WO1997031530A1 - Novel antimicrobial activity of gemfibrozil - Google Patents

Abstract

The present invention provides for a method for inhibiting growth of a bacterium which consists essentially of contacting the bacterium with a compound having a specific structure as disclosed. The compound is present in a concentration effective to inhibit growth of the bacterium. The bacterial infections to be inhibited may be those associated with Legionella pneumophila, Mycobacterium tuberculosis, Bacillus subtilis, Bacillus Megaterium, Pseudomonas Oleovorans, Alcaligenes eutrophus, Rhodococcus sp., Citrobacter freundi, Group A Streptococcus sp., Coag neg Staphylococcus aureus or Nocardia sp..

Description

Novel Antimicrobial Activity of Gemfibrozil This application is a continuation-in-part of U.S. Serial No. 08/608,712 filed February 29, 1996, the contents of which are incorporated by reference. The invention disclosed herein was made with Government support under Grant No. AI23549 and AI20516 from NIAID. Accordingly, the U.S. Government has certain rights in this invention.

Background of the Invention

Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding Sequence Listing and the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

Gemfibrozil (GFZ) is a compound that has been utilized as a drug for increasing intracellular accumulation of hydrophilic anionic agents (U.S. Patent No. 5,422,372, issued June 6, 1995) and as a lipid regulating composition (U.S. Patent No. 4,859,703, issued August 22, 1989).

Gemfibrozil has been shown to be effective in increasing the amount of cholesterol excreted in to bile. (Ottmar Leiss et al., Metabolism, 34(1): 74-82 (1985)). Gemfibrozil is described in U.S. Patent No. 3,674,836 and in The Merck

Index, 11 ed., Merck & Co., Inc. Rahway, N.J. 1989; #4280.

Gemfibrozil, a drug which therapeutically lowers triglycerides and raises HDL-cholesterol levels, previously has not been reported to have antimicrobial activity.

(Brown, 1987; Oliver et al., 1978 and Palmer et al., 1978). Summary of the Invention

The present invention provides for a method for inhibiting growth of a bacterium which consists essentially of contacting the bacterium with a compound having the structure

In the compound each of R₁, R₂ , R₃ , R₄ , R₅ and R₆ may be independently H, F, Cl, Br, I, -OH, -0R₇, -CN, -COR-,, -SR ₇, -N(R₇) -, -NR₇COR₈ , -NO - , - (CH -) _p OR ₇, -(CH₂)_pX (R₇)-, - (CH₂) XR₇ COR ₈ , a straight chain or branched, substituted or unsubstituted C₁ -C₁₀ alkyl, C ₂-C ₁₀ alkenyl, C₂-C₁₀ alkynyl, C ₃ -C ₁₀ cycloalkyl, C₃ -C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl; wherein R₇ or R₈ may be independently H, F, Cl, Br, I, -OH, -CN, -COH, -SH ₂ , -NH ₂ , -NHCOH, - (CH ₂) _p OH, - (CH ₂) _p X(CH₂), -(CH ₂) _pXCOH, a straight chain or branched, substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, C₃ -C₁₀ cycloalkyl, C₃ -C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl; wherein A may be -N₂- , -NH-, -C=C=CH ₂-, -C≡C-C - HOH- , -C≡C-CH ₂ -, -CH₂ -CH₂ -O-, -CH₂-CH₂ -CH₂-O-, -S-, -S(=O)₂-, -C=O-, -C=O-O-, -NH-C-O-, -C=O-NH-; and wherein Q, p, N and X may independently be an integer from 1 to 10, or if Q is 1 A may be a (C₁ -C₁₀ )- alkyl chain, (C₁ -C₁₀ )-alkenyl chain or (C₁ -C₁₀ )-alkynyl chain which is branched or unbranched, substituted or unsubstituted and can optionally be interrupted 1 to 3 times by -O- or -S- or -N-; or a pharmaceutically acceptable salt or ester thereof, which compound is present in a concentration effective to inhibit growth of the bacterium. In this method, A may be an (C 1 -C 10 )-alkylene chain, (C₁ - C ₁₀ )-alkyl chain, (C₁ - C₁₀ )-alkenyl chain or (C₁ -C₁₀ )- alkynyl chain which is branched or unbranched, substituted or unsubstituted and can optionally be interrupted 1 to 3 times by -O- or -S- or -N-; and wherein the ether linkage to the benzene ring may be alternatively -S-, -N- or -C-.

Brief Description of the Figures

Figure 1. MICs (minimal inhibitory concentration) for gemfibrozil were determined by incubating L. pneumophila or F4b with various concentrations of GFZ in AYE broth (microbiological media). Bacteria were present at an initial concentration of 1x10⁶ CFU's (colony forming units) /ml. Growth was turbidimetrically assessed by determining the OD at 600nm after a 48 hour incubation at 37°C.

Figure 2. MICs for probenecid were determined by incubating L. pneumophila, resuspended to 1x10⁶ CFU's/ml, with various concentrations of probenecid in AYE broth. Growth was turbidimetrically assessed by determining the OD at 600nm after 48 hours at 37°C.

Figure 3. MICs for clofibric acid were determined by incubating L . pneumophila, resuspended to 1x10⁶ CFU's/ml, with various concentrations of clofibric acid in AYE broth. Growth was turbidimetrically assessed by determining the OD at 600nm after 48 hours at 37°C.

Figure 4. Bacteria were screened for sensitivity to gemfibrozil using a zone of inhibition assay. The assay was performed by adding bacteria to a suitable nutrient agar plate, adding a disk containing gemfibrozil to the plate, and then incubating the plate at the appropriate temperature. The presence of a zone of inhibition (area around the disk where no growth occurred) was considered positive for sensitivity.

Figure 5. Twenty one clinical and CDC M. tuberculosis strains, demonstrating different drug resistant profiles, were tested for sensitivity to gemfibrozil. Disks containing a given amount of GFZ were added to each of four quadrants of a plate. Five mis of Middlebrook agar were added to each quadrant, and the drug was allowed to diffuse throughout the agar in each quadrant overnight. 100 μls of a standard dilution of each M. tuberculosis strain were added to each quadrant, and the plates were incubated for three weeks at 37°C. No growth was indicated by (-). Fewer than 50 colonies were counted; (+) 50-100 colonies; (++) 100-200 colonies; (+++) 200-500 colonies; (++++) confluent growth. Figure 6A-6B. GFZ induces large distending inclusions in a subpopulation of L. pneumophila grown in the presence of a subinhibitory concentration of GFZ. (A) Stationary phase L. pneumophila, grown in AYE, stained with Nile Blue A. Numerous nondistending granules present in the majority of the bacteria. (B) Stationary phase L . pneumophila , grown in AYE (+GFZ), stained with Nile Blue A. Numerous large, distending granules present in a subpopulation of the bacteria, other bacteria demonstrate few to no inclusions. Figure 7. Electron micrograph, 20,000x, of L. pneumophila grown to log phase on a CYE plate. Note the presence of small, non-distending inclusions.

Figure 8. Electron micrograph, 20,000x, of L. pneumophila grown on a CYE plate containing an inhibitory concentration of GFZ. Note the presence of large, distending inclusions in a subpopulation of the bacteria, and the absence of inclusions in other bacteria. Figures 9A, 9B, 9C and 9D. Demonstration of an intermediate phenotype during GFZ-induced inclusion development in L. pneumophila . Electron micrographs, 8,000x, of pelleted L. pneumophila and F4b grown in AYE broth in the presence or absence of GFZ 85 μg/ml for 4.5 hours. L. pneumophila demonstrates increased numbers of inclusions, while F4b, the GFZ semi-resistant mutant, does not. (A) L. pneumophila; no GFZ (B) F4b; no GFZ (C) L . pneumophila ; GFZ 85 μg/ml (D) F4b ; GFZ 85 μg/ml .

Figures 10A, 10B, IOC and 10D. Fatty acid compositions of wild type L. pneumophila , and the GFZ semi-resistant mutant F4b, grown in the presence or absence of a subinhibitory concentration of gemfibrozil. Fatty acid compositions were assessed by saponifying, methylating, and extracting the fatty acids present in the bacteria scraped from the plates, and then injecting the methylated fatty acids into a gas chromatograph. A step temperature program was used such that as the temperature was increased, sequentially longer chain fatty acids were released from the column and detected as peaks on the chromatogram. (A) Wild type L. pi-eumopiiila grown on CYE plates in the absence of GFZ (B) Wild type L. pneumophila grown on CYE (GFZ 30 μg/ml) plates; peaks that have decreased in size are marked by arrows, new peaks are marked by dots. (C) F4b grown on CYE plates in the absence of GFZ (D) F4b grown on CYE (GFZ 30 μg/ml) plates. Figure 11. Sensitivity of L . pneumophila and F4b to INH.

Bacterial overlays on CYE agar plates were prepared by adding 2x10⁷ bacteria to 3 mis of melted 50°C agar and pouring the mixture over 15 ml CYE agar plates. Sterile disks containing 1 mg of INH, or 250 μg of GFZ were added to the overlays, and the plates were incubated for four days. Sensitivity was assessed by measuring the diameter of the zone of inhibition, the area where bacterial growth was inhibited, surrounding the drug disks. Figure 12. Demonstration of inverse relationship between GFZ sensitivity and INH sensitivity using INH-resistant F4b revertants. INH-resistant F4b revertants were obtained by adding F4b to CYE- INH drug plates (400 μg/ml) and screening for spontaneous INH-resistant mutants after four days of incubation at 37°C. INH resistant colonies, which arose at a frequency of 1/10^-7, were picked, passed non-selectively three times on CYE, and then tested for GFZ and INH sensitivity using the zone of inhibition assay. The assay was performed by adding 2x10⁷ bacteria to 3 mis of melted 55°C agar, pouring the mixture over 15 ml CYE plates, and then adding 1 mg INH sterile disks and 250 μg GFZ sterile disks to the overlays. After a four day incubation at 37°C, the diameter of the zones of inhibition were measured. The colonies indicated by the left bar under the histogram retained the parental phenotype and thus were not revertants. The colonies indicated by the right bar under the histogram regained GFZ sensitivity as INH sensitivity was lost.

Figure 13. Sensitivity of L . pneumophila and F4b to ethionamide. Ethionamide resistance correlates with GFZ resistance in the L . pneumophila-derived mutant F4b. Sensitivity was assessed by measuring the diameter of the zone of inhibition in bacterial overlays surrounding 250 μg GFZ disks, or 500 μg ethionamide disks. Figures 14A-14B. GFZ inhibits intracellar multiplication of L . pneumophila in PMA-differentiated HL-60 cells. (A) Monolayers of PMA-differentiated HL-60 cells were infected for 2.5 hours with L . pneumophila or F4b in the wells of 96 well microtiter plates. The wells were washed to remove extracellular bacteria, and medium containing 100 μg/ml of GFZ was added to the monolayers. Bacteria were titered at different time points by lysing the monolayers and counting the total number of CFU's present in the lysate and medium. (B) Monolayers of PMA-differentiated HL-60 cells were infected with increasing concentrations of L . pneumophila in the wells of 96 well microtiter plates. After 2.5 hours GFZ was added to the wells to a final concentration of 100 μg/ml. An MTT assay was performed after a five day incubation at 37°C to assess HL-60 cell viability. Reduction of MTT by viable HL-60 cells was measured spectrophotometrically at 590nm. Figures 15A, 15B and 15C. GFZ inhibits intracellular multiplication of L . pneumophila in monocytic cells. (A) Monolayers of human peripheral blood derived monocytes were infected with L . pneumophila in the wells of 96 well microtiter plates. After 2.5 hours, the well were washed and medium containing GFZ 100 μg/ml was added to the monolayers. Bacteria were titered at different time points by lysing the monolayers and counting the total number of CFU's present in the lystate and medium of each well. (B) Monolayers of human peripheral blood derived macrophages were infected with F4b and titered for CFU's as described above. (C) Monolayers of the murine macrophage J774 cell line were infected with L. pneumophila and titered for CFU's as described above.

Figure 16. A L. pneumophila 2. lkb DNA insert, expressed from pBSK, complements the envM E. coli ts mutant and confers sensitivity to GFZ at the restrictive temperature, 42°C, on low osmolarity LB plates. ts envM E. coli containing pBSK:2.1 were grown overnight in the presence of ampicillin, and then diluted 10^-2 . 100 μl of this dilution was mixed with 3 mis of melted 55°C agar and poured over low osmolarity LB plates. Disks containing 5 mg of GFZ were added to the overlays, and the plates were incubated at 30°C or 42°C overnight. The diameter of the zones were measured to assess GFZ sensitivity.

Figure 17. GFZ inhibits intracellular multiplication of L. pneumophila in PMA-differentiated HL-60 cells. PMA-differentiated HL-60 cells were synchronously infected with L. pneumophila at an MOI of 0.01 in suspension. 100 μl aliqouts were plated in the wells of a 96 well microtiter plate. The plates were centrifuged to pellet the cells and bacteria at the bottom of the wells. The monocytes were allowed to internalize the bacteria for 2.5 hours prior to incubating with gentamicin 100 μg/ml for 0.5 hours. The gentamicin was then washed away, and the medium was replaced (+/-) GFZ 100 μg/ml. Intracellular multiplication was measured by lysing the monolayers, and titering the combined lystate and supernatant at the given time points in triplicates.

Figures 18A, 18B and 18C. Nile Blue A staining for PHB. (Fig 18A) Nile Blue A fluorescent staining (l,000x) of L. pneumophila Philadelphia 1 grown on CYE agar in the absence of GFZ. (Fig 18B) Nile Blue A fluorescent staining (l,000x) of L. pneumophila Philadelphia 1 grown on CYE agar in the presence of GFZ lOug/ml, and INH 40 μg/ml (Fig 18C) L . pneumophila Philadelphia 1 grown on CYE agar in the presence of INH 400 μg/ml. Figure 19. Fatty Acid Synthesis Pathway. There are four reactions in each cycle of fatty acid elongation in E. coli. The first is condensation of malonyl-ACP with acetoacyl-ACP or a longer chain acyl-ACP to form a ketoester. This reaction can be catalyzed by FabH, FabB, or " FabF. The second step involves NADPH-dependent reduction of the ketoester catalyzed by FabG. The third step involves dehydration of the substrate by FabA or FabZ to form a highly unstable enoyl-ACP. Reduction of the enoyl-ACP by FabI, an enoyl reductase, produces an acyl-ACP that can be utilized by the cell, or elongated by additional cycles of fatty acid synthesis. Due to the instability of the enoyl-ACP compound, inhibition of FabI has been shown to result in the accumulation B-hydroxybutyrate in cell free extracts of E. coli .

Figure 20. Synergistic Inhibition of L. pneumophila by GFZ/INH. AYE containing INH at 0, 100, 200 and 400 μg/ml was added to a series of duplicate test tubes for each INH concentration. GFZ was then added to the first tube in each INH series to a final concentration of 10 μg/ml, the MIC for

L. pneumophila, and then serially diluted two-fold, (10, 5, 2.5, 1.25 μg/ml). The final volume in each test tube was 2ml. L. pneumophila were then added to a final concentration of 1 x 10⁶ CFU's/ml. The tubes were incubated for 48 hours, and the turbidity measured by the Spec OD at 600nm.

Figures 21A-1, 21-A2 and 2IB. (Figs 21A-1 and 21A-2) Nucleotide sequence of the L. pneumophila FabI enoyl reductase homolog. (Fig 21B) Multiple amino acid sequence alignment of enoyl reductase homologs from various bacterial species.

Detailed Description of the Invention

The present invention provides for a method for inhibiting growth of a bacterium which consists essentially of contacting the bacterium with a compound having the structure

wherein each of R ₁, R₂ , R₃ , R₄ , R ₅ and R ₆ may be independently H, F, Cl, Br, I, -OH, -OR₇, -CN, -COR ₇, -SR ₇, -N(R₇) ₂, -NR₇COR₈, -NO ₂, - (CH ₂)_p OR₇, (CH ₂ ) _p X(R

(CH₂ ) _p XR ₇ COR ₈ , a straight chain or branched, substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl; wherein R₇ or R₈ may be independently

H, F, Cl, Br, I, -OH, -CN, -COH, -SH -NH -NHCOH, -(CH

₂ ) _p OH, (CH₂ ) _pX(CH₂ ), -(CH₂ ) _p XCOH, a straight chain or branched, substituted or unsubstituted C₁ -C₁₀ alkyl, C₂-C

₁₀ alkenyl, C₂ -C₁₀ alkynyl, C₃ -C₁₀ cycloalkyl, C ₁ -C ₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl; wherein A may be -N 2

-NH-, -C=C=CH -O≡C-C₂ HOH-, -C≡C-CH₂-, -CH₂ -CH₂ - O-,

CH ₂ -CH ₂ -CH₂ -S-, -S(=O)₂-, -C=O-, -C=O-O-, -NH-C=O- C=O-NH- and wherein Q, p, N and X may independently be an integer from 1 to 10, or if Q is 1 A may be a (C₁ -C₁₀ )- alkyl chain, (C₁ -C₁₀ )-alkenyl chain or (C₁ -C₁₀)-alkynyl chain which is branched or unbranched, substituted or unsubstituted and can optionally be interrupted 1 to 3 times by -O- or -S- or -N-; or a pharmaceutically acceptable salt or ester thereof, which compound is present in a concentration effective to inhibit growth of the bacterium. In this method, A may be an (C₁ -C₁₀)-alkylene chain, (C₁ -C ₁₀ )-alkyl chain, (C₁ -C₁₀ )-alkenyl chain or (C₁ -C₁₀ )- alkynyl chain which is branched or unbranched, substituted or unsubstituted and can optionally be interrupted 1 to 3 times by -O- or -S- or -N-. The ether linkage to the benzene ring may alternatively be -N-, -S- or -C-.

In one embodiment, the compound may include the following:

R₁ = R₄ = CH₃ or -OH,

R₂ = R₃ = R₅ = R₆ = H or -OH,

A = CH₂,

and Q = 3.

In one embodiment, the compound may include the following:

R₃ = Cl,

R_{1 =} R_{2 =} R_{4 =} R_{5 =} R₆ = -OH or H,

and Q = 0.

In anther embodiment, the compound may include:

R₆ = CH ( CH₃) ₂ ,

R₁ = R₂ = R₃ = R₅ = H or -OH,

and Q = 0 .

In another embodiment, the compound may include

R₃ = Cl ,

R₆ = C₂H₅ ,

R₁ = R₂ = R₄ = R₅ = H or -OH,

and Q = 0 .

The bacterium may include Legionella pneumophila, Nycojbacterium tuberculosis, Bacillus subtilis, Bacillus

Megaterium, Pseudomonas Oleovorans, Alcaligenes eutrophus,

Rhodococcus sp. , Citrobacter freundi , Group A Streptococcus sp . , Coag neg Staphylococcus aureus or Nocardia sp. The bacterium may be Legionella pneumophila . The bacterium may be Mycobacterium tuberculosis . The bacterium may be

Nocardia sp . The bacterium may be in a eukaryotic cell.

The concentration of the compound may be from about 5μg/ml to about 100μg/ml. In another embodiment, the concentration of the compound may be 20μg/ml.

The present invention also provides a method for alleviating the symptoms of a bacterial infection in a subject which consists essentially of administering to the subject an amount of a compound having the structure

wherein each of R ₁ , R₂ , R₃ , R₄ , R₅ and R₆ are as defined above. The ether linkage to the benzene ring may alternatively be -N-, -S- or -C-. The method also includes use of a pharmaceutically acceptable salt or ester thereof, which compound is present in a concentration effective to inhibit bacterial growth and thus alleviate the symptoms of the bacterial infection in the subject.

The bacterial infection may be associated with a bacterium listed above. The subject may be a human or an animal. The bacterial infection may be associated with Leprosy, Brucella or Salmonella. The concentration of the compound may be from about 5 μg/ml blood of the subject to about 180 μg/ml blood of the subject. In one embodiment, the concentration of the compound may be 90 μg/ml blood of the subject. The administration to the subject may be oral.

The present invention also provides a method of inhibiting activity of Enoyl Reductase Enzyme which includes contacting the enzyme with a compound having the structure

wherein each of R ₁ , R₂ , R₃ , R₄ , R ₅ and R₆ are as defined above. The ether linkage to the benzene ring may alternatively be -N-, -S- or -C-.

As used herein Enoyl Reductase Enzyme includes enzymes having enoyl reductase activity. Such enzymes may be bacterial enoyl reductases or eukaryotic enoyl reductases. Examples of bacterial enoyl reductases include those from the bacterium listed above. The enoyl reductase may be one of the enoyl reductases from L. Pneumophila . The enoyl reductase may be a gene product of a gene that hybridizes with moderate or high stringency with the envM gene. The enzyme may be in a bacterium. The bacterium may be Legionella pneumophila, Mycobacterium tuberculosis, Bacillus subtilis, Bacillus Megaterium, Pseudomonas Oleovorans, Alcaligenes eutrophus, Rhodococcus sp. , Ci trobacter freundi , Group A Streptococcus sp. , Coag neg Staphylococcus aureus or Nocardia sp. The bacterium may be Legionella pneumophila . The bacterium may be Mycobacterium tuberculosis . The enzyme may be in a cell. The cell may be a mammalian cell. The concentration of the compound may be from about 5μg/ml to about 100 μg/ml. The concentration of the compound may be 20μg/ml. The present invention provides for a method of altering a pathway of fatty acid synthesis in a bacterium which comprises contacting the bacteria with a compound having the structure

wherein each of R ₁, R₂, R₃, R₄, R₅ and R₆ is as defined above . The ether linkage to the benzene ring may alternatively be -N- , -S- or -C-.

The present invention provides for a method for determining whether or not a bacterium is sensitive to a compound having the structure

wherein each of R₁, R ₂ , R₃ , R₄ , R₅ and R₆ is as defined above. The ether linkage to the benzene ring may alternatively be -N- , -S- or -C-.

The present invention provides for a method of selecting a compound which is capable of inhibiting the enzymatic activity of enoyl reductase which includes: (A) ccntacting enoyl reductase with the compound; (B) measuring the enzymatic activity of the enoyl reductase of step (A) compared with the enzymatic activity of enoyl reductase in the absence of the compound, thereby selecting a compound which is capable of inhibiting the enzymatic activity of enoyl reductase. The compound may contact enoyl reductase at same site at which gemfibrozil contacts enoyl reductase. U.S. Patent No. 5,422,372 discloses a method of increasing intracellular accumulation of hydrophilic anionic agents using gemfibrizol (gemfibrozil). U.S. Patent No. 4,859,703 discloses lipid regulating compositions. U.S. Patent No. 4,891,220 discloses a method and composition for treating hyperlipidemia. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

Another embodiment of the present invention is a kit which is capable of detecting the presence of a particular organism based on the sensitivity of the organism to gemfibrozil. The present invention provides for a kit for detecting the presence of one or more organisms in a sample which comprises: (a) an agar or solution medium suitable for growth of the organism; (b) a means for testing growth of each organism in the presence and absence of gemfibrizol such that the growth of the organism or lack thereof can be detected; (c) a means for determining the growth of the organism thus detecting the presence of one or more organisms in a sample. The kit may be in form of an assay, a screening kit or a detection kit.

In one embodiment the compound of the present invention is associated with a pharmaceutical carrier which includes a pharmaceutical composition. The pharmaceutical carrier may be a liquid and the pharmaceutical composition would be in the form of a solution. In another embodiment, the pharmaceutically acceptable carrier is a solid and the composition is in the form of a powder or tablet. In a further embodiment, the pharmaceutical carrier is a gel and the composition is in the form of a suppository or cream. In a further embodiment the active ingredient may be formulated as a part of a pharmaceutically acceptable transdermal patch.

A solid carrier can include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active ingredient. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo- regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellent.

Liquid pharmaceutical compositions which are sterile solutions or suspensions can be utilized by for example, intramuscular, intrathecal, epidural, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously. The active ingredient may be prepared as a sterile solid composition which may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium. Carriers are intended to include necessary and inert binders, suspending agents, lubricants, flavorants, sweeteners, preservatives, dyes, and coatings. The active ingredient can be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents, for example, enough saline or glucose to make the solution isotonic, bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The active ingredient can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

This invention is illustrated in the Experimental Details section which follows. These sections are set forth to aid in an understanding of the invention but are not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter. EXPERIMENTAL DETAILS

Example 1: Legionella pneumophila is Sensitive to Gemfibrozil

The original experimental objective, which led to the discovery of a gemfibrozil- inhibitable target in bacteria, involved the use of gemfibrozil (GFZ) to block a eukaryotic transporter in Legionella pneumophila-infected J774 macrophages. As a control experiment, L. pneumophila was incubated with the concentration of GFZ required to inhibit the eukaryotic transporter, and it was found that growth of L . pneumophila was suppressed, which was an unexpected result. A subsequent minimum inhibitory concentration (MIC) assay demonstrated that L. pneumophila grown in AYE medium was sensitive to GFZ concentrations as low as 10 μg/ml This was unexpected since gemfibrozil, a drug which therapeutically lowers triglycerides and raises HDL-cholesterol levels, has not been reported to have antimicrobial activity. The MIC assay (Figure 1) was performed by preparing various concentrations of GFZ in AYE medium in test tubes. L. pneumophila was added to each tube to a final concentration of 1 x 10⁶ CFUs/ml. After a 48 hour incubation at 37°C, growth was assessed turbidimetrically (OD at 600nm). 10 μg/ml was the minimum GFZ concentration at which no growth occurred.

MIC assays were then performed using clofibric acid, a related fibric acid, and probenecid, a drug which inhibits anion transporter activity in J774 cells. Probenecid had a MIC of 160 μg/ml in AYE (Figure 2) , while clofibric acid had an MIC of 125 μg/ml in AYE (Figure 3). Both MICs were well above the 10 μg/ml seen with gemfibrozil. These results showed that gemfibrozil is especially effective as an inhibitor of L . pneumophila .

To determine whether gemfibrozil is bacteriocidal or bacteriostatic, L. pneumophila was grown for 48 hours in AYE medium containing varying concentrations of GFZ. Five microliter samples of each culture were plated on CYE agar plates. Growth was assessed after a four day incubation period at 37°C. The GFZ concentration at which no growth was seen on the CYE plates was 400 μg/ml. Therefore, GFZ at 10 μg/ml is bacteriostatic rather than bacteriocidal. Other commonly used antibiotics with bacteriostatic rather than bacteriocidal activity include chloramphenicol, the tetracyclines, erythromycin, and clindamycin.

Gemfibrozil Selectively Inhibits Bacteria That Synthesize Branched Chain Fatty Acids To determine whether the antimicrobial effect of gemfibrozil was specific for L. pneumophila, several strains of bacteria were screened using a zone of inhibition assay. This assay was performed by mixing 100 μl of a bacterial suspension with 3 mis of F-top agar heated to 50°C and then pouring the mixture over a suitable agar-nutrient plate. When the overlay hardened, a disk containing GFZ was placed on the overlay, and the plate was incubated at the appropriate temperature until growth was seen. A clear zone surrounding the disk, or a "zone of inhibition," indicates that the drug on the disk inhibited bacterial growth. In general, the larger the zone of inhibition, the more potent the drug on the species of bacteria being tested. Assessing zones of inhibition is a quick way of screening many bacterial species for susceptibility. A wide variety of bacteria were then screened. The results of these screens indicated that all susceptible bacteria had branched chain fatty acids in their membranes, although not all bacteria with branched chain fatty acids were susceptible (Figure 4). The susceptibility of Mycobacterium tuberculosis, which contains very long, branched chain mycolic acids, was especially interesting given the prevalence of, and the mortality associated with, this organism. Therefore, 21 strains of M. tuberculosis were tested, including pan-sensitive and multidrug resistant strains. Sensitivity of M. tuberculosis to GFZ was assessed by a placing a gemfibrozil-containing disk in the bottom of one of four quadrants of a petri dish. Five milliliters of Middlebrook agar were then poured over the disk or disks in each quadrant, and the plates were incubated overnight at room temperature to allow diffusion of the drug throughout the quadrant. A saline suspension containing M- tuberculosis at a McFarland standard of two was prepared, and then diluted 10^-2. 100 μl of this dilution was added to each quadrant, and the plates were incubated at 37°C for three weeks. The presence or number of colonies in each of the quadrants then was assessed. The GFZ concentration at which no growth was seen was considered to be the MIC. All strains were susceptible to concentrations between 100 μg/ml and 200 μg/ml of gemfibrozil (Figure 5). Although the inhibitory concentration is higher than the concentration of gemfibrozil used in humans treated for hyperlipidemia (15-30 μg/ml) , all 21 strains were susceptible to GFZ within a two-fold concentration range. No greater than a two fold difference in sensitivity was seen. This suggests that none of the presently evolved antibiotic resistance mechanisms affect sensitivity to gemfibrozil, and that it has a novel target site.

Development of a Mutant of L. pneumophila with Increased Resistance to GFZ

The discovery that L. pneumophila was sensitive to GFZ necessitated the development of a L. pneumophila-derived GFZ-resistant mutant that could be used in the transporter experiments. Efforts to obtain spontaneous mutants by plating 10⁸ wild type L . pneumophila on CYE agar plates containing GFZ were unsuccessful. Therefore, the alkylating agent ethyl methane sulfonate (EMS) was used to mutagenize the DNA of L. pneumophila cultures. Although attempts to generate a fully resistant mutant were unsuccessful, development of a semi-resistant mutant was successful. The mutant, F4b, had an MIC of 50 μg/ml GFZ. Similar attempts to generate a Bacillus subtilis gemfibrozil-resistant mutant, by either spontaneous mutagenesis or by EMS mutagenesis were completely unsuccessful. The inability to develop high-level gemfibrozil-resistant mutants in either species of bacteria suggests that gemfibrozil's target may be an essential gene product in these bacteria.

GFZ Induces the Accumulation and Expansion of Lipid-like Inclusion Bodies in L. pnuemophila Since the mechanism of action and target of GFZ was still unclear, Nile Blue A fluorescence was used to assess the morphology of L. pneumophila grown in the presence of sub- inhibitory concentrations of the drug. Nile Blue A is a water soluble basic oxazine dye that fluoresces at 460nm. This dye has greater specificity and higher affinity than Sudan Black for polyhydroxybutyrate (PHB) and does not stain glycogen and polyphosphate inclusions. As wild type L. pneumophila enter stationary phase in the absence of gemfibrozil, they tend to elongate and accumulate numerous non-distending granules (Figure 6A). However, staining of L. pneumophila grown to stationary phase in the presence of GFZ demonstrated that there was a subpopulation of bacteria with few to no inclusions, and a subpopulation of bacteria distended by large granules (Figure 6B). The ability of Nile Blue A to stain these granules indicates that they are composed of PHB or other types of polyhydroxy alkanoic acids (PHAs).

PHAs are natural polyesters of B-hydroxyacyl monomer units, three to fourteen carbons in length. Hydroxyacyl monomer units can be utilized by bacteria as a carbon source, as precursors in fatty acid synthesis, or, in some bacterial species, stored as PHA in inclusion bodies. PHA forming species include Bacillus megaterium, Pseudomonas oleovorans, Psuedomonas aeruginosa, Alcaligenes eutrophus, and some Rhodococcus sp . , Corynebacterium sp . , and Nocardia sp. strains. P. aeruginosa is not susceptible to GFZ, but does form PHA granules. Therefore, the ability to form PHA inclusions does not seem to be correlated with susceptibility. However, the ability of some species, such as P. oleovorans, to incorporate branched chain hydroxyacyl fatty acid precursors into PHA, suggests that the distending granules seen in L. pneumophila exposed to GFZ might be composed of branched chain fatty acid precursors. Since only bacteria that synthesize branched chain fatty acids are susceptible, it is possible that a metabolic block in branched chain fatty acid synthesis induced by GFZ would result in the accumulation of precursors. In bacteria capable of producing PHA inclusions, accumulation of precursors might result in their packaging and storage in PHA granules.

Experiments utilizing electron microscopy yielded confirmed the fluorescence data. Log phase L. pneumophila grown on CYE agar plates with no gemfibrozil, contained one or two small lipid-like inclusions (Figure 7). In contrast, L. pneumophila and L . pneumophila mutants that were partially resistant to GFZ, grown on CYE-GFZ drug plates, appeared as either bacilli without inclusions, or, as short, swollen bacilli (about 2x the normal diameter) packed with large inclusions (Figure 8). These results suggested that GFZ induced the accumulation of a metabolic precursor that was incorporated into the inclusion bodies seen in susceptible L. pneumophila .

A second EM experiment compared log phase growth of L. pneumophila and the GFZ resistant F4b mutant in AYE broth, in the presence and absence of a sub-inhibitory concentration of gemfibrozil. The concentration of GFZ used inhibited L. pneumophila growth in AYE, but did not inhibit F4b growth in AYE. Four and one half hours after the addition of GFZ to the log phase AYE cultures, L. pneumophila (+GFZ) (Figure 9C) accumulated granules, while L . pneumophila ( -GFZ) (Figure 9A) did not. In contrast, there was no apparent difference in the number or size of inclusions present in F4b in the presence (Figure 9D) or absence (Figure 9B) of GFZ. This experiment demonstrated an intermediate stage in inclusion accumulation in wild type L . pneumophila .

In summary, the presence of gemfibrozil induces the accumulation of inclusions in L. pneumophila , and, induces large, distending inclusions in a subpopulation of these susceptible bacteria. Additionally, the inclusions have a lipid-like morphology by EM, and stain with Nile Blue A, indicating that they may be composed of PHAs. These results suggest that the large, distending inclusions may be due to the accumulation of a precursor involved in fatty acid metabolism.

Gemfibrozil Affects the Fatty Acid Composition of L. pneumophila, but not its Semi-Resistant Derivative, F4b. If GFZ affects enzyme (s) involved in fatty acid synthesis, and inhibition of this enzyme results in the accumulation of fatty acid precursors, then exposure to GFZ should alter the fatty acid composition of GFZ-susceptible bacteria. To address this possibility, gas chromatograhy was used to compare the fatty acid profiles of L . pneumophila (Figure 10A) and the semi-resistant mutant, F4b (Figure 10C), grown in the presence and absence of sub-inhibitory concentration, of gemfibrozil. Base hydrolysis was used to saponify the fatty acids, which were then methylated, extracted, and injected into a gas chromatograph. A step temperature program was used such that as the temperature increased, progressively longer chain fatty acids were released from the column and detected as peaks on the chromatograph. The presence of gemfibrozil resulted in decreased peak areas for several typical L. pneumophila fatty acids, and, the appearance of several new fatty acids (Figure 10B). This indicated that GFZ inhibited the synthesis of several "typical" fatty acids and suggested that "new" fatty acids accumulate as a result of a metabolic backup. In Figure 10B, fatty acid peaks which decreased in the presence of GFZ are marked by downward arrows, new peaks which appeared in the presence of GFZ are marked by dots.

Importantly, the presence of gemfibrozil did not affect the fatty acid profile of F4b (Figure 10D) . This indicates that F4b resistance may be mediated by an enzyme with a lower affinity for GFZ, and, that this enzyme is involved in fatty acid synthesis. Additionally, the fatty acid profile of F4b looked quite different from that of wild type L. pneumophila, in that it had fewer peaks than L. pneumophila . Although no new peaks were detected in F4b, there were fewer peaks, and those peaks which were present, were present in different proportions than in L. pneumophila . The identity of the fatty acids has not been determined in the chromatograms since many of them were not present in the standard. A disappointing limitation was the inability to detect or identify fatty acids less than twelve carbons long using this system. Identification of shorter chain fatty acids may be useful in determining the identity of the gemfibrozil-induced inclusions. Isoniazid (INH)-resistant F4b Revertants

Since there is significant evidence supporting the hypothesis that gemfibrozil affects fatty acid metabolism in L . pneumophila, L . pneumophila and F4b were tested for sensitivity to isoniazid, a tuberculostatic drug which targets an enoyl reductase involved in mycolic fatty acid synthesis in M. tuberculosis . While wild type L. pneumophila showed no sensitivity to isoniazid, the semi-resistant mutant, F4b, was sensitive (Figure 11). This indicated that the mutation responsible for GFZ resistance might also have conferred isoniazid sensitivity. To see whether isoniazid sensitivity and gemfibrozil resistance had a reciprocal relationship (which would imply that they share the same target enzyme) INH-resistant derivatives of F4b were isolated. To do this, the isoniazid-sensitive, gemfibrozil-resistant, F4b strain was plated out on CYE drug plates containing isoniazid 400 μg/ml, and screened for spontaneous mutants resistant to isoniazid. Single colonies of spontaneous mutants arose at a rate of 1 x 10^-7. Several of these colonies were picked and purified by passage on nonselective CYE agar plates. The purified F4b derived INH-resistant strains were then tested for both isoniazid sensitivity and gemfibrozil sensitivity using the zone of inhibition assay (Figure 12).

The colonies indicated by the left bar under the histogram maintained the parental, F4b, INH-sensitive GFZ-resistant phenotype, indicating that they were not true INH-resistant revertants. Since the purification was nonselective, contaminating parental F4b bacteria may form colonies which are picked for father passage. Also, isoniazid-resistance due to up regulation of an enzyme, rather than a genetic-mediated resistance, would be lost in the absence of a selective pressure. Therefore, when the "purified" colonies are retested for isoniazid sensitivity, one expects to see colonies with either the parental phenotype or a genetically-mediated isoniazid-resistance phenotype.

The colonies indicated by the right bar under the histogram regained GFZ sensitivity as INH sensitivity was lost. The reciprocal relationship between gemfibrozil sensitivity and isoniazid sensitivity in these revertants, indicates that both drugs have the same target enzyme but different target sites. The mutation which allows gemfibrozil resistance (and probably affects substrate recognition) may result in a conformational change exposing an isoniazid sensitive site. In Mycobacteria sp. , the INH target, enoyl reductase, is encoded by inhA . Since GFZ appears to target an enzyme which can be made INH-sensitive, it is probably targeting an InhA homologous enzyme in L. pneumophila .

Example 2: Additional Evidence for an InhA Homologous Target: Differences in Sensitivity to Ethionamide

Ethionamide sensitivity provided additional evidence to support the hypothesis that the target gene in Legionella is homologous to the InhA gene. Ethionamide is a second line anti-tuberculosis drug which is thought to target the same enzyme as isoniazid in Mycobacteria sp. If GFZ is targeting the homologous enzyme in L. pneumophila , and F4b resistance to GFZ is mediated by a conformational change in the target enzyme, then sensitivity to ethionamide might also differ between L. pneumophila and F4b. Using a zone of inhibition assay, wild type L. pneumophila was twice as sensitive to ethionamide as F4b (Figure 13).

Growth of Intracellular L. pneumophila is Inhibited by Gemfibrozil

Since bacterial species such as L. pneumophila and M. tuberculosis are primarily intracellular pathogens, for an antibiotic to be effective it must affect bacterial growth within host white blood cells. For example, the drug must permeate macrophages, and have access to the intracellular compartment containing the pathogen. It is equally important that factors or nutrients provided by the host white blood cells do not bypass the metabolic step blocked by the drug. GFZ at 100 μg/ml partially inhibited growth of L. pneumophila in human monocytes, macrophages, HL-60 cells (a human leukemic cell line), and J774 cells (a mouse macrophage cell line). In these experiments, monolayers of human or mouse cells were infected for two and one half hours with wild type L . pneumophila or the GFZ semi-resistant Legionella mutant F4b and then washed to remove extracellular bacteria. GFZ was added to the medium at a concentration of 100 μg/ml, and the cells were incubated at 37°C. Bacteria were assayed at the specified time points by lysing the cells in each monolayer, combining the cell lysate with the medium from the same well, and plating for CFU's on CYE agar plates. Since L. pneumophila and F4b do not replicate in the medium, any growth inhibition measured will be due to inhibition of intracellular replication by GFZ. The presence of GFZ (100 μg/ml) did not affect HL-60 cell viability after a 5 day period, so inhibition of intracellular L. pneumophila by GFZ is not due to decreased host cell viability.

In HL-60 cells, L. pneumophila growth was inhibited by three logs over a 54 hour time period when GFZ was present in the media at a concentration of 100 μg/ml (Figure 14A). Growth of F4b, the semi-resistant mutant, was only inhibited by one log by this concentration of GFZ. GFZ inhibited intracellular growth of L. pneumophila and of F4b to about the same extent as extracellular growth in AYE medium. The ability of GFZ to protect HL-60 cells from intracellular L. pneumophila-induced lysis was assessed using an MTT assay

(Figure 14B). This assay, which measures cell viability as a function of the ability of the monolayer to reduce MTT, showed increased viability of L. pneumophila infected HL-60 cells, in the presence of GFZ 100 μg/ml, over a five day incubation period.

Growth of intracellular L. pneumophila in human monocytes was inhibited by GFZ (100 μg/ml) by one log after a 72 hour incubation period (Figure 15A). Similarly, growth of F4b in human macrophages (Figure 15B), and of L . pneumophila in mouse J774 macrophages (Figure 15C) was inhibited by one log in the presence of GFZ 100 μg/ml. These experiments demonstrate that intracellular L. pneumophila remain sensitive to growth inhibition by GFZ. A L . pneumophila 2. lkb DNA Insert Complements an E. coli ts envM Mutant and Confers Sensitivity to GFZ

Based on the above arguments, it is possible to hypothesize that GFZ affects the inhA homologous gene in L. pneumophila . Since InhA from M. tuberculosis has significant sequence similarity (40% identity over 203 amino acids) to the EnvM protein of E. coli , a cloning strategy was employed in which a temperature sensitive envM E. coli mutant, ts100, was transformed with DNA from a L. pneumophila library. A 2. lkb insert of L. pneumophila DNA, expressed from a pBluescript vector, was found to complement the EnvM ts phenotype. When the envM ts mutant was grown at the permissive temperature

(30°C), with or without the insert, it was not sensitive to

GFZ. However, when the ts envM E. coli was grown at the restrictive temperature (42°C) on low osmolarity LB plates, the ts EnvM enzyme was nonfunctional, and growth was dependent on the expression of the homologous L. pneumophila enzyme encoded by the 2. lkb DNA insert. Under restrictive conditions (42°C on low osmolarity LB plates), the ts envM E . coli strain was sensitive to GFZ indicating that the protein encoded by the 2. lkb DNA is the target, or a target, of GFZ (Figure 16).

Disks containing 5 mg GFZ were required to affect growth of tsl00envM:pBsk2.1 at 42°C. Whether this indicates differences in the substrates and products of EnvM in E. coli and L. pneumophila is uncertain. For example, if GFZ predominantly interferes with the ability of the L. pneumophila enzyme to utilize branched-chain or long chain fatty acid precursors, but does not interfere with the ability of the enzyme to utilize straight chain or short chain precursors, then GFZ would be expected to have less of an effect in E. coli which synthesizes most, if not all, of its fatty acids from B-hydroxy butyrate, a four carbon precursor of straight chain fatty acid synthesis. It has been recently demonstrated that the E. coli EnvM enzyme reduces a four carbon fatty acid crotonyl CoA substrate, while the homologous M. tuberculosis InhA enzyme will not reduce fatty acid substrates less than eight carbons long. Although untested, the homologous enzymes in E. coli and M. tuberculosis may differ significantly in their ability to accept and reduce branched chain fatty acid precursors.

Since the 2. lkb L. pneumophila insert confers GFZ sensitivity, the next step would be to sequence the envM homologous gene contained in this insert. Once the gene is sequenced it can be tagged and expressed from high copy plasmids to facilitate purification for biochemical assays. Such assays may be used to directly assess in vi tro inhibition of enzyme function by GFZ. EnvM and InhA activity have been measured in vi tro by a NADH oxidation assay. In this assay, the purified enzyme, fatty acid CoA substrate, and NADH are combined in a cuvette, and NADH oxidation is measured over time at 340 nm in a spectrophotometer. This assay may be utilized to test the purified EnvM homologous enzyme. GFZ may inhibit NADH oxidation.

Once the L. pneumophila envM homologous gene is sequenced, PCR can be used to pull out the homologous gene from the GFZ semi -resistant mutant F4b. This gene can then be transformed into wild type L. pnuemophila to see if its expression confers resistance to GFZ. Additionally the homologous protein from GFZ semi-resistant F4b can be tested for resistance to GFZ biochemically. It is possible that there is more that one enoyl reductase in L. pneumophila (E. coli contains two known enoyl reductases). The envM homologous gene can also be used to hybridize to other potential enoyl reductases in a L. pneumophila library, and potentially pull out other GFZ sensitive targets. Once the target genes are identified, site-directed mutagenesis can be used to identify the GFZ and substrate binding sites.

Discussion

In summary, a compound, GFZ, was identified which appears to inhibit fatty acid synthesis in several species of bacteria containing branched chain fatty acids. The GFZ target in L. pneumophila may be fully characterized and utilizing both genetic and biochemical approaches. Once the target has been identified, site-directed mutagenesis can be used for structure-function analysis to determine its GFZ binding site. Although the enzymatic target is found in other organisms beyond Mycobacteria, this enzyme has not been utilized as a target in any other species of bacteria. GFZ appears to have a novel and essential target site on the enzyme, since cross-resistance associated with other antibiotics has not been seen, and no high level resistant mutants have been obtained. It is possible that bacteria that do not contain branched chain fatty acids have a similar enzymatic site that can be targeted by other compounds or GFZ derivatives. Sensitivity can be tested biochemically using the NADH oxidation assay described above. Identification of the protein targeted by gemfibrozil, and the role of this protein in synthesizing fatty acids from specific precursors, and which enzymatic sites are important for these reactions, should be informative for both basic biology and for medicinal therapy. The ability of GFZ to inhibit synthesis of some, but not all fatty acid precursors in bacteria suggests it may have a similar effect in eukaryotic cells. Thus, these studies may provide insight into the mechanism by which this drug lowers blood lipids in humans. REFERENCES

Brown, W.V. Potential use of fenofibrate and other fibric acid derivatives in the climic. Am. J. Med. , 1987, 83, Suppl. 5B, 85-89.

Oliver, M.F.; Heady, J.A.; Morris, J.N. and Cooper, M.M. A co-operative trial in the primary prevention of ischaemic heart disease using clofibrate. Br. Heart J. , 1987, 40, 1069-1118.

Palmer, R.H. Prevalence of gallstones in hyperlipidemia and incidence during treatment with clofibrate and/or cholestyramine. Trans . Assoc . Am . Physicians, 1987, 91, 424-432.

Example 2: Novel Antibiotic Activity of Gemfibrozil for Mycobacterium tuberculosis and other Pathogens Abstract

The emergence of antibiotic resistant bacterial pathogens has intensified the search for new bacterial targets and for novel therapeutic agents that inhibit them. It is described herein that gemfibrozil (Lopid™) inhibits growth of Mycobacterium tuberculosis, Legionella pneumophila , and other bacteria that synthesize branched chain fatty acids.

M. tuberculosis strains that are resistant to multiple antibiotics remain sensitive to gemfibrozil. Genetic complementation assays identified an enoyl reducatse homolog in L. pneumophila as a target, suggesting that gemfibrozil inhibits bacterial growth by inhibiting an enzyme (s) involved in branched chain fatty acid synthesis.

Introduction

Gemf ibrozil (Lopid™) is widely used to treat hypertriglyceridemia and to lower LDL cholesterol levels in humans (1). Gemfibrozil also inhibits the efflux of anionic substrates, including anionic antibiotics such as norfloxacin, from J774 macrophage-like cells (2). When gemfibrozil is used in conjunction with such antibiotics, it potentiates their efficacy against the intracellular pathogen, Listeria monocytogenes, in J774 cells (2). When the effect of gemfibrozil alone was tested on L. pneumophila , another intracellular pathogen, it was discovered that it is an effective inhibitor of the intracellular growth of this bacterium in macrophage-like cells derived from the human monocytic HL-60 cell line (3)

(Fig 17). Subsequent studies in AYE medium showed gemfibrozil to be bacteriostatic at concentrations of

10ug/ml, the minimum inhibitory concentration (MIC₉₉) at which >99% of L . pneumophila growth is inhibited (4).

To determine whether gemfibrozil uniquely effects L. pneumophila, its effect were tested on 31 other bacterial species (5). Nine of the 31 were sensitive (Table 1). Human pathogens in the sensitive group in addition to L. pneumophila included M. tuberculosis, Nocardia sp . Staphylococcus aureus, and Staphylococcus epidermis . All of the susceptible species are reported to contain branched chain fatty acids, although not all bacteria containing branched chain fatty acids are susceptible to gemfibrozil (e.g. L. monocytogenes) (6).

The sensitivity of M. tuberculosis to gemfibrozil was of special interest given the prevalence, morbidity, and the mortality associated with infections by this organism. Twenty-one (21) M. tuberculosis strains were tested, three of which were sensitive to all antibiotics, and 18 of which were resistant to one or more anti-tubercular drugs (7). Growth of all strains was completely inhibited at gemfibrozil concentrations between 100-200 μg/ml, regardless of the classes of antibiotics to which they were resistant (Table 2). Transmission electron microscopy (TEM) was used to determine if growth in the presence of GFZ resulted in gross morphological changes in L. pneumophila . L . pneumophila grown on CYE plates in the presence of a subinhibitory concentration of gemfibrozil contained many large, distending, electron lucent, cytoplasmic inclusions. Subsequent studies, utilizing the fluorescent dye Nile Blue A (NBA), indicated that the inclusions were composed of polyhydroxyalkanoate (PHA) (8a). Examination of L. pneumophila grown on CYE in the presence or absence of GFZ and then stained with NBA, demonstrated that in the presence of GFZ, L . pneumophila Phil 1 acquired many large, distending, brightly fluorescent inclusions (Figure 18B), similar to the inclusions seen by EM, and in the absence of GFZ, L. pneumophila contained relatively fewer, and smaller, inclusions (9) (Figure 18A).

GC-MS analysis of PHA propyl esters prepared from L . pneumophila after hydrochloric acid propanolysis, confirmed that the granules are composed of PHB (9b). L. pneumophila grown in the presence of gemfibrozil contained XmgPHB/mg dry cell weight, while L. pneumophila grown in the absence of GFZ contained mgPHB/dry cell weight. No other hydroxyalkanoates were detected. The ability to store PHB is not correlated with gemfibrozil sensitivity, since PHB inclusions are found in several species of bacteria that are not GFZ sensitive including Pseudomonas sp. . However, since L. pnuemophila utilizes B-hydroxybutyrate as a precursor for fatty acid synthesis (8b), the observed accumulation of PHB may be the result of an imbalance or block in fatty acid synthesis resulting in the accumulation of B-hydroxybutyrate, and its subsequent incorporation into PHB granules (as shown in Figures 6A-B). It has been suggested that isoniazid (INH), an antitubercular drug, acts by inhibiting the synthesis of mycolic acids, high molecular mass a-alkyl B-hydroxy fatty acids (10, 11). The recently characterized enoyl -reductase, InhA, involved in fatty acid synthesis and presumably mycolic acid synthesis, is thought to be one of the targets for INH (10). Therefore, tests were carried out to determine whether INH altered the effect of gemfibrozil on PHB granule formation or growth of L. pnuemophila . INH (400 μg/ml) had no effect on L. pneumophila growth in AYE broth. However, L. pneumophila growth was completely inhibited in AYE broth containing 400 μg/ml of INH and a subinhibitory concentration (5 μg/ml) of gemfibrozil. INH at a lower concentration (200 μg/ml) also potentiated gemfibrozil's growth inhibitory effect on L. pneumophila, but to a lesser extent than INH 400 μg/ml (Figure 20). Synergy between INH and GFZ also was seen when L. Pneumophila was grown on CYE agar. Again, INH (400 μg/ml) did not detectably affect L. pneumophilia growth. However, when a disk containing 250 μg gemfibrozil was placed on CYE containing INH, the zone of inhibition increased from 37 mm (CYE without INH) to 47 mm [CYE with INH (400 μg/ml)]. A synergistic effect was also seen with Nycobacteria spp .

INH also potentiated the xapacity of gemfibrozil to cause accumulation of PHB granules in L . pneumophila . L . pneumophila grown on CYE plates containing up to 400 μg/ml of INH or 10 μg/ml of GFZ, did not demonstrate an increase in size or number of PHB granules (Figure 18C) relative to L . pneumophila grown on CYE (Figure 18A). However, L. pneumophila grown on CYE containing 40 μg/ml of gemfibrozil (Figure 18B) accumulated as many large PHB granules as L. pneumophila grown on CYE containing 30 μg/ml of gemfibrozil.

The observation that INH increased the sensitivity of L. pneumophila and Mycobacteria sp . , to GFZ, or vice versa, indicated that GFZ might be inhibiting an InhA enoyl reductase homolog in L. pneumophila . Inhibition of an enoyl reductase would also be consistent with the observed accumulation of polyB-hydroxybutyrate in GFZ-inhibitedL. pneumophila .

To identify a putative inhA homolog in L. pneumophila, and evaluate its role in GFZ sensitivity, a functional complementation strategy was used. Since InhA from N. tuberculosis is 32% identical in amino acid sequence to the FabI protein of E. coli , (11,16), it was reasoned that the putative GFZ-sensitive, enoyl CoA reductase of L . pneumophila, would complement an E. coli , mutant that is defective in enoyl reductase activity. A temperature sensitive fabl E. coli mutant, FT 100 (17), was transformed with DNA from a L. pneumophila library (18). A 1400 base pair insert (Figures 21A-1, 21A-2), with homology to FabI (19), complemented the fabl temperature sensitive phenotype. The predicted 268 amino acid L. pneumophila Fabl homolog is 58% identical, 78% similar to the E. coli Fabl enzyme (16), and is 31% identical, 57% similar to the M. tuberculosis InhA enzyme (11 ) (Figure 21B).

When the ts fabl E. coli mutant, FT100, is grown on LB agar plates at 30°C, the permissive temperature, it is not sensitive to GFZ regardless of the presence or absence of the complementing L . pneumophila insert (20). However, when FT100 is grown at 42°C, the restrictive temperature, the endogenous Fabl ts enzyme is nonfunctional (17), and growth is dependent on the expression of the heterologous L. pneumophila enzyme. Under these conditions, the complemented E. coli strain FT100 (pCR2.1:1400) gains sensitivity to GFZ (20), indicating that the L. pneumophila fabl homolog is sensitive to GFZ. The presence of INH 300 μg/ml in the plates, increases the sensitivity of ts100 (pBSK1400) to GFZ at 42°C. The isogenic wild type E. coli strain 101 (transformed with the pBSK vector only), demonstrates no sensitivity to either GFZ or INH, or the combination of GFZ and INH, at either 30°C or 42°C. In summary, gemfibrozil inhibits the growth of certain species of bacteria containing branched chain fatty acids. For L . pneumophila, it has been shown that GFZ is an effective inhibitor of both intracellular and extracellular growth. This result has potential clinical relevance for the intracellular pathogens L. pneumophila and M. tuberculosis, since it indicates that gemfibrozil blocks a key step in Legionella sp. metabolism that cannot be circumvented by nutrients provided by the host cell.

In L . pneumophila, an enoyl reductase homolog has been identified, that when expressed in an E. coli ts Fabl mutant, complemented for the ts phenotype and conferred sensitivity to GFZ at the restrictive temperature. High concentrations of the drug were required to see growth inhibition in this strain of E. coli . This could be due to poor permeability of the drug into E. coli , an enhanced ability of E. coli to pump out the drug, relative to L. pneumophila, or, that there may be more than one target for GFZ in L. pneumophila and the enoyl reductase homolog is only one of them.

The latter explanation would be consistent with the inability to obtain spontaneous mutants of L . pneumophila with resistance to GFZ. Over 10¹⁰ Legionella colonies have been screened without finding a single spontaneously resistant mutant. The inability to select spontaneous gemfibrozil resistant mutants suggests that either this drug affects a key metabolic pathway that cannot be circumvented, or, that GFZ has two or more targets. The partial sequence of another enoyl reductase homolog in L. pneumophila has been reported (21), and it is possible that this homolog is also inhibited by GFZ. Inhibition of an enoyl reductase homolog (s) is consistent with the observed accumulation of PHB in GFZ-inhibited L. pneumophila . Heath and Rock recently demonstrated that Fabl was the only enoyl reductase in E. coli , and that inhibition of a tsFabl enzyme in cell free extracts resulted in the accumulation of B-hydroxybutyrate (22) (Figure 19). Since PHB is synthesized by esterification of monomers of 3-hydroxybutyric acid (8), the inhibition of an enoyl reductase by GFZ would be consistent with the observed accumulation of B-hydroxybutyrate, as measured by GC-MS, and its subsequent storage as PHB in the large, Nile Blue A positive, inclusion bodies. Although all bacteria contain enoyl reductases, only two clinically used drugs, isoniazid and ethionamide, are thought to inhibit this enzyme (10, 11).

Remarkably, the use of both of these drugs is limited to M. tuberculosis infections. Isoniazid is selective for

Mycobacteria sp . (concentrations in excess of 500 μg/ml are required to inhibit the growth of other microorgansisms), and ethionamide is a relatively toxic compound, thus reserved for use as a secondary agent for M. tuberculosis (12). The current model for INH-mediated inhibition of M. tuberculosis involves activation of INH through a peroxidatic reaction mediated by the catalase peroxidase KatG (23). The activated INH product is then thought to inhibit the NADH-dependent enoyl reductase InhA enzyme in M. tuberculosis (24). However, intracellular metabolism of INH is known to generate reactive oxygen intermediates which may also be involved in INH-mediated toxicity (25). Recent evidence demonstrates that some species of Mycobacteria which are sensitive to INH have mutations causing decreased expression of the ahpC gene, a gene encoding a critical subunit of alkyl hydroperoxide reductase which reduces organic peroxides to their corresponding alcohols (26). Decreased expression of AhpC may be responsible for the hypersenstivity of M. tuberculosis to the oxidative stresses generated by the intracellular metabolism of INH (27). Additionally, INH may also deplete pools of NAD by interefering with the inhibition of NAD⁺ nucleosidase, an enzyme responsible for the conversion of NAD into nicatinamide, which is then deaminated into nicotinic acid (28). So, it is evident that the mechanism of the antimycobacterial inhibition by INH is not completely understood, and that it may be difficult to separate the proposed InhA inhibitory effect, from the proposed oxidative stress effects, or the NAD depletion effects in M. tuberculosis. The synergistic activity of INH and GFZ in the E. coli tsl00pBSK1400 mutant indicates that the L. pneumophila enoyl reductase homolog mediates senstivity to both GFZ and INH. However, GFZ is required for INH-mediated inhibition. There are several models that could explain the observed synergy between GFZ and INH. For example, a simple model might be that the binding of GFZ to the L. pneumophila enoyl reductase homolog induces a conformational change exposing an INH-sensitive site. Alternatively, that the metabolism of unique products generated by the L . pneumophila enoyl reductase homolog are sensitive to the effects of INH may be another model. Further, that INH, or it's metabolic products, inhibit a downstream gene involved in fatty acid synthesis that becomes important when the endogenous E. coli Fabl enoyl reductase is knocked out may be a third model. Since GFZ 1.) is synergistic with INH in M. tuberculosis strains, 2.) targets an InhA homolog in L. pneumophila, 3.) is not affected by mutations in katg or ahpC, and therefore does not seem to require activation by a catalase-peroxidase, as does INH, it may be a good compound to study the role of InhA-mediated inhibition in M. tuberculosis . As resistance to known antibiotics does not confer cross-resistance to gemfibrozil, it increases its value as a probe for novel antibiotic targets. Identification of these targets may also provide clues for the inhibition of their homologs by related compounds in GFZ-resistant bacteria. Given the high mortality associated with M. tuberculosis infections, the frequency in appearance of multi-drug resistant M. tuberculosis, the ability of these multidrug-resistant organisms to spread rapidly, and the high morbidity and cost associated with infections from these multidrug-resistant organisms, identification of all the enzymes targeted by gemfibrozil and the development of compounds to inhibit these enzymes would seem to be an important objective.

Table 1

Bacteria were screened for sensitivity to gemfibrozil using a zone of inhibition assay. The assay was performed by overlaying bacteria on a suitable nutrient agar plate, adding a disk containing 2mg gemfibrozil to the plate, and then incubating the plate at the appropriate temperature. The presence of a zone of inhibition (area around the disk where no growth occurred) was considered positive for sensitivity. The number in parenthesis indicates the fraction of strains tested with the listed characteristic. (*) indicates that sensitivity was determined by a simple bacterial overlay followed by the addtion of a GFZ disk, rather than by NCCLS standardized procedures. The fraction in parenthesis after each bacterial species indicates how many of the strains tested of each species demonstrated the phenotype for the respective column.

Table 2

Twenty one M. tuberculosis strains, demonstrating different drug resistance profiles, were tested for sensitivity to gemfibrozil. OADC-enriched Middlebrook agar plates with quadrants containing 0, 50, 100, or 200 μg/ml of GFZ in were prepared. 100 μls of a standard dilution of each resuspended M. tuberculosis strain were added to each quadrant, and the plates were incubated for three weeks at 37°C. No growth was indicated by (O); quadrants containing fewer than 50 colonies were counted, and the numbers given are the average of duplicate quadrants ; quadrants containing 50-100 colonies are indicated by a (+); quadrants containing 100-200 colonies are indicated by a (++); quadrants containing 200-500 colonies are indicated by a (+++); and quadrants with confluent growth are indicated by

(++++). The drug resistance profile is indicated to the left of each strain; S= streptomycin 2 μg/ml; 1= isoniazid

1 μg/ml; R= rifampin 1 μg/ml; E= ethambutol 5 μg/ml; K= kanamycin 6 μg/ml; O= ofloxacin 4 μg/ml; C= ciprofloxacin 2 μg/ml; R* = low level resistance to isonaizid at 0.2 μg/ul.

Endnotes

(1) S. M. Grundy, G. L. Vega, Am. J. Med. 83 (Suppl5b), 9 (1987); Y. Horsmans, J. P. Desager, C. Harvengt, Clinica Chimica Acta 218, 223 (1993).

(2) C. Cao, H.C. Neu, S.C. Silverstein, J Cell Biol. 115, 467a (1991); D. E. Rudin, P. X. Gao, P. X. Cao, H. C. Neu, S. C. Silverstein, J. Exp. Med. 176:1439 (1992).

(3) HL-60 cells were differentiated in Teflon wells in RPMI (1% glutamine) supplemented with 10% NHS and 0.1% PMA.

After incubation at 37°C for 48 hours, the differentiation medium was washed away, and the cells were resuspended in RPMI (1% glutamine) supplemented with 10% NHS. L. pneumophila resuspended from a two day CYE plate, were added to the suspension at an MOI of 0.001. 100 μl aliquots of the suspension were plated in the wells of a 96 well microtiter plate. The plate was centrifuged 220g x 5 min, then 880g x 10 min, to pellet the cells and bacteria together. This point was T=0. The plates were incubated at 37°C for 2.5 hours to allow internalization of the bacteria. Gentamicin was then added to each well to a final concentration of 100 μg/ml to kill any extracellular bacteria. The monolayers were then washed 2x to wash away the gentamicin, and the medium was replaced (+/-) GFZ 100 μg/ml. The first time point, T=6, was assessed by collecting the supernatant from each well, lysing the monolayers with 100 μl ofsterile water, adding each lysate to its respective supernatant, and then plating for CFU's on CYE. The lysate+supernatant was divided so that the entire well was plated at each time point. Time points were obtained at 6, 8, 10, 12 and 14 hours so that GFZ-mediated inhibition of intracellular bacteria was determined in a one step growth curve.

(4) Serial gemfibrozil dilutions were prepared in test tubes containing 2 mis of AYE broth. L. pneumophila were added to the tubes to a final concentration of 1 x 10⁶ CFU's/ml. After a 48 hour incubation at 37°C, growth was turbidometrically assessed by the OD at 600nm. To determine bacteriostatic vs bacteriocidal effect, five microliters from each culture were spotted on CYE plates. The presence or absence of growth was assessed after three days at 37°C. (5) Bacterial strains were screened following NCCLS standardized procedures by the Clinical Microbiology Dept. of Columbia-Presbyterian Hospital unless indicated by a (*). Strains indicated with a (*) by combining a 100 μl aliquot of an overnight culture with 3mls of 50°C 0.8% agar (Bacto-agar, Difco), and pouring the suspension over a suitable nutrient agar plate. A sterile disk containining 2mg of gemfibrozil (Sigma) was added to the overlay. Sensitivity was determined by the presence of a zone of growth inhibition surrounding the disk.

(6) T. Kaneda, Biol . Rev. 55, 288 (1991).

(7) Twenty one M. tuberculosis strains, including patient isolates and CDC strains, were obtained from the clinical microbiology department at Columbia-Presbyterian Hospital. Each strain was resuspended in saline to a McFarland standard of one and then diluted 100 -fold. 100 μls of the final dilution were added each of four quadrants of a Petri dish containing OADC-enriched Middlebrook agar with either 0, 50 100, or 200 μg/ml of gemfibrozil. The plates were incubated at 37°C for three weeks, after which the presence, or number of colonies, in each quadrant was assessed. The GFZ concentration at which no growth was seen was considered to be the MIC.

(8a) A. G. Ostle, J. G. Holt, Appl . Env. Microbiol . 44, 238 (1982); A. J. Anderson, E. A. Dawes, Microbiol Rev. 54, 450 (1990).

(8b) W. S. Mauchline, C. W. Keevil, Appl . Environ . Microbiol . 57, 3345 (1991); W. S. Mauchline, R. Araujo, R. Wait, B. Dowsett, P. J. Dennis, C. W. Keevil, J. Gen Microbiol . 138, 2371 (1992).

(9) A suspension of approximately 10⁸ L. pnemophila was added to CYE plates, or to CYE plates containing 30 μg/ml of gemfibrozil. The plates were incubated at 37°C for three or four days prior to Nile Blue A, fatty acid, or PHA analysis. A high concentration of wild type L. pneumophila was required in order to see growth on CYE plates containing 30 μg/ml gemfibrozil.

(9b) Quantitative determination and identification of PHB was performed by gas chromotographic analysis of PHA propyl esters obtained by hydrochloric acid propanolysis of lyophilzed bacteria. (V. Riis, W. Mai, J. Chromatog. 445, 285 (1988). Analyses were done with Hewlett Packard gas chromatograph. Electron energy. PHB amounts were calculated from a standard curve generated by using known amounts of PHB (obtained from Sigma).

(10) A. Banerjee et al . , Science 263, 227 (1994).

(11) A. Dessen et al . , Science 267, 1638 (1995).

(12) Sande MA, Mandell GL. Goodman and Gilman ' s The

Pharmacological Basis of Therapeutics (Pergamon Press, New York, 1990).

(16) A. F. Egan, R. R. B. Russel, Genet . Res . 21, 139 (1973); H. Bergler, G. Hogenauer, F. Turnowski, J. Gen Microbiol . 138, 2093 (1992); H. Bergler, et al . , J. Biol . Chem. 269, 5493 (1994).

(17) The isogenic pair of strains E. coli FT100 ( envM- ts) and E. coli FT 101 (wildtype) were derived from E. coli JP1111. (where the Ts phenotype is due to a serine to phenylalanine substitution at position 241) FT 100: Hfr gallE45 L - envM392 (Ts) relAl spoTl thi -1 trp: :Tn10 (Tet^r) FT 101: Hfr galE45 L - envM+ relAl spoTl thi -1 trp: : Tn10(Tet^r). FT 100 will not grow at 42°C on plates of rich medium containing ≤0.5% NaCl due to an osmotically repairable membrane defect. (18) The library consisted of L. pneumophila10-20kb fragments, from a partial EcoRI digest, cloned into the EcoRI site of plasmid pMMB207 (Cm^r). (19) DNA sequence determined by a ? DNA sequencer; Fabl homology determined by BLAST comparison of amino acid databases using the predicted amino acid sequence; percent identity and similarity were determined using the Gap GCG program. (20) Sensitivity determined by a zone of inhibition assay as described above utilizing a disk containing 5mg of GFZ. (21) M.S. McClain, M.C. Hurley, J.K. Brieland, N.C. Engleberg, Infect . Immun . 64, 1532 (1996). (22) R. J. Heath, CO. Rock, J. Biol . Chem. 270, 26538 (1995).

Example 3: Screen for Organsims Sensitive to Gemfibrozil or the Synergistic Combination of Gemfibrozil and Isoniazid

B . subtilis was grown overnight in LB broth. 100 μl aliqouts of 10-1 or 10-2 dilutions were then overlayed on trypticase soy broth plates containing no isoniazid, or 300 μg/ml of isoniazid. Disks containing 5.0 mg of GFZ were added to the plates and the plates were incubated overnight at 37°C. The diameter of the zones of inhibition were assesed the next day.

Candida albicans was grown overnight in SAB broth, 100 μl aliquots of 10-1 dilutions were then added to potassium acetate buffered SAB plates pH 7.0 (+/- INH 300 μg/ml), and a 5mg GFZ disk was placed on top. The plates were incubated at 30°C, and the diameter of the zones were assessed after a few days.

Four E. coli ts100 strains (contains a temperature sensitive mutation in an enoyl reducatse enzyme) were grown in LB-Amp(100 mg/ml) for 18 hours at 30°C. The culture was then diluted 10-1, and 100ul aliquots were mixed with 50°C F-top agar and poured over LB plates containing 0.3% NaCl, 100 μg/ml ampicillin, (+/- isoniazid 300 μg/ml) . A sterile disk containing 2.5mg of GFZ was added to the bacterial overlay. The plates were incubated at either 30°C, the permissive temperature, or 42 °C, the restrictive temperature, overnight. The presence/absence/diameter of zones was then assessed. tsl00:pBSK and ts100 :pCR2.1 are both vector-only controls. ts100:pBSK2.1 has the pBSK vector with a 2. lkb EcoRI insert containing the L . pneumophila Fabl homolog. ts100:pCRII1400 has the pCR2.1 vector with a 1400bp insert also containing the L. pneumophila Fabl homolog. At the restrictive temperature, expression of the L. pnemophila homolog is required for growth, as the ts100 Fabl enzyme is nonfunctional.

So, the L. pneumophila insert encoding the Fabl homolog, complemented for growth at the restrictive temperature, 42 °C. Further, growth at this temperature, which was dependent on the L. pneumophila Fabl homolog, was sensitive to GFZ. This sensitivity was enhanced in the presence of INH.

Example 4: Assays to Detect Inhibition of Enoyl Reductases in Bacteria or Fungi

In the first type of assay, fractions of crude cell extracts of various bacterial species are assayed for enoyl reductase activity, and the ability of gemfibrozil, a derivative, or an unrelated compound, to inibit enoyl reductase activity in the fraction assayed. Enoyl reductase activity is determined by spectrophotometrically measuring the rate of NADH hydrolysis at 340nm, in the presence of an acteylCoA substrate and NADH. Inhibition is determined by the ability of a given compound to inhibit NADH hydrolysis.

In the second type of assay, bacteria are grown in or on suitable nutrient media in the presence of the test compound at various concentrations. Sensitivity to the compound can be screened for by inhibition of growth in the presence of the compound. For bacteria which demonstrate sensitivity to the test compound, inhibition of an enoyl reductase, or perhaps another enzyme involved in fatty acid synthesis, can be screened for by directly assessing the inhibited culture, or growing the bacteria in a subinhibitory concentration of the test compound. The culture is then pelleted and resuspended in a 1% solution of the dye Nile Blue A. The Nile Blue A suspension is then incubated at 50μC (or at various temperatures) for 10 min, pelleted, and then resuspended and washed in a suitable buffer. The washed bacteria can be repelleted and incubated in 7.5% acetic acid to remove non-specific staining if necessary. The pellet is then washed and resuspended in a suitable buffer, and then assessed for fluoresence at an excitation wavelength of 460nm. Fluoresence can also be detected with a fluorescent microscope using a Texas Red, PI, or FITC filter lens. This assay is very flexible and can also be adapted to 96 well microtiter plates, glass slides, etc. In this adaptation, the bacteria are grown in the presence of the test compound, and then heat fixed or otherwise affixed, to the bottom of the wells, incubated with Nile Blue A, washed, destained with acetic acid, and then measured for fluoresence using a microtiter plate reader, or by eye using a microscope, with the conditions described above. Crystal violet can also be substituted for Nile Blue A for a microscopic screen.

Bacteria demonstrating increased fluoresence in the presence of the test compound can then be directly tested for inhibition of an enoyl reductase enzyme using the first type of assay.

Example 5:

Susceptibility of L. pneumophila Philadelphia 1 and a Gemfibrozil Semi-Resistant Mutant Derived From L. pneumophila Philadelphia 1 to Compounds with Structural Similarity to Gemfibrozil

In brief, chemical compounds with structural similarity to gemfibrozil were examined for their ability to inhibit wild type L. pneumophila and its gemfibrozil semi-resistant derivative, F4b. It was reasoned that compounds that inhibited wild type L. pneumophila, and inhibited F4b to a lesser degree, probably had the same target as GFZ in L. pneumophila . Therefore, 2-hydroxybenzoic acid, 3 (p4 - hydroxyphenyl ) propionic acid , 3, 4-dihydroxyphe nylpropionic acid, and 2,2-dimethyl-5-(2,5-dimethylphenoxy) pentanoic acid (GFZ) were prepared in solutions of either 250 mg/ml or 100 mg/ml. 10μl of each solution was then spotted onto sterile disks, 6 mm in diameter, and allowed to dry. Wild type L. pneumophila Phil 1, or its derivative F4b, were resuspended from 2 day plates, mixed with 50° F-top agar, and poured over CYE plates. The disks were then added to the bacterial overlays, and zones were assessed after four days. One GFZ disk was always added as an internal control. The second disk was the experimental compound. The results are tabulated below.

So, resistance to GFZ conferred resistance to 2-HBA. Since the mechanism of resistance for F4b is unknown, it is possible that resistance to both GFZ and 2-HPA is mediated by a common altered atraget. Additionally, it is possbole that the 2- substitution on the phenyl ring is responsible for the activity of both GFZ and 2-HPA. See below for structures.

In Vivo Mouse Models to Test the Synergy of GFZ and INH in L . pneumophila and M. tuberculosis Infections GFZ/INH Synergy in a L. pneumophila Infection

A/J mice are injected i.t. with L. pneumophila Philadelphia 1. Mice are anesthetized with ketamine 2.5 mg/mouse intraperitoneally, a slit is made in the skin of the ventral neck, the trachea is isolated, and a 10 μl suspension containing 106 L. pneumophila organisms is injected directly into the trachea using a 27 gauge needle. The skin incision is closed with a sterile wound clip. At 3 hrs, 11 hrs, 19 hrs, 27 hrs, 35 hrs, and 43 hrs, a 5 mg/kg dose of isonaizid and/or a 5 mg/kg dose of gemfibrozil is given i.m.; i.e. three times at day at eight hour intervals. At 43 hours, the mice are sacrificed and their lungs are removed, minced, and homogenized. The lung homogenates are serially diluted, and cultured on CYE agar containing polymixin B, cefamandole, and anisomycin for four days at 37°C. CFU's/lung are then determined.

GFZ/INH Synergy in a M. tuberculosis Infection B6D2F1 mice, 24 months of age, are injected i.t. with M. tuberculosis H37RV as described above, except that 100 CFU's are injected per mouse. At 3, 11, 19, 27, 35, 43, 51, and 58 hours, a 5mg/kg dose of isoniazid and/or a 5mg/kg dose of GFZ is given i.m.. At 58 hours, the mice are sacrificed, and the lungs and spleens removed, minced, and homogenized. The homogenates are serially diluted, and plated for CFU's on Middlebrook 7H11 agar and incubated at 37°C for twenty-one days. CFU's/organ are then determined. Example 6:INH/GFZ Synergy in Mycobacteria spp.

Various Mycobacteria species were resuspended to a McFarland 1 density standard, and then diluted 10^-2 and 10^-4. 100 μl of the 10^-2 dilution was spotted onto each of the four quadrants of OADC-enriched Middlebrook agar plates, with the exception of one quadrant which received 100 μl of the 10^-4 dilution (for the purpose of determining the CFU's/ml in the innoculum). Each quadrant varied with respect to GFZ and INH concentration; (+/-) GFZ 0, 50, 100 μg/ml and (+/-) INH 0.2, 1.0 μg/ml. The plates were incubated at 37°C for twenty one days, after which growth was assessed.

1-200 colonies were counted (the number is the average of duplicate quadrants; except for quadrants containing both INH and GFZ)

+++ indicates 201-500 colonies

++++ indicates a lawn of growth

BOLD indicates synergy

So, synergy was seen for the M. tuberculosis strains, most dramatically with the Ferrel strain. The sensitivity of most of the M. tuberculosis strains to GFZ 50 made it difficult to observe synergy, although it demonstrated how effective GFZ is in each of these strains regardless of their INH resistance profile. If a GFZ concentration of 25 μg/ml had been included, synergy may have been better assessed. If synergy is observed at a GFZ concentration of 25μg/ml, it is significant in itself since this concentration is close to the levels achievable in humans after a 300mg oral dose (15 μg/ml after 2 hours, and 5 μg/ml after 9 hours). Resistance profile for each of the M. tuberculosis strains:

R^H indicates high levels of resistance to INH

R^L indicates low levels of resistance to INH

So, the level of INH resistance, which presumabely represents different mutations conferring INH-resistance, does not affect GFZ sensitivity (+/- INH).

Broth MICs for many of the strains were also performed by adding 100μl of the 10^-2 dilution (prepared in the above experiment) to a series of three tubes containing 5mls of 7H9 broth (+/-) GFZ 0, 50, 300 μg/ml. Growth in the tubes was visually assessed every three to four days, the twenty- one day assessment is displayed below. Two additional strains, Nocardia sp. and M. fortui tum, were added to this experiment.

+++ indicates very thick growth

++ indicates less thick growth

+ indicates present, but not impressive growth

- no apparant growth

Although Nocardia demonstrates large zones on plates, it does not appear to be sensitive to GFZ50 in the 7H9 broth.

Claims

What is claimed is: 1. A method for inhibiting growth of a bacterium which consists essentially of contacting the bacterium with a compound having the structure:

wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ comprises independently H, F, Cl, Br, I, -OH, -OR₇, -CN, -COR₇ , -SR

-N(R₇ ) ₂, -NR₇ COR₈, -NO - (CH₂ ) _p OR₇, - (CH₂ ) _p X(R

(CH₂ ) _p XR₇ CO R₈ , a straight chain or branched, substituted or unsubstituted C ₁-C₁₀ alkyl, C₂ -C₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, C₃ -C₁₀ cycloalkyl, C₃ -C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl; wherein a linkage to the benzene ring may alternatively be -N-, -S-, -O- or -C-; wherein R₇R₈ may be independently H, F, Cl, Br, I, -OH, -

CN, -COH, -SH -NH₂, -NHCOH, (CH₂) _p OH, (CH₂) _p X(CH

(CH₂ ) XCOH, a straight chain or branched, substituted or unsubstituted C ₁ -C ₁₀ alkyl, C₂ -C₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, C ₃-C ₁₀ cycloalkyl, C ₃-C ₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl; wherein A may be -N₂- ,

-NH-, -C=C=CH₂-, -C=C-C₂ HOH- -CΞC-CH₂ -, -CH₂ -CH₂ -O-,

-CH₂ -CH₂ -CH₂ -O- -S-, -S(=O) ₂-, -C=O-, -C-O-O-, -NH-C=O-

, -C=O-NH-; and wherein Q, p, N and X may independently be an integer from 1 to 10, or if Q is 1 A comprises a (C₁ -C

₁₀ )-alkyl chain, (C₁ -C₁₀ )-alkenyl chain or (C₁ -C₁₀ )- alkynyl chain which is branched or unbranched, substituted or unsubstituted and can optionally be interrupted 1 to 3 times by -O- or -S- or -N-; or a pharmaceutically acceptable salt or ester thereof, which compound is present in a concentration effective to inhibit growth of the bacterium.

2. The method of claim 1, wherein A comprises an (C₁ -C₁₀

)-alkylene chain, (C₁ -C₁₀)-alkyl chain, (C₁ -C₁₀ )- alkenyl chain or (C ₁ -C ₁₀ )-alkynyl chain which is branched or unbranched, substituted or unsubstituted and can optionally be interrupted 1 to 3 times by -O- or -S- or -N-.

3. The method of claim 1, wherein

R₁ = R₄ = CH₃ or -OH,

R₂ = R₃ = R₅ = R₆ = H or -OH,

A = CH₂,

and Q = 3.

4. The method of claim 1, wherein

R₃ = Cl,

R₁ = R₂ = R₄ = R₅ = R₆ = H or -OH,

and Q = 0.

5. The method of claim 1, wherein

R₆ = CH(CH₃)₂,

R₁ = R₂ = R₄ = R₅ H or -OH, and Q = 0.

6. The method of claim 1, wherein

R₃ = Cl,

R₆ = C₂H₅,

R₁ = R₂ = R₄ = R₅ = H or -OH, and Q = 0.

7. The method of claim 1, wherein the bacterium is Legionella pneumophila, Mycobacterium tuberculosis, Bacillus subtilis, Bacillus Megaterium, Pseudomonas Oleovorans, Alcaligenes eutrophus, Rhodococcus sp. , Ci trobacter freundi , Group A Streptococcus sp. , Coag neg Staphylococcus aureus or Nocardia sp.

8. The method of claim 1, wherein the bacterium is

Legionella pneumophila .

9. The method of claim 1, wherein the bacterium is Mycobacterium tuberculosis.

10. The method of claim 1, wherein the bacterium is in a eukaryotic cell.

11. The method of claim 1, wherein the concentration of the compound is from about 5μg/ml to about 100μg/ml.

12. The method of claim 1, wherein the concentration of the compound is 20μg/ml.

13. A method for alleviating the symptoms of a bacterial infection in a subject which consists essentially of administering to the subject an amount of a compound having the structure:

wherein each of R ₁ , R ₂ , R₃ , R₄ , R₅ and R₆ may be independently H, F , Cl, Br, i, -OH, -OR₇ , -CN, -COR ₇ , -SR

₇, -N(R₇) ₂, -NR₇COR₈ , NO₂ , - (CH ₂ ) _pOR₇, (CH₂)_p X(R

₇ ) ₂ , -(CH₂ ) _p XR₇ COR₈ , a straight chain or branched, substituted or unsubstituted C ₁ -C ₁₀ alkyl, C₂ -C₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, C₃ -C₁₀ cycloalkyl, C₃ -C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl; wherein a linkage to the benzene ring may alternatively be -N-, -S-, -O- or -C-; wherein R ₇ or R₈ may be independently H, F, Cl , Br, I, -OH, -CN, -COH, -SH₂ , -NH₂ , -NHCOH, - (CH₂) _p OH, -(CH₂) _pX(CH

- (CH₂ ) _p XCOH, a straight chain or branched, substituted or unsubstituted C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, C₃ -C₁₀ cycloalkyl, C₃ -C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl; wherein A may be -N₂- , -NH-, -C=C=CH₂ -, -CΞC-C₂ HOH-, -C=C-CH₂ -, -CH₂ -CH₂ -O-, -CH₂ -CH₂ -CH₂ -O-, -S-, -S(=O) ₂-, -C=O-, -C=O-O-, -NH-C=O- , -C=O-NH-; and wherein Q, p, N and X may independently be an integer from 1 to 10, or if Q is 1 A may be a (C₁ -C-₀)- alkyl chain, (C₁ -C₁₀)-alkenyl chain or (C₁ -C₁₀)-alkynyl chain which is branched or unbranched, substituted or unsubstituted and can optionally be interrupted 1 to 3 times by -O- or -S- or -N-; or a pharmaceutically acceptable salt or ester thereof, which compound is present in a concentration effective to inhibit bacterial growth and thus alleviate the symptoms of the bacterial infection in the subject.

14. The method of claim 13, wherein A comprises an (C₁-C

1₀)-alkylene chain, (C ₁ -C ₁₀)-alkyl chain, (C ₁ -C ₁₀)- alkenyl chain or (C ₁ -C ₁₀)-alkynyl chain which is branched or unbranched, substituted or unsubstituted and can optionally be interrupted 1 to 3 times by -O- or -S- or -N-.

15. The method of claim 13, wherein

R₁ = R₄ = CH₃ or -OH,

R₂ = R₃ = R₅ = R₆ = H or -OH,

A = CH₂,

and Q = 3.

16. The method of claim 13, wherein

R₃ = Cl,

R_{1 =} R_{2 =} R_{4 =} R_{5 =} R₆ = H or -OH,

and Q = 0.

17. The method of claim 13, wherein

R₆ = CH(CH₃)₂,

R₁ = R₂ = R₄ = R₅ = H or -OH, and Q = 0.

18. The method of claim 13, wherein

R₃ = Cl ,

R₆ = C₂H₅ ,

R₁ = R₂ = R₄ = R₅ = H or -OH , and Q = 0 .

19. The method of claim 13, wherein the bacterial infection is associated with Legionella pneumophila, Mycobacterium tuberculosis, Bacillus subtilis, Bacillus Megaterium, Pseudomonas Oleovorans, Alcaligenes eutrophus, Rhodococcus sp. , Ci trobacter freundi , Group A Streptococcus sp. , Coag neg Staphylococcus aureus or Nocardia sp.

20. The method of claim 13, wherein the bacterial infection is associated with Legionella pneumophila .

21. The method of claim 13, wherein the bacterial infection is associated with Mycobacterium tuberculosis .

22. The method of claim 13, wherein the subject is a human or an animal.

23. The method of claim 13, wherein the bacterial infection is associated with Leprosy, Brucella or

Salmonella.

24. The method of claim 13, wherein the concentration of the compound is from about 5 μg/ml blood of the subject to about 180 μg/ml blood of the subject.

25. The method of claim 13, wherein the concentration of the compound is 90 μg/ml blood of the subject.

26. The method of claim 13, wherein the administration to the subject is oral.

27. A method of inhibiting activity of Enoyl Reductase Enzyme in a cell which comprises contacting the cell with a compound having the structure:

wherein each of R ₁ , R ₂ , R₃ , R₄ , R₅ and R₆ may be independently H, F, Cl, Br, I, -OH, -OR -CN, -COR -SR

-N(R₇ )₂, -NR₇ COR₈, -NO₂ ' -(CH₂ ) _p OR₇, -(CH₂ ) _p

X(R ₇ ) ₂, [CH₂ ) _p XR₇ COR₈ , a straight chain or branched, substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C 1₀ alkenyl, C₂ -C₁₀ alkynyl, C₃ -C₁₀ cycloalkyl, C₃ -C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl; wherein a linkage to the benzene ring may alternatively be -N- , -S-, -O- or -C-; wherein R₇ or R₈ may be independently H, F, Cl , Br, I, - OH, -CN, -COH, -SH₂ , -NH₂, -NHCOH, - (CH₂ ) _n OH, -(CH₂ ) X(CH ₂ ), -(CH₂ )_p XCOH, a straight chain or branched, substituted or unsubstituted C ₁ -C ₁₀ alkyl, C₂ -C₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, C₃ -C₁₀ cycloalkyl, C ₃ -C ₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl; wherein A may be -N₂- ,

-NH-, -C=C=CH₂-, -C-BC-C₂ HOH-, -CBC-CH₂ -, -CH₂ -CH₂ -O-,

-CH₂ -CH ₂ -CH ₂ -O-, -S-, -S(=O) ₂-, -C=O-, -C=O-O-, -NH-C=O-

, -C=O-NH-; and wherein Q, p, N and X may independently be an integer from 1 to 10, or if Q is 1 A may be a (C₁ -C₁₀)- alkyl chain, (C₁ -C₁₀)-alkenyl chain or (C₁ -C₁₀)-alkynyl chain which is branched or unbranched, substituted or unsubstituted and can optionally be interrupted 1 to 3 times by -O- or -S- or -N-; or a pharmaceutically acceptable salt or ester thereof, which compound is present in a concentration effective to inhibit activity of the enzyme .

28. The method of claim 27, wherein A comprises an (C₁ -C

1₀)-alkylene chain, (C₁ -C₁₀)-alkyl chain, (C₁ -C₁₀)- alkenyl chain or (C ₁ -C ₁₀)-alkynyl chain which is branched or unbranched, substituted or unsubstituted and can optionally be interrupted 1 to 3 times by -O- or -S- or -N-.

29. The method of claim 27, wherein

R₁ = R₄ = CH₃,

R₂ = R₃ = R₅ = R₆ = H or -OH,

A = CH₂,

and Q = 3.

30. The method of claim 27, wherein

R₃ = Cl,

R_{1 =} R_{2 =} R_{4 =} R_{5 =} R₆ = H or -OH,

and Q = 0.

31. The method of claim 27, wherein

R₆ = CH(CH₃)₂,

R₁ = R₂ = R₄ = R₅ = H or -OH, and Q = 0.

32. The method of claim 27, wherein

R₃ = Cl,

R₆ = C₂H₅,

R₁ = R₂ = R₄ = R₅ = H or -OH, and Q = 0.

33. The method of claim 27, wherein the enzyme is in a bacterium .

34. The method of claim 33, wherein the bacterium is

Legionella pneumophila, Mycobacterium tuberculosis, Bacillus subtilis, Bacillus Megaterium, Pseudomonas Oleovorans, Alcaligenes eutrophus, Rhodococcus sp. , Ci trobacter freundi , Group A Streptococcus sp. , Coag neg Staphylococcus aureus or Nocardia sp.

35. The method of claim 33, wherein the bacterium is

Legionella pneumophila .

36. The method of claim 33, wherein the bacterium is Mycobacterium tuberculosis .

37. The method of claim 27, wherein the enzyme is in a cell.

38. The method of claim 37, wherein the cell is a mammalian cell.

39. The method of claim 27, wherein the concentration of the compound is from about 5μg/ml to about 100μg/ml.

40. The method of claim 27, wherein the concentration of the compound is 20μg/ml.

41. A method of altering a pathway of fatty acid synthesis in a bacterium which comprises contacting the bacterium with a compound having the structure

wherein each of R ₁ , R₂ , R₃ , R₄ , R₅ and R₆ may be independently H, F, Cl, Br, I, -OH, -OR₇, -CN, -COR₇ , -SR 7 , -N(R₇) ₂ , -NR₇ COR₈ , -NO₂, -(CH₂) _p OR₇, -(CH₂) _p X(R ₇ ) ₂ , -(CH₂ ) XR₇ COR₈ , a straight chain or branched, substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C 1₀ alkenyl, C₂-C₁₀ alkynyl, C₃ -C₁₀ cycloalkyl, C₃ -C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl; wherein a linkage to the benzene ring may alternatively be -N- , -S-, -O- or -C-; wherein R₇ or R ₈ may be independently H, F, Cl , Br, I, - OH, -CN, -COH, -SH₂ , -NH₂ , -NHCOH, -(CH₂ ) _p OH, -(CH₂ ) X(CH₂ ), - (CH ₂ )_p XCOH, a straight chain or branched, substituted or unsubstituted C ₁ -C ₁₀ alkyl, C₂ -C₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, C₃ -C₁₀ cycloalkyl, C₃ -C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl; wherein A may be -N₂- , -NH-, -C=C=CH₂-, -CΞC-C ₂ HOH-, -CΞC-CH₂ -, -CH ₂ -CH₂ -O-, -CH₂ -CH ₂ -CH₂ -O-, -S-, -S(=O) ₂-, -C=O-, -C=O-O- , -NH-C=O- , -C=O-NH-; and wherein Q, p, N and X may independently be an integer from 1 to 10, or if Q is 1 A may be a (C₁ -C₁₀)- alkyl chain, (C ₁ -C ₁)₀-alkenyl chain or (C₁ -C₁₀)-alkynyl chain which is branched or unbranched, substituted or unsubstituted and can optionally be interrupted 1 to 3 times by -O- or -S- or -N- ; or a pharmaceutically acceptable salt or ester thereof, thus altering the pathway of fatty acid synthesis.

42. The method of claim 41, wherein A comprises an (C₁ -C 1₀)-alkylene chain, (C ₁ -C ₁)₀-alkyl chain, (C ₁ -C ₁₀)- alkenyl chain or (C ₁ -C ₁₀ )-alkynyl chain which is branched or unbranched, substituted or unsubstituted and can optionally be interrupted 1 to 3 times by -O- or -S- or -N-.

43. The method of claim 41, wherein

R₁ = R₄ = CH₃ or -OH,

R₂ = R₃ = R₅ = R₆ = H or -OH,

A = CH₂,

and Q = 3.

44. The method of claim 41, wherein

R₃ = Cl,

R_{1 =} R_{2 =} R_{4 =} R_{5 =} R₆ = H or -OH,

and Q = 0.

45. The method of claim 41, wherein

R₆ = CH(CH₃)₂,

R₁ = R₂ = R₄ = R₅ = H or -OH, and Q = 0.

46. The method of claim 41, wherein the bacterium is

Legionella pneumophila, Mycobacterium tuberculosis,

Bacillus subtilis, Bacillus Megaterium, Pseudomonas Oleovorans, Alcaligenes eutrophus, Rhodococcus sp. , Ci trobacter freundi , Group A Streptococcus sp. , Coag neg Staphylococcus aureus or Nocardia sp.

47. A method of inhibiting growth of a bacterium which consists essentially of contacting the bacteria with an enoyl reductase inhibitor so as to inhibit the reductase and thus inhibit the growth of the bacterium.

48. A method for determining whether or not a bacterium is sensitive to a compound having the structure:

wherein each of R ₁ , R₂ , R₃ , R₄ , R₅ and R₆ may be independently H, F, Cl, Br, I, -OH, -OR₇, -CN, -COR₇, -SR 7 , -N(R₇) ₂ , -NR₇ COR₈ , -NO ₂ , -(CH₂ ) _p OR ₇ , -(CH₂ ) _p X(R ₇ ) ₂ , -(CH₂ ) XR₇ COR₈ , a straight chain or branched, substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C 1₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C ₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl; wherein a linkage to the benzene ring may alternatively be -N-, -S-, -O- or -C-; wherein R₇ or R₈ may be independently H, F, Cl, Br, I, -OH, -CN, -COH, -SH₂ , -NH₂ , -NHCOH, -(CH₂ )_p OH, -(CH₂ )_p

X(CH ₂ ), -(CH₂ )_p XCOH, a straight chain or branched, substituted or unsubstituted C ₁ -C ₁₀ alkyl, C₂ -C₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, C₃ -C₁₀ cycloalkyl, C₃ -C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl; wherein A may be -N₂- , -NH-, -C=C=CH₂-, -C=C-C ₂ HOH-, -CΞC-CH₂ -, -CH₂ -CH₂ -O-, -CH₂ -CH₂ -CH₂ -O-, -S-, -S(=O) ₂-, -C=O-, -C=O-O-, -NH-C=O- , -C=O-NH-; and wherein Q, p, N and X may independently be an integer from 1 to 10, or if Q is 1 A may be a (C ₁ -C ₁₀ )-alkyl chain, (C ₁ -C ₁)₀-alkenyl chain or (C ₁ -C ₁)₀-alkynyl chain which is branched or unbranched, substituted or unsubstituted and can optionally be interrupted 1 to 3 times by -O- or -S- or -N-; or a pharmaceutically acceptable salt or ester thereof, which comprises contacting the bacterium with a concentration of the compound effective to inhibit growth of the bacterium if the bacterium is sensitive to the compound, thereby determining whether or not the bacterium is sensitive to the compound.

49. The method of claim 48, wherein A comprises an (C₁ -C

1₀)-alkylene chain, (C ₁ -C ₁)₀-alkyl chain, (C ₁ -C ₁₀)- alkenyl chain or (C ₁ -C ₁₀)-alkynyl chain which is branched or unbranched, substituted or unsubstituted and can optionally be interrupted 1 to 3 times by -O- or -S- or -N-.

50. The method of claim 48, wherein

R₁ = R₄ - CH₃ ,

R₂ = R₃ = R₅ = R₆ = H or -OH,

A = CH₂ or -OH ,

and Q = 3 .

51. The method of claim 48, wherein

R₃ = Cl,

R_{1 =} R_{2 =} R_{4 =} R_{5 =} R₆ = H or -OH,

and Q = 0.

52. The method of claim 48, wherein

R₆ = CH(CH₃)₂,

R₁ = R₂ = R₄ = R₅ = H or -OH, and Q = 0.

53. The method of claim 48, wherein R₃ = Cl,

^R6 ^{= C}2^H5'

R₁ = R₂ = R₄ = R₅ = H or -OH, and Q = 0.

54. The method of claim 48, wherein the bacterium is in a cell.

55. The method of claim 48, wherein the bacterium is selected from the group consisting of Legionella pneumophila, Bacillus subtilis, Caulobacter crescentus, Ci trobacter freundi , Nocardia sp. , Rhodobacter spheroides, Group A Streptococcus sp . , Coag neg Staphylococcus aureus and Mycobacterium tuberculosis .

56. The method of claim 48, wherein the concentration of the compound is from about 5μg/ml to about lOOμg/ml.

57. The method of claim 48, wherein the concentration of the compound is 20 μg/ml.

58. A method of selecting a compound which is capable of inhibiting the enzymatic activity of enoyl reductase which comprises:

(A) contacting enoyl reductase with the compound;

(B) measuring the enzymatic activity of the enoyl reductase of step (A) compared with the enzymatic activity of enoyl reductase in the absence of the compound, thereby selecting a compound which is capable of inhibiting the enzymatic activity of enoyl reductase.

59. The method of claim 58, wherein the compound contacts enoyl reductase at the site at which gemfibrozil contacts enoyl reductase.