WO2005056826A1

WO2005056826A1 - Transcription-patterned biological activity screening system

Info

Publication number: WO2005056826A1
Application number: PCT/CA2004/002114
Authority: WO
Inventors: Julian E. Davies; Michael G. Surette; Grace Yim; Huimi Wang
Original assignee: The University Of British Columbia
Priority date: 2003-12-12
Filing date: 2004-12-10
Publication date: 2005-06-23

Abstract

A screening system for screening the biological activity of unknown or uncharacterized compounds in general, or for screening the biological activity by comparing patterns of transcription generated by the unknown or uncharacterized compounds with predetermined patterns of transcription generated by known compounds with known biological activities is disclosed. A kit containing a plurality of screening systems, a method of screening compounds using the screening system, and a method of making screening systems for screening compounds with any biological activity are also disclosed.

Description

TRANSCRIPTION-PATTERNED BIOLOGICAL ACTIVITY SCREENING SYSTEM

Related Applications

[0001] This Application claims priority from US Provisional Application No. 60/528,791, filed 12 December 2003.

Technical Field

[0002] This invention relates to systems and methods of screening compounds for biological activities.

Summary of Invention

[0001] The inventors disclose a screening system for screening the biological activity of unknown or uncharacterized compounds by comparing patterns of transcription generated by the unknown or uncharacterized compounds with patterns of transcription generated by known compounds with known biological activities. The screening system can screen for compounds with any biological activity, including antibiotic, anti-viral, anti-fungal, anti-tumour, or immunosuppressive activity. The invention can also used to screen unknown compounds for activity in general.

[0002] The inventors disclose a kit containing a plurality of screening systems.

[0003] The inventors disclose methods of screening compounds using the screening system. [0004] The inventors also disclose methods of making screening systems for screening or detecting compounds with any biological activity.

Brief Description of Drawings

In drawings which are intended to illustrate embodiments of the invention and which are not intended to limit the scope of the invention:

Fig. 1 A is a comparison between growth inhibition and promoter activation (light production) by erythromycin (A and B) and rifampicin (C and D) over a range of different concentrations as measured with Etest strips placed on bacterial cell overlays.

Fig. IB is a comparison between growth inhibition and promoter activation (light production) by erythromycin (A and B) and rifampicin (C and D) over a range of different concentrations as measured with Etest strips placed on bacterial cell overlays.

Fig. 2 is a combined scatter plot of the actions of rifampicin (lug/ml) and erythromycin (5ug/ml) on the 6500 clone S. typhimurium random promoter-/wc library.

Fig. 3 is a scatter plot of the reassay of selected clones of the S. typhimurium promoter-/«x library responsive to erythromycin (left plot, ■_ =1 ug/ml, • =30 ug/ml) and rifampicin (right plot, X=0.2 ug/ml and • =

2.5 ug/ml).

Fig. 4 is a chart depicting the effect of 8 different antibiotics on a panel of 94 selected clones of the S. typhimurium promoter-/w library. Fig. 5 is a chart depicting the effect of 8 different antibiotics on 12 selected clones of the S. typhimurium promoter-/wx library.

Figs. 6A(a-aa) are luminosity images of a panel of selected clones of the S. typhimurium promoter-t α library grown in the presence of various antibiotics.

Figs. 6B(a-aa) are luminosity images of a panel of selected clones of the S. typhimurium promoter-Zzα; library grown in the presence of various antibiotics.

Figs. 7A(a-g) are luminosity images of panels of selected clones of the S. typhimurium promoter-/ια: library where panels grown in the presence of antibiotics which are structurally related or which have similar modes of action are grouped together.

Figs. 7B(a-g) are luminosity images of panels of selected clones of the S. typhimurium promoter-/røc library where panels grown in the presence of antibiotics which are structurally related or which have similar modes of action are grouped together.

Fig. 8 is a curve showing the antibiotic concentration dependence of promoter activation in an E. coli host.

Fig. 9 is a graph which illustrates the antibiotic specificity of transcription activation of different promoters in the presence of different classes of antibiotics.

Fig. 10 is a chart of promoters from some of the 82 selected clones of the S. typhimurium promoter-/tα; library. Fig. 11 A depicts images comparing the effect of bogorol on a panel of 96 clones with the effect of other antibiotics on the same panel.

Fig. 1 IB depicts images comparing the effect of bogorol on a panel of 96 clones with the effect of other antibiotics on the same panel.

Figs. 12A(a-j) are images of agar overlays of various promoter clones reacting to products produced by various microbial isolates.

Figs. 12B(a-j) are images of agar overlays of various promoter clones reacting to products produced by various microbial isolates.

Fig. 13 is a chart of promoters from 44 additional clones of the S. typhimurium promoter-/ /* library.

Description

[0005] Throughout the following description, specific details are set forth in order to provide a more thorough understanding of the invention. However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive, sense.

[0006] This invention relates to screening systems for determining the biological activities of compounds. In particular, this invention is a screening system which is used to determine the biological activities of novel or unknown compounds by comparing transcription regulation patterns generated by the novel or unknown compounds to transcription regulation patterns generated by known compounds. [0007] The screening system comprises a plurality of clones, each clone containing a different promoter cloned upstream of a reporter gene. When the clones are grown in the presence of different compounds with different biological activities, the promoters in each of the clones can be activated, repressed or unaffected by the compounds. Different compounds activate or repress different promoters to different degrees. The level of activity of the promoters is determined by measuring the activity of the reporter gene, namely by quantifying the gene product of the reporter gene.

[0008] The clones can comprise prokaryotic or eukaryotic cells.

More specifically, the clones can comprise bacterial cells. In one embodiment, the clones comprise Salmonella typhimurium cells.

[0009] The promoters can comprise prokaryotic or eukaryotic promoters that are capable of being recognized and/or activated and/or repressed in the clones. More specifically, the promoters can comprise bacterial promoters. In one embodiment, the promoters comprise S. typhimurium promoters.

[0010] The reporter gene can comprise any gene that produces a quantifiable gene product or phenotype. Preferably, the gene product or phenotype is produced or displayed in proportion to the level of expression of the reporter gene. The gene product may be directly or indirectly quantified. Many different types of reporter genes are known to persons skilled in the art, such as reporter genes which produce coloured or chromogenic products, reporter genes which produce fluorescent products, and reporter genes which produce luminescent products. In one embodiment, the reporter gene comprises a lux operon, the gene product of which produces luminscence that is quantifiable.

[0011] Known compounds generate reproducible patterns of promoter activation in a given panel of clones. Therefore, unknown or novel compounds can be screened for biological activity by growing a panel of clones in the presence of an unknown or uncharacterized compound, determining the pattern of promoter activation generated by the unknown or uncharacterized compound, and comparing the pattern of promoter activation generated by the unknown or uncharacterized compound to predetermined patterns of promoter activation generated by known compounds in the same clones.

[0012] The screening system can be used to generate patterns for many different types of compounds with different biological activities. In one embodiment of the invention, the screening system can be used to differentiate compounds with different biological activities. For example, the screening system can be used to differentiate between compounds which may have antibiotic, anti-fungal, anti-viral, anti-tumour, immunosuppressive activity, or any other biological activity.

[0013] In another embodiment of the invention, the screening system is used to characterize the chemical class and/or structural class and/or mode of action to which the uncharacterized compound belongs. Therefore, throughout this application, biological activity can also refer to any biological activity, including the chemical class, chemical structure, or mode of action of a compound. [0014] For example, the screening system can be used to identify the class of antibiotic to which an unknown or uncharacterized compound potentially belongs. The screening system can also be used to characterize the class of anti-fungal compounds, anti-viral compounds, anti-tumour compounds, immunosuppressive compounds, or any other class of compounds, to which an uncharacterized or unknown compound potentially belongs. In one embodiment of the invention, the screening system is used to identify a compound as an aminoglycoside antibiotic, an aminocyclitol antibiotic, a beta-lactam antibiotic, a lincosamide antibiotic, a macrolide antibiotic, a streptogramin antibiotic, a sulfonamide antibiotic, a fluoroquinolone antibiotic, a peptide antibiotic, a polyketide antibiotic, a fosfonic acid antibiotic, an ansamycin antibiotic, a benzoquinolone antibiotic, a nucleic acid base analogue antibiotic, or any other class of antibiotic.

[0015] In another embodiment, the invention is used to screen unknown compounds for activity in general. For example, the invention can be used to rapidly screen a large number of unknown compounds for any type of activity in general and assist in determining whether or not further characterization of the compound should be pursued.

[0016] In another embodiment, the invention is a kit comprising multiple screening systems. For example, a kit may contain one or more screening panels to screen for antibiotics, one or more screening panels to screen for anti-viral compounds, one or more screening panels to screen for anti-fungal compounds, one or more screening panels to screen for anti-tumour compounds, one or more screening panels to screen for immunosuppressive compounds, or one or more screening panels to screen for any other biological activity, or any combination of the above.

[0017] The invention also relates to methods of screening compounds for biological activity. The method comprises applying the compound to the screening system which comprises a panel of a plurality of clones each having a reporter gene linked to a promoter reactive to compounds; growing the panel of clones in the presence of the compound; determining the pattern of activity of the reporter genes in the presence of the compound; and comparing the pattern of activity generated by the compound to known patterns of activity of the reporter genes generated in the presence of known compounds with known biological activities, or determining a level of activity of the reporter genes in general.

[0018] Compounds with unknown biological activities can be rapidly screened using the screening system. The screening system is also effective at screening small quantities of compounds, such as subinhibitory concentrations of antibiotics. Therefore, only small quantities of unknown or uncharacterized compounds need to be generated for screening. The screening system can provide valuable information on the activity of a compound before a great deal of time and money are invested in isolating or synthesizing larger amounts of the compound for further testing. Further, the screening system utilizes cloned cells, and the compound that is being screened must be capable of being taken up by the clones in order to activate transcription. Therefore, if the compound affects transcription levels, the user will also know that the compound is effectively taken up by cells, which can be a desired characteristic in therapeutic compounds. Therefore, the screening system of the invention provides a simple, efficient, and rapid way to obtain large amounts of information on potentially useful compounds.

[0019] The screening system is useful for screening any unknown or uncharacterized compound. The unknown or uncharacterized compound may be derived from any natural sources, such as soil samples, marine samples, natural product libraries made from bacteria, fungi, plants, animals, or other organisms, etc. The unknown or uncharacterized compound may also be derived from compounds produced by combinatorial chemistry.

[0020] The screening system is also useful for screening unknown or uncharacterized compounds for potential synergistic activity with known compounds. It has long been known that different compounds that act on the same molecular target in cells may act in synergy (one compound activates the other) or they may antagonize each other (one compound reduces the activity of the other). These reactions are due to enhancement or competition of target binding. For example, similar compounds may bind to ribosomes and synergistically affect protein synthesis. An example is the eukaryotic protein synthesis inhibitor cycloheximide, which is inactive against bacterial protein synthesis, but which has demonstrable effects in synergizing certain bacterial antibiotics as indicated by transcription modulation (W. Tsui et al., 2004, Chemistry & Biology, Vol. 11, 1307-1316).

[0021] [0022] Such interactions are readily detected by the use of the screening system of the invention, as promoter-reporter clones can be used to identify biological activity of compounds that have an affect on a target (for example, the compounds may bind weakly to a target that affects transcription patterns of the clones), even though the compounds do not show any effect in traditional inhibition assays. The screening sytem of the invention can be used to determine a potential synergistic function of an unknown compound. For example, a compound may affect the transcription pattern of a panel of clones in a manner similar to known compounds. Therefore, the compound could be identified for further synergy testing with the other known compounds. As a result, other types of inhibitors could also be developed from the detection of synergy or antagonism of compounds acting on other molecular targets, such as topoisomerases, polymerases, etc.

[0022] The invention also provides a method for making a screening system for screening biological activity in unknown or characterized compounds according to the following steps:

1) Generating a library of promoter-reporter gene constructs;

2) Transforming the library into a eukaryotic or prokaryotic host cell capable of recognizing and activating or repressing the promoters in the library to create a set of screening clones;

3) Selecting clones from the library to form a screening panel;

4) Growing the panel of clones in the presence of known compounds;

5) Determining the pattern of activation of the promoters in the clones in the presence of the known compounds by quantifying the gene product of the reporter gene; 6) Using the patterns of activation of known compounds to detect or identify biological activity of unknown or uncharacterized compounds.

[0023] The method can be used to generate a library of promoter- reporter genes from any organism. For example, if it is desired to generate a screening system for screening biological activity of compounds in bacteria, viruses, fungi, mice, humans, or any other organism, the library can be generated from DNA derived from any of these organisms. Preferably, the DNA is total genomic DNA. In one embodiment, the library is a promoter-reporter gene library made from genomic S. typhimurium DNA. A library can also be generated with DNA made from an antibiotic resistant bacteria to screen, with high sensitivity, for compounds which have the capacity to act on antiobiotic resistant bacteria.

[0024] The method involves transforming the library into any organism that recognizes and activates or represses the promoter-reporter gene constructs. For example, a library of promoters made from eukaryotic DNA could be transfected into a library of eukaryotic cells for efficient recognition of the eurkaryotic promoter-reporter gene constructs. Alternatively, the library can be made from prokaryotic DNA and transformed into prokaryotic cells. In one embodiment, the method comprises transforming a library made from S. typhymurium DNA into S. typhymurium cells.

[0025] The method further involves selecting clones to create a panel for testing the biological activity of compounds. The clones can be selected on a number of different criteria, including degree of activation or repression of the promoter in the presence and absence of different compounds. Clones containing promoters which are always activated can be included as positive controls. Clones which are never activated can be included as negative controls.

[0026] The method can be used to create a screening system to screen for compounds with any desired biological activity by selecting clones which are activated or repressed in the presence of compounds of interest. For example, the method can be used to create a screening system to screen for compounds with antibiotic activity, anti-fungal activity, anti-tumour activity, anti-viral activity, immunosuppressive activity, or any other desired activity, by selecting clones which are activated or repressed in the presence of the desired compounds. In one embodiment, the method is used to create a screening system to screen compounds for antibiotic activity.

Examples

[0027] The following examples are intended to illustrate various embodiments of the invention and are not intended to limit the scope of the invention.

I) Bacterial strains and growth conditions.

[0028] Strains used in the study are listed in Table 1. Cultures were grown aerobically in Luria broth (LB) at 30°C or 37°C. When appropriate, kanamycin (50 μg/ml), tetracycline (20 μg/ml), erythromycin (50 μg/ml and 500 μg/ml), and rifampicin (50 μg/ml and 200 μg/ml) were added. All antibiotics were obtained from Sigma Chemical (St. Louis, MO) or from the laboratory collection.

Table 1 : Strains Used in Examples of the Invention.

Solid Media Assay

[0029] Overnight liquid LB (Difco Laboratories, Detroit, MI) cultures from single colonies of reporter strains were diluted 1: lOO-fold, inoculated into 0.7% agar, and overlaid on LB agar plates. Etest strips (AB Biodisk, Solna, Sweden), Sensi-discs (BD Biosciences, Sparks, MD), or self-made antibiotic discs were placed on the overlay. Etest strips contain precisely graduated concentrations of antibiotics that permit the accurate determination of minimal inhibitory concentrations (MICs) where the lower end of the inhibition zone intersects the strip (See Fig. 1 A and Fig. IB panels A and C). Plates were incubated at 30°C or 37°C overnight and luminescence (relative light units) was detected with a Berthold USA (Oakridge, TN) LB980 photon camera.

Liquid Media Assay

[0030] Two-fold serial dilutions of antibiotics were made in the wells of black, clear bottom 96-well plates or white 96-well plates (Thermo Labsystems, Helsinki). Overnight liquid cultures of reporter strains were diluted from 1 : 100 to 1 :300 in LB and added to the wells containing antibiotics. OD₆₂o and luminescence from each well were recorded at 37°C in a Wallac 1420 Victor multilabel counter (Perkin Elmer, Boston, MA) or a Tecan SpectraFluor Plus (Tecan, Durham, NC).

II) Screening for promoters activated by sub-inhibitory concentrations of antibiotics.

[0031] Salmonella enterica serovar Typhimurium (Salmonella typhimurium) strain ATCC 14028 was used in this study. A random promoter library was constructed by cloning genomic restriction endonuclease fragments into the expression vector pCS26 upstream of a promoterless luxCDABE operon. The library consisted of approximately 6500 clones (17x384 microtiter plates) exhibiting promoter activity under different growth conditions.

[0032] The Salmonella clones were cultured aerobically at 37°C in

LB containing kanamycin (25 μg/ml). To test the reactivity of the clones to specific antibiotics, erythromycin was added to selected cultures at a concentration of 1-30 μg/ml, and rifampicin at a concentration of 0.2-5 μg/ml. Screening was conducted using black 384-well solid bottom plates. A 384-pin replicator (V &P Scientific, San Diego, CA) was used to inoculate 384-well plates from overnight cultures. The plates were incubated at 37°C and light production was measured in a multilabel counter at 6 hours and 24 hours. Results were plotted to identify clones activated or repressed by the antibiotics. Clones which displayed a 3 -fold or greater differential response in the presence of the antibiotics were rearrayed into 384-well plates. A second found of screening was performed on the selected clones in a similar manner, except light reading were taken at 2, 4, 6, 8, and 24 hours. Clones which displayed a 3 -fold or greater differential response were rearrayed into 96 well plates. These clones were screened in similar manner to confirm responses to the antibiotics.

[0033] The results of the tests using erythromycin and rifampicin are shown as scatter plots in Figs. 2 and 3. The patterns show that these two antibiotics, at sub-inhibitory concentrations, activate (points above the diagonal) or repress (below the diagonal) many different promoters in S. typhimurium.

IIP Selection of Clones for the Screening System Panels

[0034] The 6500 clone library was next tested for activity against various aminoglycoside and macrolide antiobiotics (erythromycin at 5, 10, and 15 μg/ml, pristinamycin at 1.25, 2.5, and 5.0 μg/ml, lincomycin at 6.25, 15, and 30 μg/ml, tylosin at 6.25, 15, 30 μg/ml, azithromycin at 0.5, 1.0, and 1.5 μg/ml, telithromycin at 0.625, 1.25, and 2.5 μg/ml, spectinomycin at 5 and 22.5 μg/ml, apramycin at 10 μg/ml, gentamicin at

1 μg/ml, amikacin at 5 μg/ml, and tetracycline at 1.2 μg/ml). Clones showing differential expression of 3X or greater were chosen and rearrayed into 384-well plates. A second round of screening was done in a similar manner, except light readings were taken every 30 minutes after innoculation for 20 hours; additional readings at OD₆₂o were taken to account for possible growth effects. Clones showing differential expression of 3X or more were re-arrayed into 96-well plates. A third round of screening was done to confirm that the clones were positive for responses to antibiotics. Many other selection strategies can be used to create a panel of clones appropriate for screening a particular biological activity.

[0035] A panel of 94 clones was then selected from the aminoglycoside positive clones and macrolide positives clones (77 aminoglycoside positive clones and 17 macrolide positive clones). The clones were selected for greatest response differential and reproducibility.

The 94 clone panel was first tested for reactivity to 8 different antibiotics. Fig. 4 is a chart which summarizes the reactivity of the 94 clones to the 8 drugs (AM = amikacin, AP = apramycin, E = erythromycin, G = gentamicin, RB = ribostamycin, RF = rifamycin, SP = spectinomycin, T = tetracycline, ALL = all drugs activated transcription in the clone, + = only the indicated drug activated transcription in the clone, - = only the indicated drug did not activate transcription in the clone but all other drugs activated transcription, blank = none of the drugs activated the clone).

[0036] Upon analysis of the activity of the 94 clones, it was found that the 8 drugs could be differentiated using 12 of the 94 clones. False positive clones were removed from the panel. When no such promoter could be found, cross reactivity of each of the promoters to other antibiotics were used to differentiate the drugs. A positive control clone was selected which was reactive to all antibiotics, and a negative control clone which was not reactive any antibiotics was also selected. Fig. 5 is a chart which summarizes the reactivity of 12 selected clones to 8 different antibiotics (AM = amikacin, AP = apramycin, E = erythromycin, G = gentamicin, RB = ribostamycin, RF = rifamycin, SP = spectinomycin, T = tetracycline, lane 9 contains a negative control clone, lane 11 contains a positive control clone). Because these clones could be used to differentiate between the 8 drugs, the inventors used the method of selection of these clones to develop a panel for screening and differentiating other types of antibiotics.

[0037] A total of 26 antibiotics, representing the major structural classes of antibiotics, were selected for testing. After screening the 94 clones with the 26 drugs, 32 of the clones were removed as they produced inconsistent results. The remaining 62 clones included 46 aminoglycoside positive clones and 16 macrolide positive clones. Another 11 aminoglycoside positive, macrolide positive clones and 3 rifampicin positive clones from a separate screening were added. The final panel contained 82 clones.

[0O38] To test the 82 clone panel against 26 known antibiotics, clones were grown in LB medium at 37°C overnight in a 96 well plate. The overnight culture was inoculated into 96 well plates containing LB + antibiotics using a 96-pin replicator and incubated at 37°C. A Luminograph LB980 Photon camera was used to observe lux gene expression in each well of the plates between 16-18 hours after inoculation.

[0039] Information on the drugs tested against the panel of clones, including class, mode of action, and concentration used are indicated in Table 2 below. The optimum concentration was determined by sensitivity tests.

Table 2: Antibiotics Tested on promoter panels.

[0040] Figures 6A(a) to 6A(z) and 6B(a) to 6B(z) each depict 96- well plates containing the panel of 82 clones grown in the presence of the antibiotics listed in Table 2. Figures 6A(aa) and 6B(aa) depict a 96-well plate containing the panel of 82 clones grown only in LB without antibiotics. Between each panel, each well in the same position in each 96-well plate contains the same clone. Each panel contains a luminosity meter on the right side of the plate to assist in determining the level of light produced in each well (the upper end of the luminosity meter indicating high luminosity, the lower end of the luminosity meter indicating background, i.e. low or no luminosity). Negative activity in a well (i.e. non-activation of promoter + lux gene construct in clone) is indicated when the well is the same pattern as the background or the same pattern as the low end of the luminosity meter. Positive activity in a well (i.e. activitation of promoter+/wx gene construct in the clone) is indicated when a well contains a pattern above background. The luminosity meter can also be used to determine intensity of a reaction.

[0041] Antibiotics with similar modes of action appear to generate similar patterns of activity in the panels. The reaction of the panel of clones to antibiotics in similar classes or with similar modes of action are grouped in some of Figures 7A(a) to 7A(g) and 7B(a) to 7B(g). The common reactions of the clones to some antibiotics are summarized in the tables below. Table 3 is a summary of the reaction of the 82 clone panel to aminoglycoside antibiotics (apramycin, gentamicin, butirosin, amikacin, tobramycin, ribostamycin).

antibiotics. (+ = transcription activated in clone) Table 4 is a summary of the reaction of the 82 clone panel to macrolide antibiotics (erythromycin, azithromycin, tylosin).

(+ = transcription activated in clone) Table 5 is a summary of the reaction of the 82 clone panel to polyketide antibiotics (tetracycline, minocycline).

Table 5: Summary of reaction of 82 clone panel to polyketide antibiotics. (+ = transcription activated in clone) Even within each class of antibiotics, or among antibiotics with similar chemical structures or modes of action, the antibiotics can activate or repress individual clones which are not affected by other drugs within the same class or with similar chemical structures or modes of action. However, each class of drug also activates common clones. The tables contain clones which are commonly activated by the drugs in each indicated class. A comparison of tables 3, 4, and 5 indicates that antibiotics in each of the different classes activates a different set a promoters. Each class of antibiotics has a different mode of action. Accordingly, a compound with unknown or uncharacterised biological activity can be tested against the panel, and the pattern of reaction generated by the unknown compound can be compared with the patterns of known compounds in different chemical classes with different chemical structures or with different modes of action to determine the biological activity of the unknown compound.

IV) Identification of Cloned Promoters

[0042] Plasmid DNA was isolated from the 82 clones using the

Concert Miniprep system (Life Technologies, Rockville, MD) and sequenced using a vector primer pZEOό 5 AATCATCACTTTCGGGAA- 3 , (Qiagen Operon, Alameda, CA). Sequencing was carried out by the Marine Biotechnology Lab, National Research Council of Canada

(Halifax, Nova Scotia). The promoters were identified by comparison to the GenBank database using the NCBI standard nucleotide-nucleotide BLAST program blastN (www .ncbi.nlm.nih.gov BLAST/) and analyzed using Vector NTI software (Informax, Bethesda, MD).

[0043] The 82 clones in the panel contain reporter constructs of more than 50 known promoters. The identified promoters are shown in Fig. 10.

[0044] The promoters activated by the antibiotics regulate many different functions including genes involved in transport, virulence, DNA repair, and numerous unidentified functions.

V) Kinetics and Concentration Dependence of Activation of Promoters

[0045] The kinetics and concentration dependence of activation of some of the promoters were examined in liquid cultures. Fig. 8 is a curve showing the concentration dependence of promoter activations (ρlasl'::luxCDABE) in a rifampicin-sensitive (K802; MIC 12 μg/ml and a rifampicin-resistance (K802NR; MIC > 256 μg/ml) E. coli host. The curve shows that positive responses can be detected, in certain cases, at antibiotic concentrations 50-100 times lower than the MIC The stimulatory activity reached a maximum level near the MIC, where transcription levels were increased some 2- to 10-fold; in all cases minimal effects on growth were observed at the most effective concentration for promoter activation, and promoter activation was considerably reduced at concentrations greater than the MIC The activity of different antibiotics varied depending on the phase of bacterial growth.

[0046] Moreover, a given promoter may be activated to different degrees, depending on the antibiotic being used. This specificity appears to be determined by the biochemical mode of action of the antibiotic. Fig. 9 illustrates the antibiotic specificity of the transcription activation of different promoters in the presence of different antibiotics. Overlays of S. typhimurium promoter-lux fusions on LB agar were exposed to discs containing antibiotics (10 μg of imipenem, 10 μg of polymixin B, 10 μg of rifampicin, or 15 μg of imipenem). Light production was measured as described above, where "RLU" means relative light units. The differences in light production can also be seen in Figures 6A(a) to 6A(z) and 6B(a) to 6B(z) where a clone in one well may produce more light when grown in the presence of one antibiotic than when the clone is grown in the presence of another antibiotic. The differences in levels of light production in Figures 6A(a) to 6A(z) and 6B(a) to 6B(z) are indicated variances in colour in the wells.

VI^s) Using an Embodiment of the Screening Panels to Determine the Mode of Action of an Uncharacterized Compound

[0047] The microbial product bogorol (T. Barsby, T. Kelly, S.

Gagne, and R. Anderson, Org. Lett., 2001, Vol. 3, No. 3: 437-440) is an antimicrobial with an unknown mode of action. To help determine the possible mode of action of this compound, the compound was tested against a 96 clone panel, and its effect was compared with the effect of known compounds on the same panel of clones.

[0048] A stock solution of bogorol of 55mg/ml in DMSO was used and diluted to 55ug/ml final working solution in LB. (This concentration is based on the reported MIC). A 96 clone panel was prepared by removing 6 clones from the 82 clone panel discussed above and adding 20 new clones. Of the 6 clones that were removed, 4 have been identified as ybeL, rfaY, corE, and garD. The additional 20 clones contain the first 20 promoters identified in Fig. 13. Fig. 13 identifies 44 more promoters in total that have been cloned using the methods discussed above and that can be selected as clones for a panel. This 96 clone panel was grown in the presence of bogorol. Known drugs were also tested against this panel to compare patterns of transcription activity. The drugs and concentrations used are gentamicin (1 ug/ml), minocycline (1.25 ug/ml), erythromycin (10 ug/ml), streptomycin (2 ug/ml), tetracycline (0.5 ug/ml), and chloramphenicol (0.5 ug/ml).

[0049] Luminescence pictures were taken after 18 hours of incubation of the wells at 37°C The images are shown in Figs. 11 A and 1 IB. The results indicate that the transcription modulation pattern of bogorol showed a strong similarity to the patterns produced by aminoglycosides. Bogorol does not have a structure that is similar to aminoglycosides.

VII) Screening Unknown Compounds for Activity Using the Clones

[0050] Products from 50 strains of marine bacteria isolated from seawater, seaweed and sediment were tested for activity against various clones of the panel (strains provided by R. Anderson). These strains were isolated because they produce one or more compounds having inhibitory activity against Gram-positive S. aureus. Since the panel reporter strains respond to extremely low concentrations of active compounds, the inventors decided to test response of selected panel clones to culture supernatants from these strains.

[0051] Six promoter clones were selected which belong to different responsive groups. The clone containing the promoter "tsr" is somewhat like a positive control because it is normally responsive to most chemicals. Clones containing the promoters "fadB" and "ybhP" are aminoglycoside responsive clones. The clone containing the promoter

"STM3595" (labelled as "A12") is a rifampicin responsive clone. The clone containing the promoter "cobC" is a triclosan responsive clone. The clone containing the promoter "ybfE" is a macrolide responsive clone.

[0052] The 50 marine bacteria strains were inoculated into Muller

Hinton broth and incubated at 37°C in a shaker for 48 hours. Then 0.5ml of each strain was spread on Muller Hinton agar and incubated in room temperature for 5 days.

[0053] Each promoter-reporter was inoculated in LB containing kanamycin (25 ug/ml) and grown overnight on the shaker. Then the overnight culture is diluted 500 times into 45°C, 0.7% soft agar to make overlay plates. Ten overlay plates were made for each promoter, so in total there are 60 plates.

[0054] One centimetre diameter agar plugs were made from the microbial lawns of each of the 50 strains, after incubation for 5 days at room temperature. These plugs contain the products of marine microorganisms which grew as a microbial lawn. Six plugs were taken from each strain, and each plug placed on 6 different promoter overlays. Then the promoter overlays were grown.

[0055] Luminescence pictures were taken after 18 hours of incubation at 37°C to examine the activity of the cloned promoter in the presence of the microbial products produced by the fifty marine bacteria. The images are shown in Figs. 12A(a-j) and 12B(a-j). The results are summarized in Table 6 below. Results show that the agar plugs have up regulated and down regulated transcription of the respective promoters as shown by luminescence. As well, this suggests that some of the marine strains tested can produce biologically functional products. Furthermore, the mode of action of the products could be predictable according to their reaction to the different promoter-reporter constructs. The clones from the panels can be used as effective screens for rapidly screening numerous compounds for activity and determining which compounds to characterize further.

Table 6: Summary of transcription activity of various panel clones to marine microbial compounds.

[0056] As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the scope thereof. Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A screening system for determining a biological activity of a compound comprising a plurality of clones each having a reporter gene linked to a promoter reactive to compounds, wherein the biological activity of the compound is determined by comparing a pattern of activity of the reporter genes in the presence of the compound to pre-determined patterns of activity of the reporter genes in the presence of known compounds.

2. A screening system according to claim 1 wherein the biological activity is selected from the group consisting of antibiotic activity, anti-viral activity, anti-fungal activity, anti-tumour activity, and immunosuppressive activity.

3. A screening system according to claim 1 wherein the biological activity is antibiotic activity.

4. A screening system according to claim 3 wherein the antibiotic activity is selected from an aminoglycoside antibiotic, an aminocyclitol antibiotic, a beta-lactam antibiotic, a lincosamide antibiotic, a macrolide antibiotic, a streptogramin antibiotic, a sulfonamide antibiotic, a fluoroquinolone antibiotic, a peptide antibiotic, a polyketide antibiotic, a fosfonic acid antibiotic, an ansamycin antibiotic, a benzoquinolone antibiotic, a nucleic acid base analogue antibiotic, or any other class of antibiotic.

5. A screening system according to claim 1 wherein the reporter gene is a lux operon.

6. A screening system according to claim 1 wherein the promoters comprise bacterial promoters.

7. A screening system according to claim 1 wherein the clones comprise bacterial clones.

8. A screening system comprising any new, useful, and inventive feature, combination of features or sub-combination of features described or clearly inferred herein.

9. A kit for screening biological activity of compounds comprising a plurality of screening systems according to claim 1.

10. A kit for screening biological activity of compounds comprising any new, useful, and inventive feature, combination of features or sub- combination of features described or clearly inferred herein.

1 LA method of determining a biological activity of a compound according to the following steps: a) applying the compound to a screening system, wherein the screening system comprises a plurality of clones each having a reporter gene linked to a promoter reactive to compounds; b) determining the pattern of activity of the reporter genes in the presence of the compound; and optionally c) comparing the pattern of activity of the reporter genes in the presence of the compound to known patterns of activity of the reporter genes generated in the presence of known compounds with known biological activities.

12.The method according to claim 11, wherein detennining the pattern of activity of the reporter genes comprises measuring the level of expression of the reporter genes.

13. The method according to claim 12, wherein the reporter genes are lux genes, and measuring the level of expression of the reporter genes comprises measuring luminescence.

14. A method of detennining a biological activity of a compound comprising any new, useful and inventive step, act, combination of steps and/or act, or sub-combination of steps and/or acts described or clearly inferred herein.

15. A method of making a screening system for screening or detecting biological activity in unknown or characterized compounds according to the following steps: a) Generating a library of promoter-reporter gene constructs; b) Transforming the library into a host cell capable of recognizing and activating or repressing the promoters in the library to create a set of screening clones; c) Selecting clones from the library to form a screening panel; d) Growing the screening panel of clones in the presence of known compounds; e) Determining the pattern of activation of the promoters in the clones in the presence of the known compounds by quantifying the gene product of the reporter gene; f) Using the patterns of activation of known compounds to identify biological actitivity in unknown or uncharacterized compounds.

16. The method according to claim 15, wherein the library of promoter- reporter gene constructs is generated from bacterial promoters.

17. The method according to claim 16, wherein the library of promoter- reporter gene constructs is generated from S. typhimurium promoters.

18. The method according to claim 15, wherein the host cell is a bacterial cell.

19. The method according to claim 18, wherein the host cell is S. typhymurium.

20.The method according to claim 15, wherein the reporter gene is a lux operon.

21. The method according to claim 15, wherein the biological activity is antibiotic activity, anti-viral activity, anti-fungal activity, anti-tumour activity, or immunosuppressive activity.

22. A method of making a screening system for screening biological activity in unknown or characterized compounds comprising any new, useful and inventive step, act, combination of steps and/or act, or sub- combination of steps and/or acts described or clearly inferred herein.