o biosynthesis of the reporter molecule in the test cell, the method of the invention encompasses the detection and measurement of the amount of reporter molecule, or the detection and measurement of the signal generated by the reporter molecule.

Accordingly, the invention provides a method for 5 screening for test compounds that inhibit IF3 in bacteria comprising:

(a) contacting a test cell with a test compound for a time sufficient to allow the test compound to inhibit IF3 activity in the test cell, wherein the 0 test cell contains a reporter gene operably linked to a promoter which is expressible in the test

19 cell, and wherein the reporter gene comprises an atypical start codon; and (b) detecting the amount of reporter molecule, wherein a specific increase in the amount of reporter molecule in the test cell contacted with the test compound relative to the amount of reporter molecule in a test cell not contacted with the test compound, indicates that the test compound inhibits IF3 activity in the test cell.

The invention further provides a method for screening for test compounds that cause IF3 inhibition in bacteria comprising:

(a) contacting a test cell with a test compound for a time sufficient to allow the test compound to inhibit IF3 activity in the test cell, wherein the test cell contains a reporter gene operably linked to a promoter which is expressible in the test cell, and wherein the reporter gene comprises an atypical start codon; and

(b) detecting the signal generated by the reporter molecule, wherein a specific increase in the signal in the test cell contacted with the test compound relative to the signal generated by the reporter molecule in a test cell not contacted with the test compound, indicates that the test compound inhibits

IF3 activity in the test cell.

Since the increase in production of the reporter molecule as a result of IF3 inhibition is usually low, (i.e., less than five to ten fold) , the primary assay is designed to be sensitive enough to detect even small changes in the level of reporter molecule. In a preferred embodiment, the invention provides a very sensitive primary assay based on cell growth, in which the reporter gene in a test cell acts as a selection marker which is necessary for the survival of

20 - the test cell when the test cell is grown under the appropriate selection condition.

For example, in one type of primary cell growth assay, due to the discrimination by IF3 against mRNA comprising an atypical start codon, the test cell produces a small amount of reporter molecule, but this low level is inadequate to overcome the selection imposed on the test cell. However, in the presence of a test compound which inhibits IF3, translation initiation at atypical codons is enhanced, and the expression of reporter gene is increased to a level which allows the test cell to survive the selection and grow in the assay. Thus, the paradoxical growth of test cells indicates a rise in level of the reporter molecule in the test cells, and identifies the test compound as a candidate for further studies. Selection conditions that may be used include but are not limited to incorporation of antibiotics in the growth media. Examples of such media, not by way of limitation, include medium containing chloramphenicol, or other antibiotics to which a cloned gene or gene(s) encoding resistance to the antibiotics is available for use as the reporter gene(s) . The sensitivity of the assay can be adjusted by manipulating the strength of the selection condition. The applicable range of antibiotic concentration provides just enough selection to suppress growth of the test cell but insufficient selection to suppress growth of the test cells in which the production of the reporter gene product is enhanced. The desired antibiotic concentration can be determined empirically by titrating the growth of the test cell against dilutions of the antibiotic under various growth conditions. Alternatively, a range of concentration of antibiotics, such as a concentration gradient, may be used in the assay.

21 Accordingly, the invention provides a method for screening for test compounds that cause IF3 inhibition in bacteria comprising:

(a) culturing a test cell under a selection condition such as those mentioned above;

(b) contacting the test cell with a test compound for a time sufficient to allow the test compound to inhibit IF3 activity in the test cell, wherein the test cell contains a reporter gene operably linked to a promoter which is expressible in the test cell to overcome the selection, and wherein the reporter gene comprises an atypical start codon; and

(c) detecting growth of the test cell under the selection condition, wherein a specific increase in growth of the test cell contacted with the test compound relative to the growth of the test cell not contacted with the test compound, indicates that the test compound causes IF3 inhibition in the test cell.

For clarity of discussion, the invention is described in the subsections below by way of example of

E. coli . However, the principles may be applied to other bacteria which contain a protein that are functionally equivalent to IF3.

In a preferred embodiment, the assay uses an E. coli strain containing an antibiotic resistance gene as the reporter gene. The assay strain is plated as a lawn on solid medium containing antibiotics at an appropriate concentration. The contacting of test compounds may be effected in any vehicle and by any means using standard protocols, such as serial dilution, and the use of wells, or disks impregnated with a solution or suspension of a test compound. The amount of time allowed for the test compound to cause IF3 inhibition in the test cells may be determined

22 empirically, such as by running a time course and monitoring the effect of IF3 inhibition as a function of time. For example, the test cells may be grown in culture to reach a certain phase or density, and then the test cells are exposed c to a test compound; or alternatively, the test cells may be grown continuously in the presence of a test compound. Paradoxical growth is determined comparing growth around the well or disk containing the test compound to growth in control areas which are free of the test compound, either visually or by using an image analysis device. Comparisons 0 of test and control areas are done at the same time point.

Compounds which interfere with IF3 function are expected to result in zones of increased growth against a background of light lawn growth. As IF3 appears to be an essential factor, some compounds which completely abolish IF3 function will 5 likely result in a zone of killing at the point of application surrounded by a zone of increased growth due to diffusion of the test compound, thus creating a concentration gradient in the media. The invention provides that compounds which apparently intefere with translation, 0 specifically lincomycin, compound 1 and compound 2, can be included in the assay as a positive control. The invention further provides that lincomycin, compound 1 and compound 2 can be used as a positive control in any functional assay that uses IF3 as the target.

The preferred assay strain normally produces a very 5 low level of CAT which is insufficient to permit growth of the test cells at the concentration of chloramphenicol present in the growth media, and thus the test cells are effectively chloramphenicol sensitive (CamR . However, in the presence of a compound which inhibits IF3 and enhances 0 translation of the CAT gene, the enzyme accumulates in the test cells which become effectively chloramphenicol resistant

(CamR . Cells exposed to such a compound can be identified

23 by its growth at a level of chloramphenicol that would normally not permit growth. The appropriate level of chloramphenicol used depends in part on the strain of test cell used and the promoter that drive transcription of the CAT -. gene, and can be determined empirically. A useful range of chloramphenicol concentration for E. coli test cells is about 2 mg/1 to 40 mg/1. The preferred concentration is about 6 mg/1.

5.2. REPORTER CONSTRUCTS AND TEST CELLS OF THE INVENTION

10

The reporter constructs and test cells of the invention are employed in screening assays for the identification of compounds that inhibit IF3 activity.

In one embodiment, the invention uses a strain of test bacterial cells that comprises a reporter gene, wherein

15 the first codon of the reporter gene is an atypical start codon. To make a test strain, the native ATG start codon of a reporter gene may be mutagenized by genetic methods or recombinant DNA techniques well known in the art.

In a more specific embodiment, the test cell

_n comprises a reporter construct comprising a reporter gene which is operably linked to a promoter that facilitates transcription of the reporter gene in the test cell, and wherein the native ATG start codon of the reporter gene has been changed to an atypical start codon for initiation of translation. Although any atypical start codon may be used to

25 make the reporter gene, the ATT start codon is preferred.

Thus, the reporter gene in the reporter gene construct is expressible in the test cell.

In various embodiments, the reporter gene sequence of the invention can include any genetic sequence which 30 encodes a detectable reporter molecule and preferably, a molecule that confers a selective characteristic. The genetic

- 24 sequences encoding such reporters are well known to those of skill in the art.

Bacterial cells may be obtained from private laboratory deposits, public culture collections of microorganisms such as the American Type Culture Collection, or from commercial suppliers. It is desirable to use bacteria which have been developed for drug screening processes, and that conditions for their growth, maintenance, and manipulations are known. The design of the reporter system can, in many instances, be easily adapted for different strains of bacterial test cells.

The most preferred bacterial species that is useful as test cells is Escherichia coli . Other preferred bacterial species may include but not limited to Bacillus subtilis, Pseudomonas aeuroginosa, Staphylococcus aureus, Enterococcus faecalis, and Mycobacterium tuberculosis . Escherichia coli is useful as a model for many Gram negative bacteria. Bacillus subtilis is useful as a model for many Gram positive bacteria.

Test compounds that inhibit IF3 activity in E. coli or Bacillus subtilis test cells are expected to have a similar inhibitory effect on the translation of many pathogenic species. It is also expected that positive compounds will be effective as an antibiotic against the multidrug-resistant strains, such as β-lactam-resistant strains of E. coli .

In the present invention, the reporter gene sequence (s) may be inserted into a recombinant expression vector. The term "reporter gene constructs" refers to a plasmid, virus, phage, or other vehicle known in the art that has been manipulated by insertion or incorporation of the reporter gene sequences. Such reporter gene constructs of the invention can either be maintained episomally in the cell, or be incorporated into the genome of the test cell. The reporter gene construct comprises a promoter that is operably associated with the reporter gene sequence (s), and that

25 facilitates transcription of the reporter gene sequences in the test cell. "Operably-associated" or "operably-linked" refers to an association in which the promoter and the reporter gene sequence (s) are joined and positioned in such a ς way as to permit transcription.

The reporter gene construct may comprise other transcriptional control signals, and other useful regulatory sequences, such as terminators (e.g., terminator derived from the E. coli rrnBl operon) and functional elements, such as broad host range origin of replication. The construct may 0 also contain an origin of replication as well as specific genes which allow phenotypic selection of the transformed cells. Thus, in one embodiment, a reporter gene construct of the invention comprises starting at the 5' end, transcriptional terminators, a promoter, a ribosome binding 5 site, and the coding sequences with the first codon being an atypical start codon.

Standard molecular biology techniques can be used to construct the reporter gene sequence that comprises an atypical start codon. For example, well known techniques of site-directed mutagenesis can be applied to modifiy the last nucleotide of the typical start codon (ATG) to the atypical start codon, ATT. Any sequences comprising a ribosome binding site (the Shine-Dalgarno sequences) can be used in the reporter gene sequence, but it is preferable to use the ribosome binding site of the E. coli infC gene. Optionally, 5 the sequence located between the ribosome binding site and the atypical start codon of the E. coli infC gene may also be included. A control reporter gene construct without the atypical start codon can also be made by the same techniques. Introduction of the reporter gene construct into 0 bacterial cell DNA may be carried out by conventional techniques well known to those skilled in the art, such as transformation, conjugation, and transduction. For example,

26 where the host is E. coli , competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth and subsequently treated by the CaCl2 method using procedures well known in the art. Alternatively, MgCl2 c or RbCl could be used.

In addition to conventional chemical methods of transformation, the plasmid vectors of the invention may be introduced into a host cell by physical means, such as by electroporation or microinjection. Electroporation allows transfer of the vector by high voltage electric impulse, which creates pores in the plasma membrane of the host and is performed according to methods well known in the art. Additionally, cloned DNA can be introduced into host cells by protoplast fusion, using methods well known in the art.

The test cells may be cultured under standard 5 conditions of temperature, incubation time, optical density, plating density and media composition corresponding to the nutritional and physiological requirements of the bacteria. However, conditions for maintenance and growth of the test cell may be different from those for assaying candidate test compounds in the screening methods of the invention. Modified culture conditions and media are used to facilitate detection of the expression of a reporter molecule. Any techniques known in the art may be applied to establish the optimal conditions .

Test cell strains generated by the above-described methods for the screening assays may be expanded, stored and retrieved by any technique known in the art that is appropriate to the test cell. For example, the test cells of the invention can be preserved by stab culture, plate culture, or in glycerol suspensions and cryopreserved in a freezer (at 0 -20°C to -100°C) or under liquid nitrogen (-176°C to -196°C) .

A reporter gene is used in the invention to monitor and report IF3 activity in test cells. Preferably, the

27 reporter gene sequence encodes a protein that is readily detectable either by its presence, or by its activity that results in the generation of a detectable signal.

A reporter gene encodes a reporter molecule which is capable of directly or indirectly generating a detectable signal. Generally, although not necessarily, the reporter gene encodes RNA and detectable protein that are not otherwise produced by the test cells. Many reporter genes have been described, and some are commercially available for the study of gene regulation. See, for example, Alam and Cook, 1990,

Anal. Biochem., 188:245-254, the disclosure of which is incorporated herein by reference.

One class of reporter genes that can be used in the methods of the invention are antibiotic resistance genes, such as, but not limited to, the chloramphenicol acetyltransferase gene (CAT; Gorman et al . , 1982, Mol Cell Biol, 2:1044; Prost et al . , 1986, Gene, 45:107-111), the β-lactamase gene, and genes encoding tetracycline resistance or kanamycin resistance .

Another class of reporter genes that can be used in the methods of the invention are auxotrophic genes that allows the test cell to grow under specialized growth media.

For convenience, enzymatic molecules and light -emitting reporter molecules are preferred for the screening assays of the invention, especially in the in vi tro translation assays.

Accordingly, the invention also encompasses colorimetric and fluorometric assays.

A variety of enzymes may be used as a reporter which includes but are not limited to β-galactosidase (Nolan et al . 1988, Proc. Natl. Acad. Sci. USA, 85:2603-2607), chloramphenicol acetyltransferase, β-lactamase, β- glucuronidase and alkaline phosphatase (Cullen et al . , 1992, Methods Enzymol, 216:362-368). A variety of bioluminescent , chemiluminescent and fluorescent proteins and other molecules

- 28 can also be used as light-emitting reporters in the invention. One type of such reporter, which are enzymes and require cofactor (s) to emit light, include but are not limited to, the bacterial luciferase (luxAB gene product) of Vibrio harveyi (Karp, 1989, Biochim. Biophys . Acta, 1007:84-90; Stewart et al . 1992, J Gen Microbiol, 138:1289-1300), and the luciferase from firefly, Photinus pyralis (De Wet et al . , 1987, Mol. Cell. Biol., 7 -. 125 - 131) . Another type of light-emitting reporter, which does not require substrates or cofactors and are particularly suited for use in whole cells, includes but are not limited to the wild type green fluorescent protein

(GFP) of Victoria aequoria (Chalfie et al . , 1994, Science, 263:802-805), and modified GFPs (Heim et al . , 1995, Nature, 373:663-4; PCT publication WO 96/23810). Transcription and translation of this type of reporter gene leads to the accumulation of the fluorescent protein in test cells, which can be measured by a device, such as a fluorimeter, or a flow cytometer. Methods for performing assays on fluorescent materials are well known in the art and are described in, e.g., Lackowicz, J.R., 1983, Principles of Fluorescence Spectroscopy, New York: Plenum Press.

5.3. SECONDARY ASSAYS

In another embodiment, the invention provides secondary assays that are preferably used to further characterize the positive compounds identified in the primary assays. In addition, with modifications to improve screening throughput, the secondary assays can also be used to screen for test compounds of interest . The secondary assays described in the following sections can be used in combination with each other and in any order to elucidate the precise mode of action of a test compound on the function of IF3. The atypical start codon ATT (or AUU) is used herein as an

29 example. Many other atypical codons can also be used interchangeably with ATT or AUU in these assays.

5.3.1. IN VITRO TRANSLATION In one embodiment of the invention, an in vi tro translation assay is used to confirm the inhibitory activity of a test compound detected in the primary whole cell growth assay. The rationale of the assay is that purified IF3 inhibits translation in vi tro of mRNA comprising a AUU start codon (AUU-mRNA) but not mRNA comprising the typical AUG start codon (AUG-mRNA) . Therefore, by comparing the level of translation of a mRNA comprising an atypical codon and of the same mRNA except an AUG start codon in the presence of a test compound and IF3, the ability of the test compound to inhibit

IF3 can be confirmed. The in vi tro translation assay is carried out using cell extract comprising active wild-type IF3 which can be prepared from bacterial cells of interest. Methods for preparing such extracts for use in in vi tro translation assays are well known in the art.

For convenience, it is preferable that the mRNA encodes a reporter gene that can be readily detected and measured in vi tro, such as but not limited to enzymatic reporters and light-emitting reporters (e.g., firefly luciferase) . The ATG start codon of a reporter gene sequence can be modified to the ATT start codon by any site-directed mutagenesis methods known in the art. Any methods can be used to generate the reporter gene mRNA (i.e., the AUU-mRNA and

AUG-mRNA) , such as inserting the respective reporter genes downstream of a T3 or T7 promoter in a construct , and carrying out in vi tro transcription by use of T3 or T7 RNA polymerase .

By way of an example, the in vi tro translation assay USe in vi tro transcribed mRNA derived from a construct (pAUU-

FL) containing a DNA sequence encoding firefly luciferase (FL) which has had its ATG start codon changed to the ATT-

- 30 containing wild-type infC initiation sequences. The reporter mRNA may also comprise the ribosome binding site of E. coli infC gene. An identical construct containing the ATG rather than ATT start codon is used as a control (pAUG-FL) . These

5 mRNAs are used to prime an in vi tro E. coli translation reaction, optionally supplemented with excess E. coli IF3 and/or other components, in the presence of serial dilutions of test compounds. Protocols outlining the purification procedures for IFl, IF2 and IF3 have been published (Mortensen et al., 1991, Biochimie, 73:983-989; Calogero et al . , 1987, 0

Mol. Gen. Genet., 208:63-69; Lestienne et al . , 1982, Eur. J.

Biochem., 123:483-488; Soffientini et al . , 1994, Prot . Exp . and Purif., 5:118-124) . Levels of FL are quantified using the appropriate substrate and a luminometer. The FL in the pAUU-

FL construct is active, although as expected levels of 5 translation in vi tro are low due to the presence of an AUU initiator codon. If a test compound affects IF3 function, then an increase in translation of pAUU-FL over that seen when no compound is present is expected, which is analogous to the increase in pAUU-CAT expression seen in the primary growth o assay. The AUG-FL control should not be affected. This assay can distinguish desirable compounds from compounds that cause increased transcription of the AUU-CAT gene rather than an initiation specific effect in the primary assay.

Accodingly, the invention provides an in vi tro 5 method for screening for test compounds that inhibit the activity of IF3 comprising:

(a) incubating in a reaction comprising a reporter gene mRNA comprising an atypical start codon, a cell extract comprising IF3, and a test compound for a 0 time sufficient to allow the test compound to inhibit IF3 activity in the cell extract; and

31 (b) detecting the translation of the reporter gene mRNA in the reaction, wherein a specific increase in the translation of the reporter gene mRNA in the reaction containing the test compound relative to the translation of the reporter gene mRNA in a reaction not containing the test compound, indicates that the test compound inhibits IF3 in the reaction. Excess IF3 may be added to the cell extract in the reaction. Since the potential interactions between different initiation factors are not clearly understood at present, it is possible that a test compound may interact not only with IF3 but also with IFl or IF2. In addition to the different IFs, a test compound may interfere with IF3 function by binding the 3OS subunit and thus preventing the binding of IF3 to its functional site. These possibilities can be further elucidated by the secondary assays described below.

5.3.2. STABILITY OF INITIATION COMPLEX

As discussed above, IF3 is believed to act primarily at controlling translation ternary complex stability. In the absence of IF3, fmet-tRNA is bound stably to 30S initiation complexes comprising AUU-mRNA or AUG-mRNA. However, when formed in the presence of IF3, the 30S initiation complexes containing AUU-mRNA are very unstable. Stability of 30S initiation complexes formed with or without IF3 can be determined by measuring the loss of f [3H] Met-tRNAfmet in the presence or absence of IF3. Test compounds which interfere with IF3 activity would be expected to increase stability of fmet-tRNA bound to AUU-mRNA containing initiation complexes. Furthermore, this assay could be used to distinguish whether a drug is acting primarily on IF3 or the 3OS subunit by using fixed concentrations of the drug and titrating with initiation complexes having variable amounts of 30S or IF3.

32 5.3.3. BASE-SPECIFIC CHEMICAL PROBING OF 30S SUBUNITS

Many antibiotics protect specific nucleotides in 16S rRNA from chemical probes when they bind to ribosomes, each producing a characteristic footprint on the rRNA (Moazed and Noller, 1987, Nature, 327:389-394). Additionally, the footprints of all of the 30S ribosomal proteins on 16S rRNA have been determined using various chemical probes (Stern et al., 1989, Science, 244:783-790; Powers and Noller, 1995, RNA, 1:194-209). IF3 also protects specific nucleotides in the 30S subunit . These protected sites overlap with those protected by 50S subunits and P-site-bound tRNA consistent with a role in subunit dissociation and initiator tRNA selection (Moazed et al . , 1995, J. Mol. Biol., 248:207-210). Compounds testing positive in the primary assay can be further evaluated for their ability to footprint 16S rRNA and alter ribosomal protein or IF3 footprints. Such footprinting experiments can yield a more detailed picture of a compound's mechanism of action.

Accordingly, the invention provides a method for screening for test compounds that inhibit the activity of bacterial translation initiation factor 3 (IF3) comprising:

(a) incubating in a reaction comprising bacterial 16S ribosomal RNA, IF3, and a test compound for a time sufficient to allow the test compound to interfere with the binding of IF3 to 16S ribosomal RNA; and

(b) determining the binding characteristics of IF3 to the bacterial 16S ribosomal RNA, wherein a change in the binding characteristics of IF3 to 16S ribosomal RNA in the presence of the test compound relative to the binding characteristics of IF3 to 16S ribosomal RNA in the absence of the test compound indicates that the test compound inhibits the activity of IF3.

The binding characteristics of IF3 to 16S ribosomal RNA may be determined by exposing the reaction to various chemical probes

33 using techniques well knwon in the art. The number and position of nucleotides in the 16S ribosomal RNA which are or are not protected (i.e., the footprint) is representative of the binding characteristics of IF3 to 16S ribosomal RNA. c Without being bound by any theory, IF3 may bind and interact indirectly with 16S ribosomal RNA.

5.3.4. TOE-PRINTING ASSAY

The ability of initiation factors to select initiator tRNAs in response to codon-anticodon interactions 0 can be ascertained in a toe-print assay (Hartz et al . , 1989,

Genes Dev., 3:1899-1912). In the absence of IF3, the 30S subunit toe-prints equally well at positions corresponding to initiator fmet-tRNA binding at AUG and to elongation tRNA binding at its cognate codon. However, in the presence of 5 IF3, the initiator fmet-tRNA -AUG interaction is preferentially selected over the elongator tRNA and cognate codon. It appears that IF3 selects against all codon- anticodon interactions other than initiator fmet-tRNA -AUG, - GUG and -UUG in the toe-printing assay. Interestingly, IF3 mutants have been shown to be defective in this toe-print assay even though they appear to bind 3OS subunits normally (Sussman et al . , 1996, Mol. Micro., 21:347-360). Importantly, one of the point mutations in IF3 that decreases start codon discrimination in vivo (Sussman et al . , 1996, Mol. Micro.,

21:347-360) has been examined in the toe-print assay, and 5 found to be defective in start codon selection. Accordingly, it is expected that test compounds which perturb this aspect of IF3 function should have a similar effect in this assay. Compounds testing positive in the assays of the invention can be evaluated for their ability to interfere with IF3 mediated 0 codon-anticodon interactions through the use of this toe-print assay.

- 34 5.3.5. DIRECT BINDING ASSAYS

Direct binding of a test compound to IF3 may also be tested in the screening assays of the invention. Purified IF3 is preferred for these assays. IF3 protein may be enriched and 5 purified by a variety of methods known in the art. In one embodiment, IF3 may be purified from bacterial cell extracts, for example, by antibody affinity purification. In another embodiment, IF3 may be produced by recombinant means (Muralikrishna et al . , 1989, Gene 80:369-374). For example, in vi tro translation methods or induction of expression from a

10 bacterial expression vector containing the IF3 open reading frame may be used. In both cases the IF3 AUU start codon is replaced with an AUG start codon, so that translation of the IF3 mRNA is not inhibited by the accumulating IF3 protein. The IF3 protein may be tagged with a peptide sequence that

15 facilitates its purification and its attachment to a solid phase, for example glutathione-S-transferase (GST) or poly- histidine (p-His) .

In a direct binding assay, the IF3 protein is contacted with a test compound under conditions that allow

_n binding of the test compound. The binding may take place in solution or on a solid surface. Preferably, the test compound is previously labeled for detection. Any detectable compound may be used for labeling, such as but not limited to, a luminescent, fluorescent, or radioactive isotope or group containing same, or a nonisotopic label, such as an enzyme or

25 dye. After a period of incubation sufficient for binding to take place, the reaction is exposed to conditions and manipulations that remove excess or non-specifically bound test compound. Typically, it involves washings with an appropriate buffer, Finally, the presence of an IF3-test 30 compound complex is detected. An example of each is presented below, in sections 5.3.5.1 and 5.3.5.2 respectively. IFl

- 35 and/or IF2 may be tested in parallel as negative controls to determine the specificity of test compound binding to IF3.

5.3.5.1 SCINTILLATION PROXIMITY ASSAY c In one embodiment of the invention, direct binding may be performed using a scintillation proximity assay (described in U.S. patent no. 4,568,649). Purified IF3 is coated onto the surface of a scintillant-loaded solid phase (e.g., beads) and the solid phase are treated with a blocking agent such as albumin or serum. Radiolabeled test compounds (e.g., 33P-labelled) are then mixed with the IF-3 coated beads, under conditions that would allow specific binding of a candidate specific binding test compound to the IF-3 on the solid phase. After washings to remove excess or non-specific binding, if specific binding of a labelled test compound and 5 IF3 took place, the radiolabel is brought into close proximity to the scintillant, allowing the scintillant to emit light, which is detectable with a scintillation counter. In an alternative embodiment, an affinity capture scintillation proximity assay may be used so that binding may be performed in solution. In this assay, IF-3 is purified and labeled with an affinity label, such as biotin. Biotinylated IF-3 is then mixed with the radiolabelled test compound, under conditions that allow solution binding to occur. Biotinylated IF-3 including complexes of IF-3 and test compound are captured on streptavidin-coated scintillant-loaded beads (available from Amersham) and counted in a scintillation counter, as described above.

5.3.5.2 BINDING ON A SOLID SURFACE

In another embodiment, an affinity binding assay may 0 be performed using a purified IF3 which is immobilized to a solid support. In various embodiments, the solid support could be, but is not restricted to, polycarbonate,

36 polystyrene, polypropylene, polyethlene, glass, nitrocellulose, dextran, nylon, polyacrylamide and agarose . The support configuration can include beads, membranes, microparticles, the interior surface of a reaction vessel such c as a microtiter plate, test tube or other reaction vessel. The immobilization of IF3 can be achieved through covalent or non- covalent attachments. In one embodiment, the attachment may be indirect, i.e., through an attached antibody. In another embodiment, IF3 and negative controls are tagged with an epitope, such as glutatione S-transferase (GST) so that the attachment to the solid surface can be mediated by a commercially available antibody such as anti -GST (Santa Cruz Biotechnology) .

The test compound is labeled, to enable detection. A variety of labeling methods are available and may be used, 5 such as luminescent, chromophore, fluorescent, or radioactive isotope or group containing same, and nonisotopic labels, such as enzymes or dyes. In a preferred embodiment, the test compound is labeled with a fluorophore such as fluorescein isothiocyanate (FITC, available from Sigma Chemicals, St. 0 °Uis) *

The labeled test compound is then added to the surface and allowed to bind. After the binding reaction has taken place, unbound and non-specifically bound test compound is removed by means of washing the surface, and the remaining label may be detected by any detection method known in the 5 art. For example, if the test compound is labeled with a fluorophore, a fluorimeter may be used to detect complexes.

5.4. DETERMINATION OF MIC

The minimum inhibitory concentration (MIC) against 0 bacterial organisms is determined for each compound that is positive in both the primary assays (as described in Section 5.1) and secondary assays (as described in Section 5.3) .

37 Methods known in the art may be used such as broth microdilution testing, using a range of concentrations of each test compound (1993, National Committee for Clinical Laboratory Standards, Methods for Dilution Antimicrobial _ Susceptibility Tests For Bacteria That Grow Aerobically -

Third Edition: Approved Standard, M7-A3, which is incorporated by reference herein in its entirety) . The MIC against a variety of pathogens are determined using the same method. Pathogenic species to be tested generally include, but are not limited to: E. coli , Enterococcus faecium, Enterococcus 0 faecalis, Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus epidermis , Shigella flexneri , and Salmonella typhimurium.

5.5. CYTOTOXICITY TESTING 5 Unfortunately, toxicity does not always arise from the same mechanism of action as is responsible for growth inhibition in the targeted microorganism. Therefore, the selectivity of the target should not be assessed solely on the basis of these results. 0 Cytotoxity can be measured by methods known in the art. One such method is based on assessing growth of mammalian cells in the presence of the test compound. The assay measures the metabolic reduction by viable cells, of colorless XTT terazolium to yield orange XTT formazan, which is measurable by conventional colorimetric techniques (Weislow 5 et al. 1989, J. Natl. Cancer Inst . , 81:577-586; which is incorporated by reference in its entirety) .

5.6. ANTIBIOTIC AGENTS IDENTIFIED BY METHODS OF THE INVENTION 0 In yet another embodiment, the invention provides novel antibiotic agents discovered by the methods described above. These antibiotic agents are capable of causing IF3

38 inhibition in a bacterial cell, leading to a reduction or inhibition of bacterial growth. These agents are expected to be effective against a variety of species of bacteria, including infectious pathogenic bacteria. The invention also -. includes novel pharmaceutical compositions which comprise antibiotic agents discovered as described above formulated in pharmaceutically acceptable formulations.

In another embodiment, the invention features a method for treating a subject infected with an infectious agent by administering to that subject a therapeutically 0 effective amount of an antibiotic agent which causes IF3 inhibition in the infectious agent as determined by the assays of the invention. Such administration can be by any method known to those skilled in the art, for example, by topical application or by systemic administration. 5 In yet another embodiment, antibiotic agents of the present invention can be used to treat contaminated items, such as crops, wood, metal or plastic and the like, by methods such as, but not limited to, spraying or dusting of that agent onto the contaminated item, or impregnating that agent into the item. 0

By "therapeutically effective amount" is meant an amount that relieves (to some extent) one or more symptoms of the disease or condition in the subject. Additionally, by "therapeutically effective amount" is meant an amount that returns to normal, either partially or completely, physiological or biochemical parameters associated with or causative of a bacterial disease or condition.

5.6.1. FORMULATION

The antibiotic compounds identified by methods of 0 the invention may be formulated into pharmaceutical preparations for administration to subjects for treatment of a variety of infectious diseases. Thus, the compounds and their

39 physiologically acceptable solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, topical, dermal, vaginal, rectal administration and drug delivery device, e . g. , porous or viscous material, such as lipofoam.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner .

The compounds may be formulated for parenteral administration by injection, e . g. , by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e . g. , in ampules or in multi- dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e . g. , sterile pyrogen- free water, before use.

The pharmaceutical compositions of the present invention comprise an antibiotic compound as the active ingredient, or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier, and optionally, other therapeutic ingredients, for example antivirals. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non- toxic acids and bases, including inorganic and organic acids and bases .

5.6.2. ADMINISTRATION

For administration to subjects, antibiotic compounds discovered by using the assays of the invention are formulated

40 in pharmaceutically acceptable compositions. The compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents.

These compositions can be utilized in vivo, ordinarily in a

5 mammal, preferably in a human, or in vi tro . In employing them in vivo, the compositions can be administered to the mammal in a variety of ways, including parenterally, intravenously, subcutaneously, intramuscularly, colonially, rectally, vaginally, nasally, orally, transdermally, topically, ocularly, or intraperitoneally. 0

As will be readily apparent to one skilled in the art, the magnitude of a therapeutic dose of an antibiotic compound in the acute or chronic management of an infectious disease will vary with the severity of the condition to be treated, the particular composition employed, and the route of 5 administration. The dose, and perhaps dose frequency, will also vary according to the species of the animal, the age, body weight, condition and response of the individual subject. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, will be _ within the ambit of one skilled in the art.

Optionally, a second antibacterial compound may be used in combination with the compound identified by the method of the invention. The second antibacterial compound may be naturally occurring or synthetic. Suitable naturally occuring antibacterial compounds include, but are not limited to, 5 aminoglycosides (including but not limited to dihydrostreptomycin, gentamycin, kanamycin, neomycin, paromycin and streptomycin) ; amphenicols (including but not limited to chloramphenicol) ; ansamycins (including but not limited to rifamycin) ; β-lactams such as carbapems (including 0 but not limited to imipenem) , cephalosporins (including but not limited to cefazedone and cefroxadine) , cephamycins (including but not limited to cefbuperazone) ; monobactams

41 (including but not limited to aztreonam) , oxacephems (including but not limited to flomoxef) or penicillins (including but not limited to ampicillin, carbencillin, methicillin, penicillin N, penicillin 0 and penicillin V) ; c lincosamides (including but not limited to clindamycin and lincomycin) ; macrolides (including but not limited to carbomycin and erythromycin) ; polypeptides (including but not limited to gramicidin S and vancomycin) ; tetracyclines (including but not limited to apicycline, methacycline and tetracycline) ; and others such as cycloserine, mupirocin and 0 tuberin. Suitable synthetic antibacterial compounds include

2, 4-diaminopyrimidines (including but not limited to trimethoprim) ; nitrofurans (including but not limited to nifuradene) ; quinolones and quinolone analogs (including but not limited to enoxacin, lo efloxacin, nalidixic acid and 5 ofloxacin) ; sulfonamides (including but not limited to sulfamoxole and sulfanilamide) ; sulfones (including but not limited to diathymosulfone) ; oxazolidinones (including but not limited to linezolid) ; and others such as glycylcycines, clofoctol, hexedine, methenamine, and nitroxoline.

The "adjunct administration" of a compound identified by the method of the invention and a second antibacterial compound means that the two are administered either as a mixture or sequentially. When administered sequentially, the compound may be administered before or after the second antibacterial compound, so long as the initially 5 administered compound is still providing antibacterial activity. Any of the above described modes of administration can be used in combination to deliver the compound and the second antibacterial compound. When a compound identified by the method of the invention and a second antibacterial 0 compound are administered adjunctively as a mixture, they are preferably given in the form of a pharmaceutical composition comprising both agents. Thus, in a further embodiment of the

- 42 invention, it is provided a pharmaceutical composition comprising a compound identified by the method of the invention and a second antibacterial compound together with a pharmaceutically acceptable carrier.

5

5.6.3. TARGET INFECTIOUS AGENTS

The antibiotic compounds identified by the methods of the infection can be used to treat infectious diseases in animals, including humans, companion animals (e.g., dogs and cats), livestock animals (e.g., sheep, cattle, goats, pigs, 0 and horses), laboratory animals (e.g., mice, rats, and rabbits), and captive or wild animals.

Specifically, infectious diseases caused by bacteria including but not limited to, gram positive cocci, such as Staphylococci (e.g., S . aureus) , Streptococci (e.g., S . 5 pneumoniae, S. pyrogens, S . faecalis, S . viridans) ; gram positive bacilli, such as Bacillus (e.g., B . anthracis) , Corynebacterium (e.g., C. diphtheriae) , Listeria (e.g., L . monocytogenes) ; gram negative cocci, such as Neisseria (e.g., N. gonorrhoeae, N. Meningi tidis) ; gram negative 0 bacilli, such as Haemophilus (e.g. H. influenzae) , Pasteurella (e.g., P. mul tocida) , Proteus (e.g., P. mirabilis) , Salmonella (e.g., S . typhi murium) , Shigella species, Escherichia (e.g., E. coli) , Klebsiella (e.g., K. pneumoniae) , Serratia (e.g. S . marcescens) , Yersinia (e.g., Y. pestis) , Providencia ς species, Enterobacter species, Bacteroides (e.g., fragilis) , Acinetobacter species, Campylobacter (e.g., C. jejuni ) , Pseudomonas (e.g., P. aeruginosa) , Bordetella (e.g., B . pertussis) , Brucella species, Fracisella (e.g., F. tularensis) , Clostridia (e.g., C. perfriugens) , Helicobacter

(e.g., H. pylori ) , Vibrio (e.g., V. cholerae) , Mycoplasma 0

(e.g., M. pneumoniae) , Legionella (e.g., L. pneumophila) ,

Spirochetes (e.g. Treponema, Leptospira and Borrelia) , Mycobacteria (e.g., M. tuberculosis) , Νocardia (e.g., N.

- 43 - asteroides) , Chlamydia (e.g., C. trachomatis) , and Rickettsia species, can be treated by antibiotic drugs discovered by the methods of the invention.

5 6. EXAMPLES

The invention may be better understood by the following description of illustrative examples which are not intended to be limiting.

6.1. REPORTER CONSTRUCT 10

An Ampr high copy plasmid, pAUU-CAT, was constructed by replacing the wild-type ATG start codon of the CAT gene with the atypical codon ATT. See Figure 2. The previous use of CAT reporter constructs in the study of bacterial translation has been reported (Lee et al , 1997, J. Mol. Biol.,

15 269:732-743; Poot et al , 1996, Nuc . Acids Res., 24:3670-3676; Murgola et al , 1990, The Ribosome: Structure, Function, and Evolution (Hill et al . , eds) Washington DC: Am. Soc . Microbiol. pp.402-407). The CAT gene was amplified from pACYC184 by polynerase chain reactor. The 5' oligonucleotide

20 was designed such that the ATG codon was deleted and replaced with a PstI restriction site in the final PCR product. A DNA oligonucleotide cassette was then designed which incorporated the wild-type infC ribosome binding site (RBS) , known to be the site of regulation by IF3. This cassette was ligated in frame to the PCR amplified CAT gene using PstI. The ATT

25 region was then ligated to another oligonucleotide cassette containing the lac promoter minus its operator sequences. The omission of the unnecessary operator sequence both simplifies the system and removes one possible false target for compounds which might act by upregulating transcription. The entire 30 Lac-ATT-CAT cassette was then ligated to the PCR amplified region of the E. coli rrnB operon containing the Tl and T2 transcription terminators (Brosius et al . , 1981, J. Mol.

44 Biol., 148:107-127). The presence of these terminators assures that read through transcription initiating from elsewhere in the plasmid other than the engineered Lac promoter will be kept at a minimum. The sequence integrity of ς this construct was confirmed by DNA sequence analysis. Function of the modified CAT gene was confirmed by demonstrating Cam resistance of cells transformed with the completed pAUU-CAT construct.

6.2. FUNCTIONAL CHARACTERIZATION OF TEST CELLS 0

The responsiveness of the pAUU-CAT construct to IF3 regulation in vivo was tested in E. coli . pAUU-CAT was transformed into a strain containing an infC mutation that lowers IF3 activity (JK003) as well as the isogenic wild-type parent strain (DR599) to this mutant (Sussman et al . , 1996, 5 Mol. Micro., 21:347-360). In the absence of chloramphenicol (Cam) , on LB or ampicillin (Amp) containing plates where expression of pAUU-CAT is not required for growth, the wild- type strain grew better than the infC mutant. This result is consistent with the reported slow growth phenotype of this IF3 0 mutant. More importantly, when these strains are plated to Cam containing plates, where expression of pAUU-CAT is required for growth, the opposite result is observed. In the presence of Cam the pAUU-CAT containing infC mutant grows better than the wild- ype strain. This result is consistent with what is known about IF3 function: expression of a gene 5 initiating from an AUU initiator codon increases when IF3 function is impaired by mutation or reduced levels. As a control, JK003 and DR599 were transformed with pSE420 the parent plasmid to pAUU-CAT (Brosius, 1989, DNA 8:759-777). The results were identical to those observed for pAUU-CAT on 0 LB or LB-Amp plates. As a genetic proof of concept, DR599 and JK003 containing pSE420 were unable to grow on Cam containing plates due to the absence of the CAT gene. Compounds that

45 - impair IF3 function are expected to give analogous results: growth on Cam should be enhanced.

In the primary growth assay, a lawn of wild-type E. coli (DR599) containing pAUU-CAT is plated to LB-Cam plates. The concentration of Cam, in the range of about 6 mg/1, is sufficient to maintain a minimal level of cell viability but does not allow for dense lawn growth. Chloramphenicol concentrations between 2 mg/1 and 40 mg/1 are appropriate, depending on the strain. Test compounds are added to wells in the plates and then allowed to diffuse through the medium so that a concentration gradient is established. The plates are incubated overnight at 37°C. Compounds which interfere with IF3 function are expected to result in zones of increased growth against a background of light lawn growth. Compounds which test positive in this system are known to penetrate the cell membrane and function in the intracellular compartment. As IF3 is an essential factor, some compounds which completely abolish IF3 function will likely result in a zone of killing surrounded by a zone of increased growth due to the concentration gradient established in the media.

6.3. ASSAY REFINEMENT AND INITIAL SCREENING RESULTS

The reactions of known translational inhibitors as well as the effects of solvents, acids, bases, salts, etc. in the primary assay were determined. 3μl of test compounds were added per well. Results are discussed below, and summarized in Table 1.

46 TABLE 1. Test Compounds in Primary Assay

Compound Growth Cone Compound Growth Cone

Ampicillin - 30mg/L Lincomycin t- 75mg/L

Anisomycin - lOmg/L Lomefloxacin lOmg/L

Benzamidme - 30mg/L Nalidixic Acid 20mg/L

Chloramphenicol - lOOmg/L Neomycm Sulfate 20mg L

Chloroquine - 20mg L Norfloxacm lOmg L

Cyclohexamide - 200mg/L Nystaun 8mg/L

Dihydrostreptomyαn - 20mg/L Ofloxacin 5mg/L

Dithiothreitol - 40mg/L Pactamycm 18mg L

DMSO - 100% Paromomycin Sulfate lOmg/L

Enoxacin - lOmg L Picolinic Acid 20mg L

Erythromycin - lOOmg/L Puromycm 3mg L

Ethanol - 100% Quinacπne lOmg/L

Ethidium Bromide - 5mg/ Rifampicin lOmg/L

EDTA - 0 5M Sodium Azide 2%

Fusidic Acid - 20mg/L Sodium Dodecyl Sulfate 20mg/L

Gentamycin Sulfate - 20mg/L Spectinomycm 200mg/L

Gerodizol - 0 5μM Stains- All 20mg/L

Gramicidin - 5mg/L Streptomycin 400mg/L

Hygromycm B - 20mg/L Tetracycline 20mg/L

Kanamyαn - 20mg L Tπton X-100 + /- 100%

Compound 1 + 30mg/L

Compound 2 + SOmp T.

Lincomycin gave a positive result in the primary assay. Lincomycin inhibits peptidyl transferase assays in vi tro by interfering with P site (peptidyl -tRNA site) and/or A site (aminoacyl-tRNA site) binding. Lincomycin has not been implicated in initiation inhibition. However, its effects in vivo are not well understood and our results would seem to indicate that further studies regarding its mechanism would be useful .

Accordingly, the invention provides the use of lincomycin, compound 1, and/or compound 2 as positive controls in any assays that are designed for identifying putative IF3 inhibitors, including the primary and secondary assays of the invention.

Most of the remaining compounds tested did not give positive results, though many inhibited growth. TritonX-100 gave a very faint positive result but this effect disappeared when plates with slightly higher doses of chloramphenicol were used (> 5mg/l) . Water and DMSO had no effect on the assay.

- 47 The latter is important, as DMSO is used as a solvent in many chemical compound libraries.

6.4. SCREENING A CHEMICAL COMPOUND LIBRARY

5 The primary assay has been used to screen 5520 compounds. Thirty hits have been identified, corresponding to a hit rate of 0.67%. Of these, 9 were medium or strong hits and 21 were weak hits. Hits are categorized using a system which incorporates scores for compounds which result in increased growth (1, weak; 2, medium; 3, strong) and increased

10 growth (1, 2 and 3) plus killing zones (10, small; 20, medium;

30, large) . Weak but questionable growth was flagged to distinguish it from the above scoring system. A representative result from the primary assay is shown in Figure 3. This plate was used in screening six 96-well plates 15 on an LB agar-Cam plate. Wells are formed in the agar, and three μl of solution, typically containing l-3μg of test compound, was added per well. A positive reaction can occur at a point away from the well, where the concentration of compound is actually much lower than that of the sample applied to the well. The two clear hits observed in this experiment are indicated. Hit number 1 received a score of 2 indicating medium growth. Hit number 2 received a score of 12 (2+10) indicating medium growth and a small killing zone. At present no preferential treatment is given to compounds receiving different scores in the primary assay (i.e. 2 or

25 12) . Positive controls (lincomycin) were included on the plate although their growth are not yet visible. Typically plates are scored prior to the emergence of the positive controls. Plates were re-examined at a later time to confirm the emergence of the positive control and score weak hits not 30 visible earlier if applicable. An alternative positive control which may be used is compound 1 or compound 2.

48 This protocol can be applied to screening a chemical compound library containing compounds stored in the 96-well plate format. Each plate typically contains 80 unknown compounds and 12 solvent control wells. As seen in Figure 3, six 96-well plates can be screened on one primary assay plate. Therefore, in the current format, 480 test compounds can be screened in each primary assay plate. Hit compounds will be characterized in secondary assays. The most promising compounds will be evaluated against a panel of pathogenic bacteria and in cytotoxicity tests.

Such chemical compound library can be catalogued in a database which is fully supported by programs for substructure, chemical abstract and patent searching.

Compounds identified as described herein will be used as leads for chemical optimization to improve potency and selectivity as well as pharmacokinetic, drug metabolism and safety profiles.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

49

Citas de patentes
Patente citada Fecha de presentación Fecha de publicación Solicitante Título
US5523288 *22 Sep 19944 Jun 1996Xoma CorporationMethod of treating gram-negative bacterial infection by administration of bactericidal/permeability-increasing (BPI) protein product and antibiotic
Otras citas
Referencia
1 *GRONER, ET AL.: "CISTRON SPECIFIC TRANSLATION CONTROL PROTEIN IN ESCHERICHIA COLI.", NATURE NEW BIOLOGY, LONDON, GB, vol. 239., 6 September 1972 (1972-09-06), GB, pages 16 - 19., XP002921378, ISSN: 0090-0028
2 *LEE-HUANG S., ET AL.: "SPECIFIC INHIBITORS OF MS2 AND LATE T4 RNA TRANSLATION IN E. COLI.", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 49., no. 02., 1 October 1972 (1972-10-01), US, pages 371 - 376., XP002921377, ISSN: 0006-291X, DOI: 10.1016/0006-291X(72)90420-2
3 *OLSSON C. L., ET AL.: "PHYSIOLOGICAL EFFECT OF TRANSLATION INITIATION FACTOR IF3 AND RIBOSOMAL PROTEIN L20 LIMITATION IN ESCHERICHIA COLI.", MOLECULAR AND GENERAL GENETICS., SPRINGER VERLAG, BERLIN., DE, vol. 250., 1 April 1996 (1996-04-01), DE, pages 705 - 714., XP002921379, ISSN: 0026-8925, DOI: 10.1007/s004380050124
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