Antimicrobial activity of gemfibrozil and related compounds and derivatives and metabolites thereof

[0003] Throughout this application, various publications are referenced by a number in brackets. Full citations for these publications may be found listed by number at the end of the specification immediately preceding 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.

[0004] Gemfibrozil (GFZ) is a compound that has been utilized as a drug for increasing intracellular accumulation of hydrophilic anionic agents (U.S. Pat. No. 5,422,372, issued Jun. 6, 1995) and as a lipid regulating composition (U.S. Pat. No. 4,859,703, issued Aug. 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. Pat. 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).

[0005] The present invention provides for a method of inhibiting activity of an enoyl reductase enzyme in a cell which comprises contacting the cell with a compound having the structure:

[0006] wherein each of R₁, R₂, R₃, R₄, R₅and R₆is independently selected from the group consisting of: —H, —F, —Cl, —Br, —I, —OH, —OR₇, —CN, —COR₇, —SR₇, —N(R₇)₂, —NR; —COR₈, —NO₂, —(CH₂)_p—OR₇, —COSR₇, —COOH, —CONH₂, —NH₂, a straight chain or branched, substituted or unsubstituted C₁-C₁₀alkyl, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl;

[0007] wherein L is alternatively —N—, —S—, —C— or —C—;

[0008] wherein R₇is independently selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —CN, —COH, —SH₂, —NH₂, —NHCOH, —(CH₂)_pOH, a straight chain or branched, substituted or unsubstituted C₁-C₁₀alkyl, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl;

[0009] wherein A is selected from the group consisting of —N₂—, —NH—, —C═C═CH₂—, —C≡C—C₂HOH—, —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—;

[0010] wherein Q is independently 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 is optionally interrupted 1 to 3 times by —O— or —S— or —N—;

[0011] wherein X is —CO₂—, —CH═CH₃, phenyl, substituted phenyl, or heteroaryl, —O-phenyl(CH₃)₂, —C(CH₂)₂—CO—NH₂, —C(CH₂)₂—COOH;

[0012] or a pharmaceutically acceptable salt or ester thereof, which compound is present in a concentration effective to inhibit activity of the enzyme.

[0013] FIG. 1. Comparison of fatty acids synthesized by E. coli[5], and L. pneumophila[6, 7]. The number immediately preceding the colon refers to the number of carbons, the number following the colon refers to the number of double bonds, the superscript number refers to the location of the double bonds. a=anteiso; i=iso; OH=hydroxy.

[0014] FIG. 2. Fatty acid synthesis in E. coli. Initiation of fatty acid synthesis occurs with the condensation of acetyl-CoA with malonyl-ACP, or, conversion of acetyl-CoA to acetyl-ACP prior to condensation with malonyl-ACP. Subsequent elongation occurs though the sequential addition of two carbon units using malonyl-ACP as the donor. Elongation occurs through a four step process in which the first step, condensation, is mediated by β-ketoacylsynthase (FabB, FabF, FabH); the second step, reduction, is mediated by β-ketoacyl reductase (FabG); the third step, dehydration, is mediated through β-hydroxyacyl dehydratase (Fab Z); and the fourth and final step, reduction, is mediated through enoyl reductase (FabI). Unsaturated fatty acids are synthesized by the diversion of β-hydroxydecanol-ACP to FabA which catalyzes the formation of a double bond and then returns the unsaturated fatty acid to the cycle. The double lines indicate points where compounds act to inhibit fatty acid synthesis. The compounds are: DZB, diazoborines; ETH, ethionamide; INH, isoniazid; CER, cerulenin; TLM, thiolactomycin; NAG, 3-decenoyl-N-acetylcysteamine.

[0015] FIG. 3. GFZ inhibits intracellular multiplication of L. pneumophilain phorbol myristate acetate-differentiated HL-60 cells. HL-60 cells were differentiated into macrophages by treatment with PMA for 48 hours, plated as a monolayer in the wells of a microtiter plate, and synchronously infected with L. pneumophila(final multiplicity of infection of 0.01). After 2 hrs, the cells were washed to remove extracellular bacteria, overlaid with fresh RPMI-2 mM GLN-10% NHS without or with GFZ (100 μg/ml), and incubated at 37° C. At the times indicated, the cells and medium were harvested and assayed for L. pneumophila. Illustrated is an experiment typical of three performed. Each point is the average of three separate wells (+/−) the SEM.

[0016] FIG. 4. GFZ protects HL-60 cells from the cytotoxic effects of an L. pneumophilainfection over a five day incubation period. HL-60 cells were differentiated and infected in microtiter wells as described in Materials and Methods. GFZ was added to a final concentration of 0 or 100 μg/ml 2.5 hours post-infection. After 5 days, the dye MTT was added, and the A₅₇₀of each well was measured. The A₅₇₀value is proportional to the number of viable macrophages in the wells. Each point is the average of six separate wells (+/−) the SEM. This experiment is representative of three experiments, all of which yeilded similar results.

[0017] FIG. 5. GFZ inhibits the first round of intracellular multiplication of L. pneumophilain PMA-differentiated HL-60 cells. PMA-differentiated HL-60 cells at a concentration of 4×10⁶cells/ml were synchronously infected with L. pneumophilaat an MOI of 0.01 in suspension. 100 μl aliquots 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 at 37° C. prior to incubating with gentamicin 100 μg/ml for 0.5 hours 37° C. The gentamicin-containing medium was then washed away, and replaced with fresh medium without or with GFZ 100 μg/ml. Intracellular multiplication was measured by lysing the monolayers, and titering the combined lysate and supernatant at the times indicated. Each point is the average (+/−) the SEM of three separate cultures. This experiment is representative of three such experiments.

[0018] FIG. 6. GFZ inhibits L. pneumophilagrowth within human monocyte-derived macrophages. Human blood monocytes, maintained in culture for five days, were suspended in fresh medium and infected with L. pneumophilaat a MOI of 0.01. 100 μl of the infection mixture was aliquoted to the wells of a 96 well plate, pelleted at 220 g and 880 g to pellet the bacteria and macrophages, and incubated for 2.5 hr at 37° C. to allow the cells to internalize the bacteria. 100 μl of fresh medium containing 2× the final drug concentration was then added to each well, and incubated at 37° C. At the indicated times, the cells and medium were harvested and assayed for L. pneumorhilaCFUs. GFZ=gemfibrozil; BZF=bezafibrate; CFA=clofibrate. Each point represents the average of 3 wells (+/−) the SEM. This experiment is representative of three.

[0019] FIG. 7. Structures of the fibric acids used in these experiments; gemfibrozil (GFZ), clofibrate (CFX), bezafibrate (BZF), fenofibrate (FNF). Note that GFZ, CFA, BZF, were used as the free acids, while FNF was used as the isopropyl ester.

[0020] FIG. 8. 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 2 mg gemfibrozil to the plate, and incubating the plate at the appropriate temperature until bacterial growth was apparent. The presence of a zone of inhibition was considered positive for sensitivity. The fraction in parenthesis after each bacterial species indicates how many of the strains tested of each species was sensitive to GFZ.

[0021] FIG. 9. GFZ zones of inhibition for S. aureusand S. epidermidison LB versus TSB agar medium. Four ATTC S. aureusstrains and one S. epidermidisstrain were grown overnight in Brain-Heart Infusion (BHI) broth. The cultures were diluted to 10⁷and 10⁶CFUs/ml in BHI, and 100 μl aliquots of each dilution were plated on LB or TSB agar in duplicate. The plates were incubated at 37° C. overnight prior to measuring the diameter of the zones in mm.

[0022] FIG. 10. Susceptibility of M. tuberculosisto GFZ. Twenty seven M. tuberculosisstrains, demonstrating different drug resistance profiles, were tested for sensitivity to gemfibrozil. OADC-enriched Middlebrook agar plates with quadrants containing 0, 100, or 200 μg/ml of GFZ in were prepared. 100 μls of a standard dilution of each resuspended M. Tuberculosisstrain in sterile water was 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 number of colonies in 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 drugs to which each strain are resistant are indicated to the left of each strain; S=streptomycin 2 μg/ml; I=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^L=Low level resistance to isoniazid at 0.2 μg/μl, but sensitive to isoniazid at higher concentrations.

[0023] FIG. 11. GFZ inhibits the growth of M. tuberculosisstrains in 7H9 broth. Approximately 10⁷bacteria were added to 5 mls of Middlebrook 7H9 broth with glycerol and incubated at 37° C. After 21 days the cultures were visually assessed for turbidity.

[0024] FIG. 12. F4b has increased resistance to GFZ in an HL-60 intracellular infection assay. HL-60 cells were differentiated into macrophages with PMA, plated as a monolayer in the wells of a microtiter plate, and synchronously infected with L. pneumophilaF4b or L. pneumophilaPhiladelphia 1 (final multiplicity of infection of 0.01). After 2 hrs, the cells were washed to remove non-phagocytosed and non-adherent bacteria, overlaid with fresh RPMI-2 mM GLN-10% NHS without or with GFZ (100 μg/ml), and incubated at 37° C. in a humidified atmosphere containing 95% air and 5% CO₂. At the times indicated, the cells and medium were harvested and assayed for L. pneumophila. Illustrated is an experiment typical of two performed. Each point is the average of three separate wells (+/−) the SEM.

[0025] FIG. 13. Structural analogs of GFZ tested for activity against wild type L. pneumophilaPhill and L. pneumophilaF4b by a zone of inhibition assay. Salicylate=SAL; 4-hydroxyphenylpropionic acid (4-HPA); 3,4-hydroxyphenylpropionic acid (3,4-HPA); gemfibrozil (GFZ).

[0026] FIG. 14. Structural analogs of GFZ demonstrate antibacterial activity against L. pneumophila(P1) and the L. pneumophiladerived mutant F4b. Sterile disks containing either 2.5 mg or 1.0 mg of GFZ, 3(p⁴-hydroxyphenyl)propionic acid (4-HPA), 3,4-dihydroxyphenyl propionic acid (3,4-HPA), and 2-hydroxybenzoic acid or salicylate (SAL) were added to BCYE plates overlaid with L. pneumophilaPhiladelphia 1 or F4b and incubated at 37° C. After four days the diameters of the zones were measured.

[0027] FIGS. 15A-D. Effect of GFZ on the accumulation of electron-lucent inclusions by L. pneumophilaPhiladelphia 1. (A and B) Electron micrographs of L. pneumophilagrown for three days on CYE agar at 37° C. without (10,000×) (A) or with (20,000×) (B) GFZ (30 μg/ml). (C and D) Fluorescence micrographs (1000×) of Nile Blue A stained L. pneumophilagrown for three days on CYE agar at 37° C. without (C) or with (D) GFZ (30 μg/ml).

[0028] FIG. 16. Effect of GFZ on 3-HB content of L. pneumophila. L. pneumophilawere grown on CYE agar with GFZ (30 μg/ml) or without GFZ for 3 days at 37° C., harvested and lyophilized. 0.16 mg of benzoic acid (internal standard) was added to 40 mg of each lyophilized sample, and the corresponding propyl esters were formed by hydrochloric acid propanolysis. The amount of 3-HB in each L. pneumophilasample was calculated from a standard curve generated by derivitizing known amounts of 3-HB and benzoic acid to their corresponding propyl esters. Both the standard curve and the L. pneumophilaanalyses were performed in triplicate using three sets of independently prepared samples for each.

[0029] FIG. 17. Putative interactions between the fatty acid synthesis pathway and the PHB synthesis pathway. There are four reactions in each cycle of fatty acid elongation. The first is condensation of malonyl-ACP with acetyl-ACP or a longer chain acyl-ACP to form a ketoester. In E. coli, this reaction can be catalyzed by FabH, FabB, or FabF. In organisms that synthesize branched chain fatty acids such as Legionella sp., butyryl-ACP, rather than acetyl-ACP, is often preferred for condensation with malonyl-ACP for initiation of fatty acid synthesis. The second step involves the NADPH-dependent reduction of the ketoester catalyzed by FabG. The third step involves dehydration of the substrate by FabA or FabZ to form enoyl-ACP. Reduction of the enoyl-ACP by FabI, an enoyl reductase, produces an acyl-ACP that can be further elongated by additional cycles of fatty acid synthesis or utilized by the cell for phospholipid synthesis. Due to the instability of the enoyl-ACP compound, inhibition of FabI has been shown to result in the accumulation β-hydroxybutyryl-ACP in E. coli. In L. pneumophila, inhibition of fatty acid synthesis could result in accumulation of acetyl-CoA or butyryl-CoA. Both are primers for fatty acid synthesis in bacteria, like L. pneumophila, which synthesize branched chain fatty acids. Alternatively, PhaG from Psuedomonas putida is known to catalyze the conversion of β-hydroxybutyryl-ACP into β-hydroxybutyryl-CoA, a substrate for PHB synthesis. L. pneumophilamay contain a homolog of this P. putidaenzyme.

[0030] FIG. 18. PHB synthesis pathway. PHB synthesis from acetyl-CoA generally occurs through a three step biosynthetic pathway. The first step involves condensation by the β-ketothiolase, PhbA, of two acetyl CoA molecules to form acetoacetyl-CoA. The second step involves the formation of 3-hydroxybutyryl-CoA (3-HB-CoA) by the acetoacetyl-CoA dehydrogenase, PhbB. The final step involves polymerization of the 3-HB-CoA monomers by the PHB synthase, PhbC. However, certain species of bacteria have the ability to utilize substrates from other metabolic pathways such as fatty acid synthesis and β-oxidation to form PHA. For example, PhaG of Pseudomonas putida, mediates the conversion of the fatty acid synthesis intermediate 3-HB-ACP to (R)-3-HB-CoA [110], while PhaJ, identified in Aeromonas caviae, mediates the hydration of enoyl-CoA substrates from the β-oxidation pathway [111]. Completion of β-oxidation results in the formation of acetyl-CoA which can be utilized as a substrate for either fatty acid synthesis or PHB synthesis. Accumulation of acetyl CoA, due to inhibition of either the tricarboxylic acid cycle or fatty acid synthesis, could result in increased incorporation of acetyl-CoA into PHB. The lightly shaded circles represent cycles which either synthesize or utilize substrates, represented by the dark shaded boxes, putatively involved in PHB synthesis in L. pneumophila.

[0031] FIG. 19. PHB synthesis pathway. PHB synthesis from acetyl-CoA generally occurs through a three step biosynthetic pathway. The first step involves the condensation of two acetyl CoA molecules to form cetoacetyl-CoA by the β-ketothiolase, PhbA. The second step involves the formation of 3-hydroxybutyryl-CoA 3-HB-COA) by the acetoacetyl-CoA dehydrogenase, PhbB. The final step involves the polymerization of the monomer unit 3-HB-CoA by PHB synthase, PhbC. However, certain species of bacteria have the ability to utilize substrates from other metabolic pathways to form PHB. For example, PhaG of Pseudomonas putida, mediates the conversion of the fatty acid synthesis intermediate 3-HB-ACP to (R)-3-HB-CoA [110], while Alcaligenes eutrophushas been demonstrated to utilize exogenous butyrate, presumably following its derivitization to butyryl-CoA, oxidation to crotonyl-CoA, and hydration to (R)-3-HB-CoA [109]. PhaJ, identified in Aeromonas caviae, mediates the hydration of enoyl-CoA substrates from the β-oxidation pathway [111]. The double lines indicate pathways, which when blocked, may increase the utilization of these intermediates by the PHB synthesis pathway.

[0032] FIG. 20. Fatty acid biosynthesis in bacteria. Precursors such as acetyl-CoA, butyryl-CoA, isobutyryl-CoA, valeryl-CoA and 2-methylbutyryl-CoA are utilized by various bacterial species for condensation with malonyl-CoA to initiate fatty acid synthesis. Subsequent elongation occurs though the sequential addition of two carbon units using malonyl-CoA as the donor. Elongation occurs through a four step process in which the first step, condensation is mediated by β-ketoacyl synthase; the second step, reduction is mediated by β-ketoacyl reductase; the third step, dehydration, is mediated through β-hydroxyacyl dehydratase; and the fourth and final step, reduction, is mediated through enoyl reductase. Additional enzymes mediating reactions allowing for fatty acyl modifications include β-hydroxydecanol dehydrase, which catalyzes double bond formation, and cyclopropyl fatty acid synthase, which catalyzes the formation of cyclopropane fatty acids. Regulation of fatty acid composition is achieved by mechanisms including multiple isoforms of enzymes with different substrate specificities and regulatory characteristics }[5], temperature sensitivity }[10], and transcriptional control }[114].

[0033] FIG. 21. GFZ inhibits ¹⁴C-acetate incorporation into lipids in L. pneumophila. Increasing concentrations of GFZ (10, 25, 50, or 100 μ/ml) were added to L. pneumophilaPhiladelphia 1 cultures in the presence of ¹⁴C-acetate. At the indicated times, 100 μl aliquots were TCA precipitated and analyzed for ¹⁴C-acetate content by scintillation spectrometry. The experiment was repeated three times and the results shown here are of one experiment, representative of the three performed.

[0034] FIG. 22. F4b, a partially GFZ-resistant L. pneumophilavariant displays increased resistance to GFZ-mediated inhibition of ¹⁴C-acetate incorporation into lipids. GFZ at the indicated concentrations (25, 50, 75, 100 μg/ml) was added to a L. pneumophilaF4b in AYE medium containing ¹⁴C-acetate (5 μCi/ml). The bacteria were incubated at 37° C. for the indicated times. 100 μl aliquots were TCA precipitated and analyzed for ¹⁴C content by scintillation spectrometry. 50% inhibition of ¹⁴C-acetate incorporation relative to the control, was achieved at a GFZ concentration of 100 μg/ml (0.4 mM). This experiment was performed only once. It is consistent with the finding that F4b has an MIC value approximately five times higher than that of wild type L. pneumophila.

[0035] FIG. 23. GFZ decreases ¹⁴C-acetate incorporation into chloroform/methanol extracts of L. pneumophilalipids. GFZ (10-100 μg/ml) was added to L. pneumophilacultures in the presence of ¹⁴C-acetate. After 60 min incubation at 37° C., the bacteria were pelleted and extracted with chloroform/methanol/double distilled H₂O (1:1:0.9). The extracts were assayed by scintillation spectrometry and autoradiography of TLC plates. By autoradiography, ¹⁴C-acetate incorporation into the various lipid species appeared equally inhibited at every GFZ concentration tested. Lane 1: GFZ 0 μg/ml; Lane 2 GFZ 10 μg/ml; Lane 3 GFZ 25 μg/ml; Lane 4 GFZ 50 μg/ml; Lane 5 GFZ 100 μg/ml. Liquid scintillation counting of duplicate samples indicated that 50% inhibition, relative to the control, was achieved at a GFZ concentration of 10 μg/ml, or 40 uM. This experiment was performed only once. It is consistent with the TCA precipitation results reported earlier in FIG. 21.

[0036] FIG. 24. Comparison of TCA precipitation and chloroform/methanol extraction as a measure of GFZ inhibition of ¹⁴C-acetate incorporation in L. pneumpchilalysates. GFZ or cerulenin at 0.4 mM or 2.0 mM concentrations was added to L. pneumophilalysates in the presence of ¹⁴C-acetate (5 μCi/ml), total volume 500 μl, and incubated at 37° C. for one hour. 200 μl of each incubation mixture was TCA precipitated, and 300 μl was extracted with chloroform/methanol/double distilled H₂O (1:1:0.9). The amount of ¹⁴C-acetate incorporated into TCA precipitable or chloroform/methanol extractable material in each incubation mixture was measured by scintillation counting and is expressed as a percent of the uninhibited control. This experiment was performed once. It is consistent with the data from while cells (FIG. 21).

[0037] FIG. 25. Comparison of the effects of GFZ, bezafibrate, and cerulenin on ¹⁴C-acetate incorporation in L. pneumophilalysates. GFZ, cerulenin (CER), or bezafibrate (BZF) at 2 mM, or EDTA at 10 mM, was added to L. pneumophilalysates in the presence of ¹⁴C-acetate (10 μCi/ml) in triplicate and incubated at 37° C. in a final reaction volume of 100 μl. One lysate was heated in a boiling water bath for ten minutes prior to the addition of ¹⁴C-acetate. At 10, 20, and 30 minutes, the reactions were TCA precipitated and pelleted. The TCA precipitate was then extracted with chloroform/methanol/double distilled H₂O (1:1:0.9). The chloroform/methanol extracts were assayed by scintillation spectrometry to determine relative rates of ¹⁴C-acetate incorporation into lipids. The experiment was repeated twice with similar results. The data shown are from a representative experiment.

[0038] FIG. 26. Structures of the GFZ analogs tested for inhibition of ¹⁴C-acetate incorporation into L. pneumophilawhole cells or lysates. SAL=salicylate; PAS=paraaminosalicylate; ASAL=acetylsalicylate; INH=isoniazid; CFA=clofibrate; 4-HPA=4-hydroxypropionate; 3,4-HPA=3,4-hydroxypropionate; GFZ=gemfibrozil; TEVA (B-H)=TEVA analogs.

[0039] FIGS. 27A-B. Ability of GFZ analogs to inhibit ¹⁴C-acetate incorporation into the TCA precipitates of intact L. pneumophila. TEVA analogs (A-H) were added to L. pneumophilain AYE medium containing ¹⁴C-acetate at a concentration of 0.4 mM. At various time points 100 μl aliquots of the cultures were TCA precipitated and analyzed for ¹⁴C-acetate content by scintillation spectroscopy.

[0040] FIGS. 28A-B. TEVA analogs C and D inhibit fatty acid synthesis in L. pneumophilacultures. Increasing concentrations of (FIG. 28A) analog C and (FIG. 28B) analog D were added to a L. pneumophilaPhiladelphia 1 cultures in the presence of ¹⁴C-acetate. At the indicated time points, 100 μl aliquots were TCA precipitated and the precipitates were analyzed for ¹⁴C-acetate content by scintillation spectrometry. 50% inhibition, relative to the control, was achieved at a concentration of 40 μM. Data presented are for one experiment using triplicate samples.

[0041] Ability of commercially available GFZ analogs to inhibit ¹⁴C-acetate incorporation into the TCA precipitates of intact L. pneumophila. Analogs were added to L. pneumophilain AYE medium containing ¹⁴C-acetate at a concentration of 0.5 mM. At various time points 100 μl aliquots of the cultures were TCA precipitated and analyzed for ¹⁴C-acetate content by scintillation spectroscopy.

[0042] FIG. 29. Sites of drug-mediated inhibition of fatty acid synthesis in E. coli. Initiation of fatty acid synthesis occurs with the condensation of acetyl-CoA with malonyl-ACP, or, conversion of acetyl-CoA to acetyl-ACP prior to condensation with malonyl-ACP. Subsequent elongation occurs though the sequential addition of two carbon units using malonyl-ACP as the donor. Elongation occurs through a four step process in which the first step, condensation, is mediated by β-ketoacyl synthases (FabB, FabF, FabH); the second step, reduction is mediated by β-ketoacyl reductase (FabG); the third step, dehydration, is mediated by β-hydroxyacyl dehydratase (Fab Z); and the fourth and final step, reduction, is mediated by enoyl reductase (FabI). Unsaturated fatty acids are synthesized by the diversion of β-hydroxydecanol-ACP to FabA which catalyzes the formation of a double bond and then returns the unsaturated fatty acid to the cycle. The double lines indicate points where compounds act to inhibit fatty acid synthesis. The compounds that inhibit at each site are; DZB, diazoborines; ETH, ethionamide; INH, isoniazid; TCN, triclosan; CER, cerulenin; TLM, thiolactomycin; NAG, 3-decenoyl-N-acetylcysteamine.

[0043] FIG. 30. Sequence alignment of enoyl reductases from L. pneumophila, E. coli, S. typhimurium, H. influenza, and M. tuberculosis(SEQ ID NOS:1-5). Completely conserved residues are indicated by bold type, while highly conserved residues are indicated by an asterisk. The figure was produced using the Clustal W program [143].

[0044] FIG. 31. Effect of Temperature and GFZ on growth of E. Colitransformed with an L. Pneumophilaenoyl reductase homolog.

[0045] FIG. 32. Effect of oleate and palmitate supplementation on the growth of E. coliFT100 and FT101 strains at 30° C. versus 43° C., without or with GFZ 500 μg/ml. An isogenic pair of E. colistrains, FT100 and FT101, transformed with vector-only (pCR2.1), or vector containing the L. pneumophilaenoyl reductase gene (pCK1), were grown at 30° C. in LB(Km)(—NaCl) broth to late log/early stationary phase. Cultures were serially diluted, plated on low osmolarity (0.2% NaCl) LB(Km) plates without (−) or with (+) GFZ 500 μg/ml, and without or with 0.3 mM palmitate (PA) or oleate (OA) alone or in combination. The plates were incubated for 2 days at 30° C. or 43° C., and the colonies counted. Solid bars=control; shaded bars=GFZ 500 μg/ml.

[0046] FIG. 33. Alignment of the FabX and FabT enoyl reductases from L. pneumophila(SEQ ID NOS:6 and 7).

[0047] FIG. 34. SDS-PAGE analysis of His-tagged L. pneumophilaFabX and FabT. 400 ml cultures of E. coliBL21 DE3 transformed with the pET15b:FabX, pET15b:FabT, or pET15b:FabI vectors were grown in LB-Amp broth at 37° C. to an OD of 1.0 at 600 nm. IPTG was added to a final concentration of 1 mM and the cultures were incubated for an additional three hours. Bacterial pellets were harvested by centrifugation and lysed by sonication. The His-tagged proteins were purified by nickel column chromatography. The purified products were eluted from the column with 400 mM imidazole. Aliquots were analyzed for purity by SDS-PAGE. 1=protein standard; 2=L. pneumophilaFabX lysate; 3=flow through after washing the FabX column with 40 mM imidazole; 4=400 mM imidazole eluate of the FabX column; 5=L. pneumophilaFabT lysate; 6=flow through after washing the FabT column with 40 mM imidazole; 7=400 mM imidazole eluate of the FabT column; 8=E. coliFabI lysate; 9=flow through after washing the FabI column with 40 mM imidazole; 10=400 mM imidazole eluate of the FabI column.

[0048] FIG. 35. Comparison of the specific activities of L. pneumophilaFabT and FabX, and of E. coliFabI, for crotonoyl-CoA. His-tagged FabX, FabT, or FabI, was incubated with increasing concentrations of crotonoyl-CoA (CCA) in the presence of excess NADH. The rate of NADH hydrolysis was assessed for each enzyme for each concentration of CCA by measuring the decrease in absorbance over time at A₃₄₀nm. Specific activities were calculated. The specific activities for FabX and FabT are the average for two experiments (+/−) the SEM. The specific activity for was only measured once.

[0049] FIG. 36. Structures of the enoyl reductase substrates and inhibitors.

[0050] FIGS. 37A-C. GFZ-CoA is a competitive inhibitor of enoyl reductase activity for L. pneumophilaFab X and FabT, and M. tuberculosisInhA using dodecenoyl-CoA (DCA) as a substrate. Panel a=FabX; panel b=FabT; panel c=InhA. Reaction mixtures containing 100 mM NaPO₄pH 7.4, 100 μM NADH, and enoyl reductase, were combined with varying concentrations of DCA and GFZ. The ability of GFZ-CoA to inhibit the enoyl reductase activity was assessed by measuring the change in absorbance over time at A₃₄₀nm as NADH was oxidized to NAD⁺. Concentrations of GFZ-CoA μM) utilized are indicated in bold type next to the corresponding plot. Inhibition was competitive with regard to the DCA substrate.

[0051] FIG. 38. Flow chart of the methods utilized for the ³H-GFZ metabolic labeling studies.

[0052] FIG. 39.280 nm absorbance tracing for the T=120 minute >10 kDA FPLC sample. 500 ml of the <10 kDa filtrate was loaded onto a Superose™ FPLC column and run at 0.5 ml/min with a paper speed of 0.5 mm/min. Each square is 2 mm².

[0053] Therefore, the large boxes correspond to 5 mls or 10 minutes, while the small boxes correspond to 1 ml or 2 minutes. 0.5 ml fractions were collected post 280 nm detection. The injection is on the right side of the tracing and the four major peaks are numbered in order of appearance. The radioactive fractions are indicated as dots along the X-axis.

[0054] FIG. 40. FPLC analysis of cytoplasmic extracts of L. pneumophilaincubated with ³H-GFZ. L. pneumophilawere incubated with ³H-GFZ in AYE broth at 37° C. Samples were collected at the times indicated. The samples were pelleted, washed, lysed, and filtered through 30 and 10 kDa Centricon filters. The filtrate was applied to a Superose12™ FPLC column. Fractions were collected and CPMs assessed by liquid scintillation spectrometry. Percentage of CPMs in each of the three major FPLC peaks relative to total CPMs for samples harvested at the time points indicated.

[0055] FIG. 41. HPLC of the ³H-GFZ standard. ³H-GFZ (1 μCi) dissolved in 100% EtOH was applied to a reverse phase C18 μBondapack™ column. 0.5 ml fractions were collected every thirty seconds and assessed for CPMs by liquid scintillation spectrometry.

[0056] FIG. 42. HPLC of the GFZ-CoA standard. 500 μl of a mixture of CoA (30%) and GFZ-CoA (70%) dissolved in water at a concentration of 500 μg/ml was added to a reverse phase C18 μBondapack™ column. 0.5 ml fractions were collected every thirty seconds and analyzed at OD=262 nm. Fractions eluting at 5-7 min (fractions #10-14) represent GFZ-CoA; the fraction eluting at 4 min (fraction #8) represents CoA.

[0057] FIGS. 43A-D. Summary of the HPLC data from FPLC fraction #53. Filtered lysates of L. pneumophilaincubated at 37° C. with ³H-GFZ for the indicated length of time were applied to an FPLC column and fractions were collected. Fraction #53 from the third peak to elute from this FPLC column was applied to a reverse phase HPLC column. Fractions from the HPLC column were collected and assessed by liquid scintillation counting. Fractions eluting from 12.5-15 minutes (fractions #25-30) contained radioactivity and co-chromatographed with ³H-GFZ as determined earlier (FIGS. 8-5). a) T=5 min; b) T=30 min c) T=60 min; d) T=180 min.

[0058] FIGS. 44A-D. Summary of the HPLC data from FPLC fraction #45. Filtered lysates of L. pneumophilaincubated with ³H-GFZ for the indicated length of time were applied to an FPLC column and fractions were collected. Fraction #45 from the second peak to elute from the FPLC column was applied to a reverse phase HPLC column and fractions were collected and assessed by liquid scintillation spectrometry. Fractions collected from 3-4 minutes (fractions #6-8) contained radioactivity and co-chromatographed with the breakdown product in the 3H-GFZ stock as determined earlier (FIGS. 8-5). Fractions collected from 12.5-15 minutes (fractions #25-30) also contained radioactivity and co-chromatographed with GFZ as determined earlier. a) T=5 min; b) T=30 min c) T=60 min; d) T=180 min.

[0059] FIGS. 45A-D. Summary of the HPLC data from FPLC fraction #38. Filtered lysates of L. pneumophilaincubated with ³H-GFZ for the indicated length of time were applied to an FPLC column and fractions were collected. Fraction #38 from the first peak to elute was applied to a reverse phase HPLC column and fractions were collected and assessed by liquid scintillation counting. Fractions collected from 3-4 minutes (fractions #6-8) contained radioactivity and co-chromatographed with the contaminant in the ³H-GFZ preparation as determined earlier (FIG. 42). Fractions collected from 12.5-15 minutes (fractions #25-30) contained radioactivity and co-chromatographed with GFZ as determined earlier. Fractions collected from 6-8 minutes (fractions #12-16) contained radioactivity and co-chromatographed with GFZ-CoA as determined earlier. a) T=5 min; b) T=30 min c) T=60 min; d) T=180 min.

[0060] FIG. 46. Percentage of CPMs in the putative ³H-GFZ-CoA peak, relative to total CPMs for each sample, increases as the incubation period of L. pneumophilawith ³H-GFZ increases.

[0061] The present invention provides for a method of inhibiting activity of an enoyl reductase enzyme in a cell which comprises contacting the cell with a compound having the structure:

[0062] wherein each of R₁, R₂, R₃, R₄, R₅and R₆is independently selected from the group consisting of: —H, —F, —Cl, —Br, —I, —OH, —OR₇, —CN, —COR₇, —SR₇, —N(R₇)₂, —NR₇—COR₈, —NO₂, —(CH₂)_p—OR, —COSR₇, —COOH, —CONH₂, —NH₂, a straight chain or branched, substituted or unsubstituted C₁-C₁₀alkyl, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl;

[0063] wherein L is alternatively —N—, —S—, —O— or —C—,

[0064] wherein R₇is independently selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —CN, —COH, —SH₂, —NH₂, —NHCOH, —(CH₂)_pOH, a straight chain or branched, substituted or unsubstituted C₁-C₁₀alkyl, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl;

[0065] wherein A is selected from the group consisting of —N₂—, —NH—, —C═C═CH₂—, —C≡C—C₂HOH—, —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—;

[0066] wherein Q is independently 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 is optionally interrupted 1 to 3 times by —O— or —S— or —N—;

[0067] wherein X is —CO₂—, —CH═CH₃, phenyl, substituted phenyl, or heteroaryl, —O-phenyl(CH₃)₂—C(CH₂)₂—CO—NH₂, —C(CH₂)₂—COOH;

[0068] or a pharmaceutically acceptable salt or ester thereof, which compound is present in a concentration effective to inhibit activity of the enzyme.

[0069] In one embodiment, A is selected from the group consisting of (C₁-C₁₀)-alkylene chain, (C₁-C₁₀)-alkyl chain, (C₁-C₁₀)-alkenyl chain or (C₁-C₁₀)-alkynyl chain which is branched or unbranched, substituted or unsubstituted and is optionally interrupted 1 to 3 times by —O— or —S— or —N—.

[0070] In another embodiment of the present invention

[0071] R₁=R₄=CH₃or —OH,

[0072] R₂=R₃=R₅=R₆=H or —OH,

[0073] A=CH₂,

[0074] and Q=3.

[0075] In another embodiment of the present invention,

[0076] R₃=Cl,

[0077] R₁=R₂=R₄=R₅=R₆=H or —OH,

[0078] and Q=0.

[0079] In another embodiment of the present invention,

[0080] R₆=CH(CH₃)₂,

[0081] R₁=R₂=R₄=R₅=H or —OH,

[0082] and Q=0.

[0083] In another embodiment of the present invention,

[0084] R₃=Cl,

[0085] R₆=C₂H₅,

[0086] R₁=R₂=R₄=R₅=H or —OH,

[0087] and Q=0.

[0088] In another embodiment, the enzyme is in a bacterium or the enzyme is in an eukaryotic cell. In one embodiment, the cell is a yeast cell, the cell is a fungus, the cell is a plant cell, or the cell is a protozoan cell.

[0089] In one embodiment, the concentration of the compound or the metabolite thereof is from about 5 μg/ml to about 200 μg/ml. In a preferred embodiment, the concentration of the compound is 100 μg/ml. In another preferred embodiment, the compound is administered at a concentration of 150 micrograms/ml/kg body weight.

[0090] The present invention also provides for a method of selecting a compound which inhibits the enzymatic activity of enoyl reductase which comprises: (A) contacting enoyl reductase with the compound or a metabolite thereof; (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 or the metabolite thereof, thereby selecting a compound or metabolite thereof which inhibits the enzymatic activity of enoyl reductase.

[0091] In one embodiment, the metabolite is a CoA metabolite. In another embodiment, the metabolite is an ACP metabolite. One of skill in the art would know of other metabolites which would be produced or generated during the fatty acid synthetic pathway.

[0092] The present invention provides a method of selecting a compound which inhibits the enzymatic activity of enoyl reductase which comprises: (A) contacting enoyl reductase with the compound or metabolite thereof, wherein the compound or metabolite thereof contacts enoyl reductase at the site at which gemfibrozil contacts enoyl reductase;(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 or metabolite thereof, thereby selecting a compound which inhibits the enzymatic activity of enoyl reductase.

[0093] The present invention also provides for a method for inhibiting growth of a bacterium which consists essentially of contacting the bacterium with a compound having the structure:

[0094] wherein each of R₁, R₂, R₃, R₄, R₅and R₆is independently selected from the group consisting of: —H, —F, —Cl, —Br, —I, —OH, —OR₇, —CN, —COR₇, —SR₇, —N(R₇)₂, —NR₇—COR₈, —NO₂, —(CH₂)_p—OR , —COSR₇, —COOH, —CONH₂, —NH₂, a straight chain or branched, substituted or unsubstituted C₁-C₁₀alkyl, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl;

[0095] wherein L is alternatively —N—, —S—, —O— or —C—;

[0096] wherein R₇is independently selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —CN, —COH, —SH₂, —NH₂, —NHCOH, —(CH₂)_pOH, a straight chain or branched, substituted or unsubstituted C-C₁₀alkyl, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl;

[0097] wherein A is selected from the group consisting of —N₂—, —NH—, —C═C═CH₂—, —C≡C—C₂HOH—, —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—;

[0098] wherein Q is independently 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 is optionally interrupted 1 to 3 times by —O— or —S— or —N—;

[0099] wherein X is —CO₂—, —CH═CH₃, phenyl, substituted phenyl, or heteroaryl, —O-phenyl(CH₃)₂, —C(CH₂)₂—CO—NH₂, —C(CH₂)₂—COOH;

[0100] or a pharmaceutically acceptable salt or ester thereof, which compound is present in a concentration effective to inhibit growth of the bacterium.

[0101] In one embodiment, A is selected from the group consisting of (C₁-C₁₀)-alkylene chain, (C₁-C₁₀)-alkyl chain, (C₁-C₁₀)-alkenyl chain or (C₁-C₁₀)-alkynyl chain which is branched or unbranched, substituted or unsubstituted and is optionally interrupted 1 to 3 times by —O— or —S— or —N—.

[0102] In one embodiment of the present invention, the bacterium is Legionella pneumophila. In another embodiment, the bacterium is Nocardia sp. In another embodiment, the bacterium is Staph auereous. In another embodiment, the bacterium is Mycobacterium tuberculosis.

[0103] In one embodiment, the bacterium is in a eukaryotic cell.

[0104] In another embodiment, the concentration of the compound is from about 5 μg/ml to about 100 μg/ml, or in a more preferred embodiment, the concentration is from 20 μg/ml to 100 μg/ml.

[0105] The present invention also provides for a method for alleviating the symptoms of a bacterial infection in a subject which comprises administering to the subject an amount of a compound having the structure:

[0106] wherein each of R₁, R₂, R₃, R₄, R₅and R₆is independently selected from the group consisting of: —H, —F, —Cl, —Br, —I, —OH, —OR₇, —CN, —COR₇, —SR₇, —N(R₇)₂, —NR₇—COR₈, —NO₂, —(CH₂)_p—OR₇, —COSR₇, —COOH, —CONH₂, —NH₂, a straight chain or branched, substituted or unsubstituted C₁-C₁₀alkyl, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl;

[0107] wherein L is alternatively —N—, —S—, —O— or —C—;

[0108] wherein R₇is independently selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —CN, —COH, —SH₂, —NH₂—NHCOH, —(CH₂)_pOH, a straight chain or branched, substituted or unsubstituted C₁-C₁₀alkyl, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl;

[0109] wherein A is selected from the group consisting of —N₂—, —NH—, —C═C═CH₂—, —C≡C—C₂HOH—, —C≡C—CH₂—, —CH₂—CH₂—O—, —CH₂—CH₂—CH₃—O—, —S—, —S(═O)₂—, —C═O—, —C═O—O—, —NH—C═O—, —C═O—NH—;

[0110] wherein Q is independently 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 is optionally interrupted 1 to 3 times by —O— or —S— or —N—;

[0111] wherein X is —CO₂—, —CH═CH₃, phenyl, substituted phenyl, or heteroaryl, —O-phenyl(CH₃)₂, —C(CH₂)₂—CO—NH₂, —C(CH₂)₂—COOH;

[0112] or a pharmaceutically acceptable salt or ester thereof, which compound is present in a concentration effective to alleviate the symptoms of a bacterial infection in the subject.

[0113] In one embodiment, the bacterial infection is associated with Legionella pneumophila, Mycobacterium tuberculosis, Bacillus subtilis, Bacillus Megaterium, Rhodococcus sp., Staph epidermidis, Group A Streptococcus sp., Coag neg Staphylococcus aureusor Nocardia sp.

[0114] In another embodiment, the bacterial infection is associated with Legionella pneumophila. In another embodiment, the bacterial infection is associated with Mycobacterium tuberculosis.

[0115] In another embodiment, the effective concentration of the pharmaceutically acceptable compound is about 100 micrograms/ml.

[0116] In another embodiment, the subject is a human or an animal. In another embodiment, the bacterial infection is associated with Leprosy, Brucella or Salmonella.

[0117] In another embodiment the concentration of the compound is from about 5 μg/ml blood of the subject to about 200 μg/ml blood of the subject. In another embodiment the concentration of the compound is 100 μg/ml blood of the subject. In another embodiment the administration to the subject is oral, subcutaneous, intraveneous or intramuscular.

[0118] The present invention provides for a method of altering (inhibiting or enhancing) a biochemical pathway of fatty acid synthesis in a bacterium which comprises contacting the bacterium with a compound having the structure

[0119] wherein each of R₁, R₂, R₃, R₄, R₅and R₆is independently selected from the group consisting of: —H, —F, —Cl, —Br, —I, —OH, —OR₇, —CN, —COR₇, —SR₇, —N(R₇)₂, —NR₇—COR₈, —NO₂, —(CH₂)_p—OR₇, —COSR₇, —COOH, —CONH₂, —NH₂, a straight chain or branched, substituted or unsubstituted C₁-C₁₀alkyl, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl;

[0120] wherein L is alternatively —N—, —S—, —O— or —C—;

[0121] wherein R₇is independently selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —CN, —COH, —SH₂, —NH₂, —NHCOH, —(CH₂)_pOH, a straight chain or branched, substituted or unsubstituted C₁-C₁₀alkyl, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl;

[0122] wherein A is selected from the group consisting of —N₂—, —NH—, —C═C═CH₂—, —C≡C—C₂HOH—, —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—;

[0123] wherein Q is independently 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 is optionally interrupted 1 to 3 times by —O— or —S— or —N—;

[0124] wherein X is —CO₂—, —CH═CH, phenyl, substituted phenyl, or heteroaryl, —O-phenyl(CH₃)₂, —C(CH₂)₂—CO—NH₂, —C(CH₂)₂—COOH;

[0125] or a pharmaceutically acceptable salt or ester thereof, which compound is present in a concentration effective to alter the pathway of fatty acid synthesis in the bacterium.

[0126] In one embodiment, the compound has the structure:

[0127] In another embodiment, the compound has the structure:

[0128] In another embodiment, the compound has the structure:

[0129] In another embodiment, the compound has the structure:

[0130] The present invention provides for a method of inhibiting activity of an enoyl reductase enzyme in a cell which comprises contacting the cell with a compound having the structure:

[0131] wherein R₂is selected from the group consisting of: —H, —F, —Cl, —Br, —I, —OH, —OR₇, —CN, —COR₇, —SR₇, —N(R₇)₂, —NR₇—COR₉, —NO₂, —(CH₂)_p—OR₇, —COSR₇, —COOH, —CONH₂, —NH₂, a straight chain or branched, substituted or unsubstituted C₁-C₁₀alkyl, C₂C₁₀alkenyl, C₂-C₁₀alkynyl, C₃-C₁₀cycloalkyl, C₃C₁₀cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl;

[0132] wherein Q is independently an integer from 1 to 10;

[0133] wherein R₇is independently selected from the group consisting of —H, —F, —Cl, —Br, —I, —OH, —CN, —COH, —SH₂, —NH₃, —NHCOH, —(CH₂)_pOH, a straight chain or branched, substituted or unsubstituted C₁-C₁₀alkyl, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl;

[0134] or a pharmaceutically acceptable salt or ester thereof, which compound is present in a concentration effective to inhibit enoyl reductase enzyme in the cell.

[0135] 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.

[0136] The present invention also provides for a pharmaceutical composition comprising a compound or metabolite thereof having any one of the structures shown or described hereinabove and a pharmaceutically acceptable carrier.

[0137] 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 200 μg/ml blood of the subject. In one embodiment, the concentration of the compound may be 100 μg/ml blood of the subject. The administration to the subject may be oral.

[0138] 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.

[0139] 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., Citrobacter freundi, Group A Streptococcus sp., Coag neg Staphylococcus aureusor 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.

[0140] 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) 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. The compound may contact enoyl reductase at same site at which gemfibrozil contacts enoyl reductase. U.S. Pat. No. 5,422,372 discloses a method of increasing intracellular accumulation of hydrophilic anionic agents using gemfibrizol (gemfibrozil). U.S. Pat. No. 4,859,703 discloses lipid regulating compositions. U.S. Pat. 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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 onohydric 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] The emergence of multiply antibiotic-resistant bacterial pathogens (i.e. M. tuberculosisand S. aureus) has prompted the search for new or unrecognized antibiotic targets in bacteria. Most currently used antibiotics act by blocking bacterial protein, DNA or RNA synthesis, and/or cell wall assembly. However, as demonstrated by the ability of isoniazid and ethionamide to inhibit InhA [1,2], an enoyl reductase of M. tuberculosis[3], bacterial enzymes involved in fatty acid synthesis are also potential antibiotic targets.

[0149] While bacterial and mammalian cells use the same general pathways and mechanisms to synthesize fatty acids, bacterial fatty acid synthases differ from their mammalian counterparts in a number of respects. For example, mammalian fatty acid synthase is a type I synthase, a homodimer composed of a single polypeptide encoding seven distinct enzymatic functions. Type I synthases perform all of the reactions required for the synthesis and elongation of fatty acids in mammals [4]. Bacterial fatty acid synthases are most commonly type II syntheses. Type II synthases are dissociated fatty acid synthase systems composed of individual proteins encoded by distinct genes. Within this system, multiple isozymes of a given protein often exist which catalyze the same basic chemical reaction but differ in substrate specificity and regulation [5].

[0150] Bacteria synthesize many fatty acids not synthesized by human cells (i.e. branched chain fatty acids, dihydroxy fatty acids). The presence of these fatty acids is hypothesized to allow bacteria to maintain membrane fluidity and function upon exposure to a variety of environmental insults including variations in temperature and osmolarity. Drugs that block synthesis of these unique bacterial fatty acids, by inhibiting bacteria-specific enzymes, may block bacterial growth without having a detrimental effect on mammalian cells. Accordingly, isoniazid and ethionamide act by inhibiting an enoyl reductase involved in the synthesis of mycolic acids, very long chain fatty acids synthesized by M. tuberculosis, but not by human cells.

[0151] The findings reported here indicate that gemfibrozil (GFZ), a commonly prescribed and well-tolerated hypolipidemic agent, inhibits an L. pneumophilaenoyl reductase, and has antibiotic activity against a wider spectrum of bacteria than isoniazid. Our findings suggest that bacterial enoyl reductases may be more useful targets for novel antibiotics than previously recognized.

[0152] Differences in Bacterial and Mammalian Fatty Acid Synthesis

[0153] Bacterial fatty acid synthases differ from their mammalian counterparts in a number of respects, providing potential targets for antimicrobial therapy. For example, human or mammalian fatty acid synthase (FAS) is a Type I synthase. In general, Type I synthases are multifunctional proteins which perform all or many of the reactions required for the synthesis and elongation of fatty acids in mammals [4]. In contrast, bacterial FAS are most commonly Type II. Type II synthases are dissociated systems composed of individual proteins encoded by distinct genes. Within this system, multiple isozymes of a given protein often exist which catalyze the same basic chemical reaction but differ in substrate specificity and regulation [5].

[0154] Not surprisingly, the products synthesized by bacterial Type II synthases are more varied and complex than those synthesized by mammalian Type I syntheses. The end products of Type I mammalian fatty acid synthases are generally palmitate, a sixteen carbon saturated fatty acid (C_16:0) myristate (C_14:0), and laurate (C_12:0). In contrast, bacterial Type II FAS systems synthesize a complex assortment of fatty acids the profiles of which can differ greatly among species of bacteria (FIG. 1). The synthesis of these fatty acids is hypothesized to allow bacteria to maintain constant membrane fluidity and function upon exposure to a variety of environmental pressures including variations in temperature and osmolarity.

[0155] The bacterial enzymes involved in the synthesis of these specialized fatty acids generally perform the same basic reactions as those performed by mammalian fatty acid synthases, but have widely different substrate specificities and regulatory characteristics. However, there are some enzymatic functions which are specific to bacteria including the formation of unsaturated fatty acids during elongation, and the formation of cyclopropyl, hydroxylated, and ω-alicyclic fatty acids [8]. Drugs that block the synthesis of unique bacterial fatty acids by inhibiting the utilization of bacteria-specific substrates or bacteria-specific enzymes, may block bacterial growth without having a detrimental effect on mammalian cells. Accordingly, the clinically effective and prescribed drugs isoniazid and ethionamide act by inhibiting an enoyl reductase involved In the synthesis of mycolic acids, very long chain fatty acids which are synthesized by M. tuberculosis, but not by human nest cells [1].

[0156] Fatty Acid Synthesis in Bacteria

[0157] Both bacteria and mammalian cells utilize acetyl-CoA as the building block for fatty acid synthesis. Bacteria acquire acetyl-CoA from the decarboxylation of pyruvate when grown on sugars, and from β-oxidation when grown on fatty acids. In mammalian cells, acetyl-CoA is derived largely from the tricarboxylic acid (TCA) cycle. High concentrations of ATP and NADH in the mitochondria inhibit the TCA enzyme isocitrate dehydrogenase resulting in the accumulation of citrate. Citrate diffuses from the mitochondrion into the cytosol where it is converted to acetyl-CoA and then to malonyl-CoA for fatty acid elongation, or to acetyl-ACP for the initiation of fatty acid synthesis.

[0158] In E. coli, the initiation or elongation of straight chain fatty acid synthesis can occur through three different condensation reactions; condensation of an acetyl-CoA with malonyl-ACP, condensation of acyl-ACP with malonyl-ACP, and the decarboxylation of malonyl-ACP to acetyl-ACP followed by condensation with malonyl-ACP. All three condensation reactions form acetoacetyl-ACP (FIG. 2) [5]. The condensation step is the only irreversible step in fatty acid synthesis in Type II systems. E. colihas three different β-ketoacyl synthases with overlapping substrate specificities. However, β-ketoacyl synthase I [9] catalyzes an essential step in unsaturated fatty acid metabolism, that cannot be catalyzed by the other two syntheses. β-ketoacyl synthase II [10] is involved in the thermal regulation of fatty acid composition, and β-ketoacyl synthase III [11] catalyzes the initial condensation reaction in the pathway. in mammalian cells, straight chain fatty acid synthesis is initiated through the condensation of acetyl-ACP and malonyl-CoA by the β-ketoacyl synthase domain of the fatty acid synthase enzyme.

[0159] β-ketoacyl synthase activity also mediates the synthesis of branched chain fatty acids by the condensation of branched chain precursors with malonyl-CoA at the initiation step of fatty acid synthesis. The primer sources for the branched chain fatty acids are generally 2-keto-acids derived from the branched chain amino acids, valine, leucine, and isoleucine [12,13]. Branched chain fatty acids are synthesized by many species of bacteria and by the sebaceous glands of mammalian skin.

[0160] The next step in both straight and branched chain fatty acid elongation involves the reduction of the β-ketoacyl-ACP by β-ketoacyl-ACP reductase to a β-hydroxybutyryl-ACP. Dehydration by β-hydroxyacyl dehydrase yields crotonoyl-ACP. This reaction is very inefficient, and the ratio of substrate to product is generally 9:1 [14]. in E. coli, there are two dehydrase enzymes which can catalyze this step [15]. One is involved in the elongation of saturated fatty acids, and the other serves as the branch point for the synthesis of unsaturated fatty acids.

[0161] The final step in the fatty acid elongation cycle involves the reduction of the enoyl-ACP substrate to generate an acyl-ACP by enoyl reductase. The acyl-ACP is either the end product of fatty acid synthesis, or, serves as the starting material for subsequent cycles of fatty acid elongation. Since the concentration of the enoyl substrate is very low, enoyl reductase is thought to “pull” successive cycles forward. For this reason it is thought to be the rate-limiting enzyme in fatty acid synthesis [14].

[0162] Inhibition of phospholipid synthesis due to increased levels of the stationary phase alarmone ppGpp in bacteria, leads to the accumulation of acyl-ACPs and inhibition of fatty acid synthesis [16]. In a reconstituted fatty acid synthesis assay, the addition of palmitoyl-ACP to the reconstituted system resulted in the accumulation of malonyl ACP and 3-hydroxybutyryl-ACP (3-HB-ACP), presumably by inhibiting of enoyl reductase and β-ketoacyl synthase III. However, 3-HB-ACP accumulated first, and at higher concentrations than malonyl-ACP, indicating that enoyl was the relevant target of palmitoyl-ACP's inhibitory effect [17].

[0163] The accumulation of long chain acyl-CoAs occurs during bacterial growth in the presence of long chain fatty acids. Exogenous long chain fatty acids are converted to their CoA thioesters by E. coli, and are either used as substrates for β-oxidation, or are preferentially incorporated into phospholipids following conversion to their ACP derivatives [18-20]. Long chain acyl-CoAs have been shown to directly inhibit enoyl reductase activity and to bind to the global transcriptional regulator FadR. The interaction of long chain acyl-CoAs (derived from oleate or palmitate) with FadR releases FadR from DNA, stimulates β-oxidation and fatty acid transport into E. coli, and inhibits three genes involved in fatty acid synthesis in these bacteria; fabA, β-hydroxydecenoyl dehydrase, the enzyme responsible for unsaturated fatty acid synthesis, fabb, one of three β-ketoacyl synthases, and the enoyl reductase fabl [21-24].

[0164] Inhibitors of Fatty Acid Synthesis

[0165] Several compounds that inhibit enzymes in fatty acid synthesis have been described. Cerulenin is an inhibitor of fatty acid synthesis in prokaryotes and eukaryotes that acts on β-ketoacyl synthase to inhibit the condensation of an acyl-ACP or an acetyl-CoA with malonyl-ACP [25]. However, since cerulenin inhibits both bacterial and mammalian fatty acid synthesis, it is not clinically useful as an antimicrobial, but is being pursued as a chemotherapeutic agent to treat cancers which over express FAS [26,27]. Thiolactomycin, is a specific inhibitor of Type II bacterial β-ketoacyl synthases [28] and is active against many species of Gram-positive and Gram-negative bacteria [29]. However, resistance is frequently acquired [30,31]. 3-decanoyl-N-acetylcysteamine (NAG) is an inhibitor of β-hydroxydecenoyl thioester hydrase, a bacterial enzyme catalyzing the synthesis of unsaturated fatty acids [32,33]. This compound inhibits unsaturated fatty acid synthesis in bacteria including E. coli, but not in mammalian cells.

[0166] Several compounds have been reported to interfere with bacterial enoyl reductase activity including isoniazid, ethionamide, triclosan and related compounds, and diazoborines Of all the compounds that inhibit enzymes in bacterial fatty acid synthesis, only two, isoniazid and ethionamide, are useful as drugs and only for the treatment of mycobacterial infections [34].

[0167] Isoniazid and ethionamide inhibit the InhA enoyl reductase enzyme of M. tuberculosis[1-3,35]. Mutations in InhA, at or near residues involved in NADH binding, confer resistance to these compounds [1,2] as do mutations affecting the intracellular levels of NADH [36]. Isoniazid is a prodrug. Kat G, a catalase-peroxidase enzyme of M. tuberculosis[37] catalyzes the formation of an activated isonicotinic acyl radical which interacts with NADH bound at the active site of the InhA enzyme [2]. The carbonyl carbon of the isonicotinic acyl group covalently attaches to the carbon at position four of the nicotinamide ring, replacing the 4S hydrogen of NADH involved in hydride transfer during the reduction of an enoyl substrate. The complex inactivates the enzyme since it displaces the side chain of Phe¹⁴⁹allowing it to form an aromatic ring stacking interaction with the pyridine ring of the isonicotinic group. This conformational change increases the affinity of the complex for the enzyme, such that it is not released. Mutations which decrease the affinity of InhA for NADH may protect the enzyme by promoting the binding of acyl-ACP substrates before NADH binds. The binding of an acyl-ACP substrate does not allow the bulkier activated isonicotinic acyl radical access to the active site.

[0168] Isoniazid also has been reported to inhibit the M. tuberculosisfatty acid synthesis β-ketoacyl synthase enzyme, KasA. Mutations in the amino-acid sequence of the KasA protein, were identified in INH-resistant clinical strains of M. tuberculosisthat lacked other known mutations conferring resistance to INH.

[0169] Diazoborines, another group of enoyl reductase inhibitors, exhibit antibacterial activity against most species of Gram-negative bacteria [38] by a similar but distinct mechanism to that of INH. The boron atom in diazoborine forms a covalent bond with the 2-hydroxyl oxygen of the nicotinamide ribose of NADH, generating a bi-substrate analog. The bicyclic rings of the diazoborines form a face to face interaction with the nicotinamide ring allowing extensive π—π stacking interactions. Crystallographic studies show that this bi-substrate analog binds non-covaiently, but tightly, to E. coliFabI enoyl reductase [39], interfering with the access of the reduced pyridine nucleotide (NADH or NADPH) to E. coliFabI's catalytic site. The activity of this class of compounds is dependent on the presence of the boron substituent, which is toxic for mammalian systems [38].

[0170] Triclosan, a topical antiseptic, not approved for oral administration, appears to inhibit E. colienoyl reductases by a mechanism similar to the diazoborines [40,41]. The phenolic hydroxyl group forms a hydrogen bond (not covalent as for the diazoborines) with the 2-hydroxyl oxygen of the nicotinamide ribose of NADH. The phenol ring of triclosan forms a face to face interaction with the nicotinamide ring allowing extensive π—π stacking interactions. Homologous mutations in the E. colifabi and M. tuberculosisinhA genes, confer resistance to diazoborines, triclosan, or isoniazid, consistent with a NADH-dependent mechanism of inhibition.

[0171] In conclusion, while the basic mechanisms of fatty acid synthesis between the mammalian Type I synthases and the bacterial Type II synthases are conserved, significant differences exist. These differences should be exploitable for the creation of new classes of antibiotics. The need for antibiotics that inhibit bacterial growth by mechanisms other than those used by current antibiotics is increasing as the number of bacterial species resistant to multiple drugs grows. The findings that isoniazid and ethionamide [1], the diazoborines [42], and triclosan [40] all act by inhibiting enoyl reductases suggest that this key regulatory enzyme in fatty acid biosynthesis is an excellent antimicrobial target.

[0172] Gemfibrozil (Lopid™) is well known as a hypolipidemic agent that lowers LDL and triglyceride levels in humans. The mechanism(s) by which GFZ exerts this effect is unresolved. GFZ has also been reported to inhibit organic anion transport in mouse J774 macrophages [43]. Although the endogenous substrates for this transporter have not been identified, it is known that anionic compounds, including Lucifer Yellow, fluorescein, penicillin and the fluoroquinolone antibiotics ciprofloxacin and norfloxacin, are efficiently secreted by J774 macrophages by GFZ inhibitable transporters [43-46].

[0173] Inhibitors of anion efflux should increase the intracellular concentration of anionic antibiotics, thus increasing the efficacy of a given oral or intravenous dose for intracellular pathogens. Addition of GFZ in combination with norfloxacin, reduced by fourfold the concentration of norfloxacin required to block intracellular growth of Listeria monocytogenesin mouse J774 macrophage-like cells [46]. This was consistent with previous findings in which treatment of J774 cells with GFZ increased the intracellular concentration of norfloxacin in the J774 cells fourfold [44].

[0174] L. monocytogenesgrows in the cytoplasm of macrophages, Other intracellular pathogens reside in specialized membrane-bound intracellular compartments. For such pathogens, increasing the concentration of antibiotics in the cytosol may have no effect if the concentration of the antibiotic is not increased in the pathogen-containing compartment. Alternatively, if the antibiotic readily penetrates the pathogen-containing compartment, then increases in the cytoplasmic concentration of the given antibiotics should potentiate the antimicrobial effect of the antibiotic. Since GFZ exerts the latter effect on fluoroquinolone antibiotics it was desirable to evaluate the effect cf GFZ in combination with these antibiotics against intracellular pathogens that grew within membrane-bound compartments in macrophages. We began with Legionella pneumophila, an intracellular pathogen responsible for up to percent of all community-acquired pneumonias requiring hospitalization [47].

[0175] L. pneumophilais an environmental pathogen most commonly found in water sources such as shower heads, water towers and air conditioning condensers. Aerosolization of contaminated water sources allows the bacteria to be inhaled into the lungs where it infects alveolar macrophages. L. pneumophilaenters macrophages within phagosomes produced as a result of a process known as coiling phagocytosis }[48]. The Legionella-containing phagosomes go through a unique series of modifications such that acidification is avoided, and mitochondria, smooth vesicles, ribosomes and rough endoplasmic reticulum are recruited to their periphery [49-52]. In human macrophages, or in the macrophage-like cells of the HL-60 human myelocytic cell line, bacterial replication generally begins within eight hours of bacterial uptake [52,53]. Twenty four to thirty six hours after infection, the cells round up, undergo either bacterially induced lysis or apoptosis [53-55], and release the expanded population of intracellular bacteria for subsequent rounds of infection.

[0176] While testing the ability of GFZ to increase the efficacy of fluoroquinolone antibiotics in a L. pneumophilainfection model, I discovered that GFZ alone inhibited the intracellular growth of L. pneumophilain peripheral-blood monocyte derived human macrophages and in the phorbol myristace acetate (PMA)-differentiated macrophage-like cells of the HL-60 human promyelocytic cell line. Results: Addition of GFZ to a final concentration of 0.4 mM (100 μg/ml) to the medium of PMA-differentiated HL-60 cells infected with L. pneumophilaresulted in inhibition of L. pneumophilaintracellular growth (FIG. 3). To determine whether growth inhibition was due to a cytotoxic effect of GFZ on the HL-60 cells, cellular viability assays were performed on uninfected and infected HL-60 cells. The MTT assay was used, which assesses cellular respiration via the reduction of a tetrazolium dye to an insoluble blue formazan in viable cells [53,56,57] to test HL-60 viability. GFZ at 100 μg/ml did not affect the viability of uninfected HL-60 cells, even when the cells were incubated or five days. Furthermore, the presence of GFZ in the medium protected infected HL-60 cells from L. pneumophila'scytolytic effects even at an infection multiplicity of 0.1 bacteria/cell (FIG. 4). Therefore, it was possible that GFZ was directly inhibiting the growth of L. pneumophila.

[0177] To assess whether GFZ had the potential to directly inhibit L. pneumophilagrowth, a minimum inhibitory concentration (MIC) assay was performed. L. pneumophilawas added to ACES-buffered yeast extract (AYE) liquid medium containing varying concentrations of GFZ. The MIC which inhibited 90% of L. pneumophilagrowth was 10 μg/ml of GFZ. At all concentrations tested (10-200 μg/ml), GFZ was bacteriostatic for L. pneumophilaPhiladelphia 1, as determined by plating 5 pl aliquots of the MIC cultures on charcoal yeast extract (CYE) and visually assessing growth after four days of incubation at 37° C.

[0178] L. pneumophiladoes not grow in human plasma or in mammalian tissue culture medium so it was unlikely that GFZ blocked L. pneumophilagrowth by affecting extracellular bacteria. Nonetheless, a single-round growth assay was developed to confirm that GFZ had access to, and was inhibiting the growth of intracellular L. pneumophilawithin HL-60 cells. In this assay gentamicin was added to the medium two hours after L. pneumophilainfection to kill any remaining extracellular bacteria. After a 30 minute incubation, the gentamicin-containing medium was washed away and replaced with fresh medium containing GFZ at a final concentration of 0.4 mM (100 μg/ml). Inhibition of bacterial growth was seen within 8 hours after infection, well within the 24-36 hour time period required for L. pneumophilato lyse these cells [50,54,55] (FIG. 5).

[0179] This experiment shows that GFZ inhibits the growth of L. pneumophilaintracellularly. L. pneumophilaalso grows intracellularly in primary human blood monocyte-derived macrophages [54,58]. In accordance with the results found in the HL-60 cell line, GFZ at a concentration of 0.4 mM (100 μg/ml), inhibited the intracellular growth of L. pneumophilain human macrophages (FIG. 6). GFZ at 0.4 mM was optimal since no further reduction in L. pneumophilacolonies was observed with GFZ 0.6 mM.

[0180] Although the previous experiments demonstrated that growth inhibition was not due to a cytotoxic effect in HL-60 cells, it was still possible that GFZ was inhibiting growth through a “fibric acid-mediated” effect on the macrophages. GFZ belongs to a class of fibric acids including clofibrate, fenoflbrate, and bezafibrate, all of which lower serum triglyceride levels though an unresolved mechanism. These analogs are also known to stimulate peroxisome proliferator activated receptors (PPARs) in rats, although this effect does not occur in humans. PPARs are transcriptional activators which affect genes in several metabolic pathways including cholesterol biosynthesis [59-61] and the β-oxidation of fatty acids [62,63].

[0181] To test whether GFZ inhibition of intracellular L. pneumophilagrowth occurred through a fibric acid mediated effect on host cell gene expression, other fibric acids were examined (FIG. 6). When bezafibrate was added to human macrophages infected with L. pneumophilaat a concentration of 1 mM, no inhibition of L. pneumophilagrowth was observed. Clofibrate, another structural analog of GFZ FIG. 7), was able to slow L. pneumophilagrowth although a concentration of 1 mM was required. Fenofibrate precipitated out of solution at a concentration of 0.1 mM, so it was not studied in this system. The ability of these fibric acids to inhibit L. pneumophilagrowth in AYE broth was assessed by MIC assays. While GFZ has an MIC of 10 μg/ml, clofibrate was found to have an MIC of 100 μg/ml, and bezafibrate an MIC of 250 μg/ml. Fenofibrate precipitated out of solution at a concentrations above 100 μg/ml, and did not inhibit L. pneumophilagrowth at this concentration.

[0182] This is the first report of GFZ's antibiotic activity. It was highly surprising and serendipitous to find that a compound used safely in man for over twenty years to lower triglycerides, inhibited bacterial growth. This suggested zhat GFZ might be inhibiting a bacterial target which is not expressed in humans or is resistant to GFZ in humans.

[0183] The mechanism by which GFZ exerts its hypolipidemic effect is unknown. At least seven different pathways in lipid metabolism have been shown to be affected by GFZ, either in vivo or in cell culture. The seven pathways affected by GFZ are: 1) GFZ stimulates lipoprotein lipase activity, cleaving fatty acids from triglycerides In the VLDL fraction of the plasma; 2) GFZ increases the activity of lipoprotein lipase [61,64]; 3) GFZ stimulates intracellular triglyceride synthesis [65] possibly mediated through an increase in acyl-CoA synthetase expression [66]; 4) GFZ promotes the transcription of several enzymes involved in the β-oxidation of fatty acids through the stimulation of peroxisome proliferator activated receptors (PPARs), although peroxisome proliferation has only been demonstrated in rats [62,63]; 5) GFZ decreases microsomal fatty acid elongation [67,68]; 6) GFZ stimulates the synthesis of Apo A-1, the major protein in HDL [59,69] and 7) GFZ decreases the synthesis of cholesterol [60]. Any or all of these mechanisms could be involved in lowering serum lipids.

[0184] Although GFZ inhibits intracellular L. pneumophilagrowth within macrophages, the concentration required to inhibit growth within these cells (100 μg/ml) is ten-fold higher than that required to inhibit growth in AYE broth (10 μg/ml). The finding that a lower concentration of an antibiotic is required to inhibit extracellular versus intracellular growth of a bacterial pathogen is not uncommon. Other antibiotics which inhibit L. pneumophilagrowth, both extracellularly and intracellularly are erythromycin, doxycycline, and rifampin. All three of these antibiotics inhibit extracellular growth of L. pneumophilaat lower concentrations than intracellular growth (e.g. erythromycin at 0.2 μg/ml versus 1.25 μg/ml, doxycycline at 0.4 μg/ml versus 0.8 μg/ml, and rifampin at 0.002 μg/ml versus 0.1 μg/ml [70-73]. For an antibiotic to inhibit the intracellular growth of a bacterial pathogen, it must enter the host cell, enter the compartment that contains the pathogen, and be retained within that compartment in an active form [74].

[0185] Therefore, with respect to L. pneumophila, several explanations could account for the difference in MICs. First, in work not described here, the addition of 10% serum to AYE increased GFZ's MIC from 10 μg/ml to 30 μg/ml, suggesting that serum contains factors capable of binding GFZ, thereby reducing its effective concentration. Charcoal is known to have a similar effect such that the MICs of antibiotics for L. pneumophilaon ACES-buffered charcoal yeast extract (ABCYE) agar [75], range from two to twenty fold higher than the MIC values found on buffered potato starch yeast extract (BSYE) agar. The latter contains potato starch instead of charcoal [76]. Second, GFZ may be transported out of the host cell cytoplasm, or, out of the ribosome-studded compartment in which L. pneumophilagrows, thus limiting the amount available to inhibit intracellular L. pneumophila. Third, the host cell may metabolize GFZ to either a less active compound, or, a compound that has limited access to the L. pneumophila-containing compartment. Fourth, L. pneumophilamay be more resistant to GFZ when growing intracellularly. Barker et al. reported that intracellular growth enhances resistance to antibiotics in L. pneumophila[77]. Fifth, metabolites provided by the host cell may reduce GFZ's inhibitory effect.

[0186] We draw several conclusions from these experiments. First, a fibric acid mediated effect on host cell transcription is not likely to be the cause of GFZ's ability to inhibit the intracellular growth of L. pneumophilasince bezafibrate was totally ineffective and clofibrate much less effective than GFZ at inhibiting intracellular growth (FIG. 6). Second, the ability of GFZ to inhibit the growth of L. pneumophilain AYE broth at a concentration of 10 μg/ml demonstrates that GFZ has a direct inhibitory effect on L. pneumophila. The ability of GFZ to inhibit intracellular growth strongly suggests that GFZ has access to the unique intracellular compartment in which L. pneumophilaresides. Further work with ³H-GFZ is required to confirm this suggestion. Third, metabolites provided by the macrophages do not circumvent the capacity of GFZ to inhibit the intracellular growth of L. pneumophila.

[0187] While many potential antibiotics are often highly effective against pathogens in laboratory conditions, they often fail in animal infection models and/or clinical trials due to unexpected toxicity, or a lack of effect, in a mammalian host. Since GFZ has been used therapeutically for years, there is a large body of literature confirming its safety, even at much higher levels that those used to treat hyperlipidemia [78,79].

[0188] In humans, GFZ is reported to achieve a serum concentration of 12-15 μg/ml within 2 hours following a single oral dose of 450-600 mg [78,79]. These levels were sustained for an additional four hours, but fell to 5 μg/ml by 9 hours after administration [78-81]. Although the MIC (10 μg/ml) for L. pneumophilain nutrient broth is within the serum levels achieved for the treatment of hypertriglyceridemia, a concentration of 100 μg/ml was required to inhibit intracellular L. pneumophilagrowth. However, plasma drug levels are proportional to the dose [82], so a serum GFZ concentration of 100 μg/ml could be achieved through higher dosage. A serum concentration of 100 μg/ml in mice would only require 3.24 mg/mouse per day (1.08 mg every eight hours) well below the LD₅₀of 250 mg/20 gm mouse per day for mice [78,79].

[0189] In summary, these experiments revealed that GFZ is inhibitory for L. pneumophilagrowth in AYE broth and within macrophages. This finding has relevance for the use of GFZ to treat bacterial infection in humans since GFZ is known to have relatively low toxicity in humans, even with long term use as a therapeutic agent for hypertriglyceridemia. Further studies are needed to assess the potential therapeutic use of GFZ, or related analogs, in the treatment of human bacterial infections.

[0190] Bacterial and Growth Conditions. L. pneumophiliaPhiladelphia 1 serogroup 1 was a generous gift from Dr. Marcus Horowitz [83]. L. pneumopholawas grown in ACES-buffered yeast extract (AYE) broth [71] that lacked bovine serum albumin at 37° C. with aeration, or, on N-[2-acetomido]-2-aminoethane sulfonic acid (ACES)-buffered charcoal yeast extract (ABCYE) agar plates [75], at 37° C. in the presence or absence of GFZ. GFZ and ACES were purchased from Sigma. All other media components were purchased from Fisher.

[0191] HL-60 Intracellular L. pneumophilainhibition assays. Tissue culture medium Roswell Park memorial Institute (RPMI) 1640 tisue culture medium was obtained from JRH Biosciences, Lenexa, Kans.; L-glutamine (GLN) from Mediatech, Herndon, Va., and phorbol 12-myristate 13-acetate (PMA) from Sigma. and normal human serum (NHS) (Ultraserum™) from Gemini Bio-Products, Calabasas, Calif. Promyelocytic HL-60 cells were differentiated into macrophage-like cells by incubation with 10 ng/ml PMA in RPMI with 2 mM GLN and 10% NHS in Teflon wells at 37° C. for 24 hours. These cells were washed, resuspended at 4×10⁶cells/ml in RPMI-2 mM GLN-10% NHS, and mixed with L. pneumophilaPhil 1 (4×10⁴CFU/ml) which had been grown for 2 days on ABCYE plates (final multiplicity of infection of 0.01). 100 μl aliquots of the suspension were plated in each well of a 96 well microtiter plate. The plates were centrifuged to pellet the cells and bacteria and incubated at 37° C. for 2.5 hours to allow phagocytosis of the L. pneumophila. 100 μl of fresh medium with or without 2× the final GFZ concentration, was added to the wells, and the plates were incubated at 37° C. At the times indicated, the cells and medium were harvested and assayed for L. pneumophilaCFUs as described [58].

[0192] For the one-step assay, following pelleting of HL-60 cells and bacteria, the plates were incubated for 2.5 hrs to allow the HL-60 cells to internalize the bacteria. Gentamicin (100 μg/ml) was added for 0.5 hrs to kill extracellular bacteria. The cells were washed to remove gentamicin, overlaid with 200 μl fresh RPMI-2 mM GLN−10% NHS without or with GFZ (100 μg/ml), and incubated at 37° C. At the times indicated, the cells and medium were harvested and assayed for L. pneumophilaCFUs as described [58]. L. pneumophilais unable to grow in tissue culture medium, so any increase in colony forming units reflects intracellular multiplication [54]. Data from the experiments are expressed as the average (+/−) the S.E.M (n=3).

[0193] HL-60 Cytotoxicity Assay.

[0194] HL-60 cells (4×10⁵cells per well) were differentiated in the wells of a 96 well microtiter dish by incubation for two days at 37° C. in an atmosphere of 95% air 5% CO₂with 10 ng/ml PMA in RPMI-2 mM GLN−10% NHS. Adherent cells were washed two times with RPMI-2 mM GLN and then incubated with RPMI-2 mM GLN−10% NHS (+/−) 100 μg/ml GFZ. 5-fold serial dilutions of L. pneumophlain RPMI, were added to the wells at multiplicities of 0.5, 0.1, 0.02, 0.004, and 0.0, starting with 2×10⁵bacteria added per well. After a 5 day incubation at 37° C., the dye MTT ((3-4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma), was added to each well at a concentration of 500 μg/ml. In this assay the A₅₇₀is proportional to the number of viable macrophages in the wells [58]. The microtiter dishes were incubated for 4 hrs at 37° C., the culture medium was aspirated, and the reduced formazan dye was suspended in 100 μl of 0.04 M HCl-1% sodium dodecyl sulfate in isopropanol. The A₅₇₀values of six separate wells, that had been seeded with 4×10⁵HL-60 cells infected with L. pneumophilainfected at the same multiplicity of infection and incubated with or without GFZ, were averaged to determine macrophage viability.

[0195] Human Peripheral Blood Monocyte-derived Macrophage Assays.

[0196] Human leukocytes were purified by layering buffy coats on Histopaque-1077 and centrifuging at 400×g for 15 min. The leukocyte layer was washed 3× in RPMI 1640-2 mM GLN, resuspended in RPMI 1640-2 mM GLN−20% heat-inactivated human serum, added to 75 cm²flasks and incubated at 37° C. in an atmosphere of 95% air, 5% CO₂to allow adhesion of monocytes. After two hours the nonadherent cells were removed by washing and fresh RPMI 1640-2 mM GLN−20% heat-inactivated human serum, was added to the adherent monolayers. Following 12 hours incubation, the medium was replaced with PD buffer containing 5 mM EDTA, and the flasks were incubated at 37° C. 5% CO₂for 30 minutes to detach the monocytes. The detached monocytes were pelleted at 400×g for 15 minutes, and resuspended in RPMI 1640-2 mM GLN+30% heat-inactivated human serum, and stored in Teflon wells at 37° C. in humidified incubators in an atmosphere of 95% air and 5% CO₂. Human monocyte derived macrophages maintained in culture for five days were resuspended in fresh RPMI 1640-2 mM GLN+10% normal human ultraserum containing L. pneumophilaat an MOI of approximately 0.001. 100 μl aliquots containing 4×10⁵monocyte-derived macrophages and 1×10²L. pneumophilawere pelleted in the wells of a 96 well microtiter plate and incubated at 37° C. for 2.5 hours to allow phagocytosis of the L. pneumophila. 100 μl of fresh medium with or without 2× the final concentration of GFZ, clofibrate or fenofibrate, was added to each well. The plates were incubated at 37° C. in humidified incubators in an atmosphere of 95% air and 5% CO₂. At the indicated times, the cells and medium were harvested and assayed for L. pneumophilaCFU's.

[0197] Antimicrobial Susceptibility Testing.

[0198] For determination of MICs, triplicate cultures of log phase L. pneumophilasuspensions, final concentration 2×10⁶CFU/ml, were incubated in AYE broth containing two-fold serial dilutions of GFZ, fenofibrate, clofibrate, or bezafibrate for 48 hrs at 37° C. Growth was assessed by the optical density at A₆₀₀. Bacteriostatic effect was determined by incubating L. pneumophilasuspensions, final concentration 2×10⁶CFU/ml, in AYE broth containing two-fold serial dilutions of GFZ (10-200 μg/ml) for 48 hrs at 37° C. 5 μl from each culture was spotted on ABCYE agar and incubated at 37° C. for three days. L. pneumophilagrew in all spots indicating that GFZ was bacteriostatic, not bacteriocidal.

[0199] Gemfibrozil, Lopid™, a compound prescribed for hypertriglyceridemia in humans, was discovered to be an inhibitor of L. pneumophilagrowth in AYE broth (MIC₉₀=10 μg/ml) and in macrophages (100 μg/ml). The discovery that gemfibrozil (GFZ) inhibited the growth of L. pneumophilasuggested that GFZ might inhibit additional bacterial species. GFZ demonstrated activity against 33% of the bacteria screened, including Mycobacterium tuberculosis, Nocardia sp., Staphylococcus aureus, and Staphylococcus epidermidis. Two yeast species, Sacchromyces cerevisiaeand Candida albicanswere also found to be susceptible to GFZ.

[0200] The susceptibility of M. tuberculosiswas of particular interest since M. tuberculosisclaims more lives, roughly 3 million people per year, than any other single infectious disease in the world [84]. While the reported number of new cases of tuberculosis in the United States is declining [85], it is nearly impossible to eradicate the disease since it can remain dormant and undetected in immunocompetent hosts for years. On average at least 5% of immunocompetent hosts will develop active disease in their lifetimes [86]. The rate is significantly higher for those who are, or become, immunocompromised [87,88].

[0201] Despite the magnitude of the problem, no new primary anti-tuberculosis medicines have been developed since the 1960's [89]. The emergence and rapid spread of multiple drug resistant M. tuberculosisstrains has led to renewed interest in the development of compounds to treat and control this deadly organism. Unfortunately, significant mortality due to multiple drug resistant bacteria may no longer be limited to M. tuberculosis.

[0202] Screening of Various Species of Bacteria and Yeast for GFZ-mediated Growth Inhibition.

[0203] L. pneumophilaPhiladelphia 1 serogroup 1 was sensitive to growth inhibition by GFZ. To determine whether GFZ was specific for this strain of L. pneumophilawe tested its effect on 38 other Legionella sp. strains using a zone of inhibition assay. A disk containing 250 μg of GFZ was added to a CYE agar plate overlaid with the test bacterium. The absence of bacterial growth in the area adjacent to the disk, or a “zone of inhibition,” indicated sensitivity to GFZ. All 39 Legionella sp. strains were sensitive by this assay.

[0204] Additional nonpathogenic bacterial species were obtained from Dr. David Figurski and tested with the same assay. The finding that four of the eight strains tested demonstrated sensitivity to GFZ led to a collaboration with the Clinical Microbiology Department of Presbyterian Hospital to screen randomly-selected clinical strains of bacterial and fungal pathogens. The screen was performed by adding a sterile disk containing 2 mg of GFZ to a nutrient agar plate overlaid with the test pathogen. Eleven of the thirty one bacterial species tested, or 33%, demonstrated susceptibility to GFZ (FIG. 8). A variation of this assay was utilized for screening the mycobacterial strains in that disks containing 2 mg GFZ were embedded in 5 mls of nutrient agar prior to overlaying with bacteria.

[0205] Human pathogens in the susceptible group in addition to L. pneumophilaincluded M. tuberculosis, Nocardia sp., S. epidermidis, and S. aureus. All of the susceptible species are reported to contain branched chain fatty acids }[13], although not all bacteria containing branched chain fatty acids demonstrated susceptibility to GFZ (e.g. L. monocytogenes) [13,46].

[0206] S. aureusand S. epidermidissusceptibility to GFZ on standard laboratory medium was fairly low, only a narrow zone of Inhibition was observed (e.g. 2-5 mm). To see if nutrients supplied by the medium might “rescue” S. aureusand S. epidermidisfrom the effects of GFZ, four strains of S. aureusand one strain of S. epidermidiswere each plated on LB, a nutrient-rich medium, and TSB, a relatively nutrient-poor medium. The zones of inhibition were significantly larger on the TSB plates (e.g. 10-20 mm) (FIG. 9).

[0207] Nocardia sp. susceptibility was notable in that GFZ produced large zones of inhibition, e.g. 40-60 mm, by the disk assay. It was also noted that the GFZ zone of inhibition assay appeared to be an effective method of rapidly differentiating Nocardia sp. from atypical mycobacteria, all of which were resistant to GFZ on standard laboratory media.

[0208] Saccharomyces cerevisiaeand Candida albicanswere also found to be susceptible to GFZ when grown on SAB medium buffered to a pH of 7. No zone of inhibition was observed with unbuffered medium, as GFZ is insoluble at an acid pH.

[0209] MIC Determination for M. tuberculosis

[0210] The susceptibility of M. tuberculosisto GFZ was of special interest given the prevalence, morbidity, and mortality associated with infections by this organism. Therefore, we tested the GFZ susceptibility of 27 M. tuberculosis strains, 22 of which were resistant to one or more anti-tubercular drugs, by plating M. tuberculosisstrains on nutrient medium containing 0, 50, 100, or 200 μg/ml of GFZ. Growth of all M. tuberculosisstrains was completely inhibited by 100-200 μg/ml GFZ, regardless of their resistance to other antibiotics (FIG. 10). Comparable results were obtained when M. tuberculosiswas added to 7H9 broth containing GFZ at concentrations of 50 or 300 μg/ml (FIG. 11).

[0211] EMS Mutagenesis

[0212] To search for genes involved in GFZ susceptibility/resistance, and thereby identify its mechanism of inhibition, GFZ resistant mutants of L. pneumophilawere sought. Over 10¹²CFUs of wild type or EMS-mutagenizec L. pneumophilawere plated on CYE plates containing 50 μg/ml of GFZ. No spontaneous mutants were obtained. Only one EMS-mutagenized L. pneumophiiavariant, F4b, was identified. F4b had an MIC₉₀of 50 μg/ml in an AYE MIC assay, compared to 10 μg/ml GFZ for the wild type L. pneumophilaparent strain. F4b also demonstrated increased resistance to GFZ in an intracellular infection assay in HL-60 cells (FIG. 12).

[0213] GFZ Analog Assays

[0214] Previous results from the intracellular and broth assays testing the effect of fibric acids on L. pneumophila, indicated that structural analogs of GFZ, such as clofibric acid, had a modest inhibitory effect on L. pneumophilagrowth. Therefore, several GFZ analogs were obtained (FIG. 13) and tested by a zone of inhibition assay against L. pneumophilaand the partially GFZ-resistant variant F4b (FIG. 14). Two of the analogs, 4-HPA and 3,4-HPA, were equally inhibitory for both wild type L. pneumophilaand the partially GFZ-resistant variant F4b. Of note was the finding that both wild type L pneumophilaand the F4b variant were more sensitive to 2-hydroxybenzoic acid (salicylic acid) than GFZ. Aside from GFZ, F4b demonstrated increased resistance to 2-hydroxybenzoic acid, also known as salicylate.

[0215] The random bacterial screen demonstrated that GFZ was active against a number of bacteria, notably M. tuberculosis, Nocardia sp. S. aureusand S. epidermidisall of which can cause infections which may be difficult to treat. M. tuberculosis, an acid-fast bacillus, latently infects up to one third of the world's population and is the leading cause of death in humans from a single infectious agent [84]. M. tuberculosisprimarily infects the lungs, although extrapulmonary infection, with dissemination throughout the body also occurs. Nocardia sp. are gram-positive, weakly acid-fast, higher bacteria, also known as actinomycetes, that form branches similar to fungii, but induce a neutrophilic inflammatory response similar to other bacteria, and demonstrate susceptibility to antibiotics. Nocardia sp. primarily infects the lungs, resulting in a pneumonia but can spread to the skin and brain where it forms abscesses. Six to twelve months of antibiotic treatment may be required to cure Nocardia infections.

[0216] S. aureus, a gram-positive clustering coccus, often colonizes the nose, and is responsible for numerous serious infections including endocarditis, osteomyelitis, toxic shock syndrome, and pneumonia. Strains of this organism have been identified that are resistant to all known antibiotics. S. epidermidisis the primary agent associated with infections of prosthetic devices including heart valves, hip and joint replacements, and indwelling catheter lines. S. epidermidisis also associated with urinary tract infections in sexually active women.

[0217] L. pneumophila, a gram-negative bacilli, is the third or fourth most common cause of community-acquired pneumonia, and is a frequent cause of nosocomial pneumonia. Infection with L. pneumophilais associated with significant morbidity, mortality, and hospital costs.

[0218] C. albicans, a pathogenic fungi, is associated with oral, esophageal, intestinal, and vaginal infections in immunosuppressed patients, and commonly with vaginal infections in an immunocompetent population.

[0219] The observation that all susceptible bacteria (and yeast) contain branched fatty acids in their membranes indicates that GFZ may be inhibitory for bacteria capable of synthesizing a complex array of membrane fatty acids. Additional support for the involvement of fatty acids in GFZ inhibition is provided by the observation that addition of cleate to the medium of GFZ-inhibited S. cerevisiae, rescues these yeast from the inhibitory effects of GFZ. Addition of cleate to the medium also rescues S. cerevisiaefrom the antibiotic cerulenin, an inhibitor of fatty acid synthesis in yeast, bacteria, and mammalian cells [90,91].

[0220] Similarly, growth inhibition in E. colimediated by drugs or by ts mutations affecting fatty acid synthesis, can be bypassed in many cases by the addition of fatty acids to the bacteriologic medium such. as oleic acid (C_18:1), a mono-unsaturated sixteen carbon fatty acid, and palmitic acid (C_16:0), a saturated sixteen carbon fatty acid [32,92]. The observation that S. aureusshowed increased susceptibility to GFZ on TSB medium as compared to the nutrient rich LB medium, suggests that LB supplies metabolite(s), possibly fatty acids, that are able to bypass the effect of GFZ.

[0221] It is important to note that the M. tuberculosisassays were performed in the presence of oleic acid. Standard mycobacterial medium utilizes oleic acid (or Triton) as a detergent to prevent “cording” of the bacteria. It is possible that if oleic acid is left out of the medium, M. tuberculosiswill exhibit greater susceptibility to GFZ. However, testing the ability of GFZ to inhibit the growth of M. tuberculosiswithin infected human macrophages may be a more relevant approach.

[0222] Therefore, if the bacterial strains screened for GFZ-susceptibility (FIG. 9) were re-tested on medium lacking fatty acids, additional susceptible strains might be identified. The GFZ-sensitivity of bacteria tested on nutrient agar free of fatty acids may better correlate with the susceptibility of these bacteria to GFZ in vivo.

[0223] Other factors besides the presence of fatty acids may contribute to the presence or size of a zone of inhibition adjacent to a GFZ disk. The ability to observe a zone of inhibition for S. cerevisiaeand C. albicanswas dependent on buffering the medium to a pH of 7. GFZ solubility in aqueous solution decreases as pH decreases. Therefore, to ensure diffusion of the drug through the medium, and to prevent acidification of the medium during growth, it was necessary to buffer the pH. The effects of pH on GFZ sensitivity was not examined with any other medium or pathogen.

[0224] The zone of Inhibition surrounding a GFZ disk represents the area in which the concentration of GFZ is high enough to inhibit bacterial growth. If GFZ is not very soluble in a given medium, or is tightly bound by proteins in the medium (e.g. albumin), the rate of diffusion from the disk may be slowed, resulting in a short and steep concentration gradient. The rate of diffusion is also affected by the thickness of the agar plate since the drug diffuses in three dimensional in agar, i.e., the thicker the plate, the smaller the zone. Nonetheless, for screening purposes, zones of inhibition afford a rapid and easy method by which to assess the presence, but not the extent, of GFZ sensitivity.

[0225] The observation that M. tuberculosisstrains that are resistant to multiple conventionally used antibiotics were as sensitive to GFZ as M. tuberculosisstrains that are sensitive to these antibiotics suggests that GFZ may be a lead compound for identifying antibiotics that can inhibit the growth of multiply drug resistant M. tuberculosis. The relative impermeability of the cell well accounts for the majority of the drug resistance in M. tuberculosisstrains. However, many potential drug resistance determinants, including β-lactamases, aminoglycoside acetyl transferases, and many potential drug efflux systems, are encoded in its genome [93]. Whether any of the chromosomally encoded potential drug resistance determinants confers increased resistance to GFZ is unknown.

[0226] Resistance to drugs can occur by several mechanisms. Drug resistance may result from the overuse of a drug, thereby selecting for organisms that grow despite the presence of the drug. The sporadic use of drugs, which often happens in unobserved TB therapy, selects for increasingly resistant bacterial populations. Subinhibitory concentrations of a drug also enhance the outgrowth of drug-resistant mutant strains, and encourage the spread of plasmids encoding drug resistance mechanisms from one species to another. Unfortunately, the development of resistance to one drug, often confers resistance to other drugs within the same class.

[0227] Our inability to identify spontaneous or EMS-generated GFZ resistant mutants suggests either that the L. pneumophilatarget cannot be readily altered to confer resistance to this drug, or, that GFZ can affect more than one enzyme or pathway in L. pneumophila. The ability to inhibit multiple targets in an organism is a desirable property for an antibiotic.

[0228] The observation that GFZ inhibited only 33% of the bacteria screened suggests that GFZ has a narrow spectrum of ant-microbial activity. While narrow spectrum antibiotics are less likely to generate the revenues desired by large pharmaceutical companies, they have the potential advantage of targeting the primary agent of disease without inhibiting the body's normal flora. Antibiotics with these characteristics should limiting the generation and spread of drug resistant bacteria. The finding that GFZ inhibited twenty three drug resistant strains of M. tuberculosis, Nocardia sp. and S. aureus, encouraged us to continue research in this area, especially with regard to the mechanism(s) of GFZ-mediated bacterial inhibition.

[0229] Antimicrobial Susceptibility Testing

[0230] For determination of bacterial susceptibility, bacterial suspensions were overlaid on suitable nutrient agar plates, a sterile paper disk containing 2 mg of GFZ was added, and the plates were incubated at 37° C. until a lawn of bacterial growth was observed. Bacteria were classified as susceptible if there was a zone of growth inhibition of more than 2 mm surrounding the disk. Non-pathogenic bacterial strains were graciously provided by D. Figurski, College of Physicians and Surgeons, Columbia University, NYC, N.Y. Pathogenic bacterial strains (except for L. pneumophila) used for GFZ susceptibility testing were clinical isolates obtained from Dr. P. Della Latta, Director Clinical Microbiology, Presbyterian Hospital, and generously screened following NCCLS standardized procedures by the Clinical Microbiology Dept. of Columbia-Presbyterian Hospital. A sterile disk containing 2 mg of gemfibrozil (Sigma) was added to the overlay. Sensitivity was determined by the presence of a zone of growth inhibition surrounding the disk.

[0231] M. tuberculosisSusceptibility Assay.

[0232] 100 μl of a standard dilution of each of 5 drug-sensitive and 22 drug-resistant M. tuberculosisstrains were tested for GFZ-susceptibility on 5 ml quadrants of solid Middlebrook 7H10 medium (Baltimore Biological Labs) supplemented with oleic acid, dextrose, catalase, and albumin (OADC) obtained from ifco, Detroit, Mich., containing 0, 50, 100, or 200 μg/ml of GFZ. Standard dilutions were prepared by resuspending each strain to a McFarland standard of one, approximately 10⁸organisms, and diluted 100-fold. Strains were obtained from the Clinical Microbiology Dept. of Columbia-Presbyterian Hospital. Plates were scored following incubation for three weeks at 37° C. For broth assays, approximately 10⁷bacteria (100 μl of a Mc Farland standard of 1) was added to BBL Prepared Culture Media (Beckton Dickenson) containing 5 mls of Middlebrook 7H9 broth with glycerol. The cultures were incubated for 21 days at 37° C. after which turbidity was visually assessed for growth.

[0233] EMS Mutagenesis.

[0234] Log phase L. pneumophilain AYE broth were incubated with 15 μl EMS for 15 minutes at 37° C., pelleted, washed twice, resuspended in 1 ml AYE, titered for CFUs, diluted 1:10 in AYE and grown overnight at 37° C. EMS exposure resulted in approximately 40% and 70% viability in two independent experiments. Following overnight growth, the EMS-mutagenized L. pneumophilawere grown on CYE plates at 37° C. for two days, harvested, and suspended at Approximately 10¹⁰CFU's/ml in AYE broth. 100 μl aliquots were plated on CYE agar containing 50 μg/ml GFZ. Putative —SFZ-resistant colonies were passed once on CYE agar without GFZ, and then on CYE agar containing GFZ 50 μg/ml. Only one GFZ-resistant colony was obtained after the second passage on CYE agar containing GFZ 50 μg/ml. Well over 10¹²colonies were screened for resistance to GFZ.

[0235] Effect of GFZ in the Growth of the L. pneumophilaF4b Variant in HL-60 Cells.

[0236] Tissue culture medium RPMI 1640 was obtained from JRH Biosciences, Lenexa, Kans.; L-glutamine (LGN) was obtained from Mediatech, Herndon, Va., and phorbol 12-myristate 13-acetate (PMA) was obtained from Sigma. Normal human serum (Ultraserum™) was obtained from Gemini Bio-Products, Calabasas, Calif. Promyelocytic HL-60 cells were differentiated into macrophage-like cells by incubation with 10 ng/ml PMA in RPMI with 2 mM GLN and 10% NHS in Teflon wells at 37° C. for 24 hours. These cells were washed, resuspended at 4×10⁶cells/ml in RPMI-2 mM GLN-10% NHS, and mixed with L. pneumophilaF4b (4×10⁴CFU/ml) which had been grown for 2 days on ABCYE plates (final multiplicity of infection of 0.01). 100 μl aliquots of the suspension were plated in each well of a 96 well microtiter plate. The plates were centrifuged to pellet the cells and bacteria and incubated at 37° C. for 2.5 hours to allow phagocytosis. 100 μl of fresh medium (+/−) 2× the final GFZ concentration, was added to the wells, and the plates were incubated at 37° C. At the times indicated, the cells and medium were harvested and assayed for L. pneumophilaCFUs as described [58]. Data from the experiments are expressed as the average (+/−) the S.E.M (n=2).

[0237] Gemfibrozil (Lopid™), a drug used to treat hyperlipidemia due to high serum triglycerides, inhibits the growth of pan-sensitive and multiple-drug resistant Mycobacterium tuberculosis, of Legionella pneumophila, the causative agent of Legionnaire's disease, and of several other bacterial pathogens and yeast. The finding that GFZ (100-200 μg/ml) inhibited four pan-sensitive and twenty two drug resistant strains of M. tuberculosiswithin a two-fold concentration range, suggested that GFZ might have a novel mechanism of action.

[0238] The inability to generate GFZ-resistant L. pneumophilamutants by EMS mutagenesis (supra), made it difficult to search for resistance mechanisms and putative targets. Therefore, the morphology of L. pneumophilafollowing incubation with GFZ was examined by transmission electron microscopy (TEM) to determine whether GFZ had a visible effect on cell structure that might provide a clue to its mechanism of action.

[0239] Analysis of L. pneumophilaIncubated on CYE Agar in the Presence or Absence of GFZ by Transmission Electron Microscopy.

[0240] By TEM, L. pneumophilagrown on CYE agar in the absence of GFZ, contained occasional, small, electron-lucent cytoplasmic inclusions similar to those reported by others (FIGS. 15A-D) [94,95]. However, when L. pneumophilawere grown on CYE agar containing 30 μg/ml of GFZ, two populations were observed. The bacteria either appeared distended by numerous, large, electron-lucent, cytoplasmic inclusions, or had a marked absence of electron-lucent inclusions (FIG. 15B). The small electron-lucent inclusions found in L. pneumophilagrown in the absence of GFZ are reported to contain polyhydroxybutyrate (PHB), polymers of 3-hydroxybutyric acid (3-HB) [96,105].

[0241] Although 10 μg/ml of GFZ inhibits growth of 10⁶L. pneumophilaCFUs/ml in AYE broth, and of individual colonies on CYE agar, higher concentrations of GFZ are required to inhibit growth of a high density L. pneumophilainnoculum (10⁹-10¹⁰CFUs) on CYE agar. When a large number of L. pneumophilais added to CYE plates containing 30 μg/ml of GFZ, the bacteria grew to form a sparse lawn. This lawn provided sufficient L. pneumophilafor analysis. The ability of a large innoculum to grow on CYE medium containing GFZ at 3× the broth MIC is consistent with reports that an innoculum size affects the apparent MIC of an antibiotic for a given bacteria [76,97]. Additionally, the presence of charcoal in CYE plates increases the MIC of many antibiotics. Substitution of potato starch for charcoal generally results in lower MIC values although this technique was not used here [76].

[0242] Nile Blue A Staining of L. pneumophilaGrown in the Presence or Absence of GFZ.

[0243] Nile Blue A (NBA), a fluorescent dye which stains PHB granules in bacteria [98], was used as a preliminary screen to assess whether the large electron-lucent granules seen by electron microscopy, contained PHB. Nile Blue A staining allowed the simultaneous visualization of larger populations of bacteria. L. pneumophilagrown on CYE agar in the presence or absence of GFZ 30 μg/ml, was harvested and stained with NBA. L. pneumophilagrown in the presence of GFZ contained many large, distending, brightly fluorescent NBA-stained inclusions (FIGS. 15A-D), similar to the inclusions seen by EM. L. pneumophilagrown in the absence of GFZ contained fewer, and smaller, NBA-stained inclusions than L. pneumophilagrown in the presence of GFZ (FIGS. 15A-D). Thus, consistent with the EM findings, GFZ treated L. pneumophilacontained populations of bacteria in which the cytosolic space was filled with NBA-stained material and populations of bacteria containing few if any NBA-stained inclusions.

[0244] Gas Chromatography-mass Spectrometry (GC-MS) of L. pneumophilaGrown in the Presence or Absence of GFZ.

[0245] Although Nile Blue A is reported to be a specific stain for PHB, as opposed to glycogen, polyphosphates, and other bacterial inclusions, it had not been shown to be specific for polyhydroxybutyrate versus other polyhydroxyalkanoates.

[0246] While L. pneumophilais reported to contain PHB granules, some bacteria are known to respond to alterations in growth conditions by varying the composition of their granules, (i.e. polymerization of short chain carboxylic acid monomers other than 3-HB) [99,100]. Therefore, GC-MS was used to assess the composition of the inclusions. L. pneumophilagrown in the presence or absence of GFZ was subjected to hydrochloric acid propanolysis [101] to transesterify 3-hydroxyalkanoate monomers with propanol to form the corresponding propyl esters. Propyl esters were extracted into dichloroethane and analyzed by GC-MS. A fifty-fold increase in the amount of 3-HB-propyl ester was observed in L. pneumophilagrown for three days on CYE agar in the presence of GFZ, relative to L. pneumophilagrown on CYE agar in the absence of GFZ (FIG. 16). The difference between the presence and absence of GFZ for PHB was analyzed by a student's t-test and found to be significant with t=−6.05, corresponding to a p<0.05 for three independent experiments.

[0247] TEM and Nile Blue A staining of L. pneumophilagrown in the presence of a subinhibitory concentration of GFZ indicated that GFZ induced the accumulation of PHB-containing granules in a majority of the bacteria. However, populations of bacteria lacking significant accumulation of PHB were also detected. In experiments not reported, log phase L. pneumophilacontained a lower percentage of PHB than stationary phase bacteria. Since the viability of the population of GFZ-treated L. pneumophilalacking large PHB was not assessed, it is unclear whether these bacteria were growing and “resistant” to the concentration of GFZ used, or were dead and therefore unable to form inclusions.

[0248] L. pneumophilais not unique in its ability to form granules containing PHB. Several bacterial species are reported to accumulate granules containing polyhydroxyalkanoates (PHA), including Alcaligenes eutrophus, Bacillus megatarium, Pseudomonas oleovorans, Pseudomonas aeruginosa, and some Rhodococcus, Corynebacterium, and Nocardia strains [102,103]. Since GFZ did not inhibit the growth of Pseudomonas aeruginosa, the ability to form PHA granules is not correlated with GFZ susceptibility.

[0249] PHAs are natural polyesters composed of, up to 1,000 b-hydroxyacyl monomer units, 3 to 14 carbons in length. The function of these stored PHAs may be to act as an oxidizable substrate during oxygen limitation, as a carbon and energy source, or as a protective mechanism against the degradation of cellular components such as RNA and protein during nutrient deprivation [102,103]. Since L. pneumophilaare able to utilize exogenous 3-HB as a carbon source [96], it is likely that their PHB stores contribute to their ability to maintain ATP content and survive in tap water for many months [104,105].

[0250] PHAs usually accumulate in bacteria in response to phosphorous, oxygen, nitrogen, or iron limitation in the presence of a carbon source [105,106]. However, to the best of our knowledge, these conditions were not present in our experiments. We noted however, that PHB synthesis and fatty acid synthesis utilize similar precursors and intermediates. Therefore, we hypothesized that the accumulation of PHB was due to a GFZ-mediated inhibition of fatty acid synthesis (FIG. 17), resulting in the accumulation of fatty acid precursors and intermediates before the block, and their subsequent shunting into PHB synthesis (FIG. 18).

[0251] Bacteria that synthesize branched chain fatty acids, such as L. pneumophila, can utilize both acetyl CoA and butyryl CoA as fatty acid precursors [13] [107] [108]. Condensation of two acetyl-CoA molecules is often the first step in PHB synthesis. Butyryl-CoA has been shown to be incorporated into PHB without degradation to acetyl CoA. Presumably this occurs following oxidation of butyryl-CoA to crotonyl-CoA, and hydration to (R)-3-HB-CoA by enzymes involved in β-oxidation [109]. Additionally, an enzyme PhaG in P. putidacan mediate the conversion of 3-HB-ACP, an intermediate in fatty acid synthesis, into 3-HB-CoA, the monomer unit for PHB synthesis. Experiments described in supra are consistent with the hypothesis that GFZ causes the accumulation of fatty acid intermediates in L. pneumophilaand that these intermediates are likely to be responsible for the GFZ-induced accumulation of PHB reported in this study.

[0252] TEM of L. pneumophila.

[0253] L. pneumophilawas grown for two days on CYE plates, harvested, and resuspended to a concentration of 10¹⁰-10¹¹CFU's/ml in AYE. 10⁹-10¹⁰bacteria were added to CYE agar medium without or with GFZ (30 μg/ml). The plates were incubated for three days at 37° C. The bacteria were harvested, pelleted and fixed by resuspension in 2.5% glutaraldehyde for 45 minutes at 25° C., rinsed in 0.1 M NaPO₄buffer (pH 7.5) and stored in this buffer overnight, rinsed 2× in 0.1M cacodylate buffer (pH 7.35), postfixed in 1% osmium tetroxide, rinsed in cacodylate buffer pH 7.35, rinsed in 0.1 M NaAc buffer (pH 6.0), stained in 1% uranyl acetate in 0.1 M NaAc buffer (pH 6.0) for 2.5 hours at 4° C., dehydrated through graded alcohols, embedded in epoxy resin, sectioned into 600 Å sections with a Sorvall MT6000 ultramicrotome, and mounted on 200 mesh copper grids. The sections were stained with uranyl acetate for 15 minutes followed by lead citrate for 10 minutes and examined by TEM using a JEOL1200EX electron microscope.

[0254] Nile Blue A Staining of L. pneumophila.

[0255] L. pneumophilawere grown for two days on CYE plates, harvested, and resuspended at a concentration of 10¹⁰-10¹¹CFU's/ml in AYE. 10⁹-10¹⁰bacteria were added to CYE agar medium without or with GFZ (30 μg/ml). The plates were incubated for three days at 37° C. The bacteria were harvested, resuspended in water, smeared on a glass slide and heat fixed. The slides were incubated in a 1% aqueous solution of Nile Blue A for 10 minutes at 55° C., washed with tap water, destained for 1 minute in 8% aqueous acetic acid, and air dried. The smears were remoistened with water, covered with a No. 1 glass cover slip, and examined by fluorescence microscopy at 460 nm.

[0256] Gas Chromatography-mass Spectrometry.

[0257] Identification and quantitation of 3-hydroxybutyrate (3-HB) was performed by gas chromatography-mass spectrometry (GC-MS). 3-HB propyl esters were formed for analysis by hydrochloric acid propanolysis of lyophilized bacteria using benzoic acid as an internal standard [101]. Hydrochloric acid, propanol, benzoic acid, and dichloroethane (DCE) were all obtained from Sigma. In brief, L. pneumophilawere grown on CYE agar in the absence or presence of a subinhibitory concentration of GFZ (30 μg/ml) for three days, harvested, and lyophilized. 2 mls DCE, 2 mls acidified propanol, and 200 μl of internal standard solution (0.8 mg/ml of benzoic acid in 1-propanol) were added to 40 mg of lyophilized bacteria and the mixture was incubated for 2 hrs at 100° C. After cooling to room temperature, the sample was washed with 4 mls water. The DCE-propanol phase was reserved and stored at 4° C. A standard curve was constructed by converting known quantities of 3-HB (Sigma) and benzoic acid to their propyl esters, using hydrochloric acid propanolysis, as before. Prior to analysis, propyl esters in dichloroethane were dried under a stream of nitrogen and resuspended in an equivalent volume of ethyl acetate. A 2 μl aliquot of the DCE propanol extract of each sample was analyzed after splitless injection into a Hewlett Packard 5987A GC/MS equipped with a DB-1 fused-silica capillary column (30 m×0.2 mm) using helium as the carrier gas. The temperatures of the injector and the source were 220° C. and 200° C., respectively. The column temperature program started at 40° C. for 1 min, and increased at a rate of 8° C./min to 200° C. Samples were ionized by electron impact (70 eV). The abundance of ions at m/e 105 was used to quantitate benzoic acid, while the abundance of ions at m/e 87 was used to quantitate 3-HB.

[0258] Previous work demonstrated that growth of several bacterial species and yeast strains was inhibited by GFZ. The observation that M. tuberculosisstrains encoding resistance to many different antibiotics were sensitive to GFZ, suggested that GFZ inhibited M. tuberculosis, and likely other bacteria, by a novel mechanism. The observation that GFZ stimulated PHB accumulation, led to the hypothesis that GFZ inhibited an enzyme in bacterial fatty acid synthesis resulting in the accumulation of precursors or intermediates which were shunted into PHB synthesis (FIG. 19). Experiments are described herein demonstrating the ability of GFZ to inhibit ¹⁴C-acetate incorporation into bacterial fatty acids. These findings confirm that GFZ targets bacterial fatty acid synthesis.

[0259] Fatty acids are synthesized by the successive addition of malonyl-ACP to a primer-ACP or a fatty acyl-ACP (FIG. 20). Malonyl-ACP is synthesized from acetyl-CoA which is itself a product of β-oxidation of fatty acids, or, of decarboxylation of pyruvate during growth on sugar. Many bacteria can also utilize exogenous acetate as a growth substrate. Acetate from the medium diffuses into the cytoplasm where it is converted to acetyl-CoA. Acetyl Co-A is subsequently used for oxidation via the tricarboxylic acid (TCA) cycle, for replenishment of intermediates of the TCA cycle, for leucine biosynthesis, and for lipid biosynthesis [112]. While L. pneumophilais capable of oxidizing acetate [96,113], ¹⁴C-acetate added to L. pneumophilacultures is primarily incorporated into the lipid fraction [113]. Therefore, ¹⁴C-acetate is useful as a tracer of fatty acid synthesis in this bacterium.

[0260] Fatty acid synthesis in most bacteria and plants is carried out by discrete, separable enzymes, which are collectively described as a Type II fatty acid synthetase (Type II FAS) system [5]. In contrast, fatty acid synthesis in mammalian cells is carried out by a homodimer of a single polypeptide encoding seven distinct enzymatic functions, characteristic of a Type I fatty acid synthetase (Type I FAS) [4]. Differences between the human and bacterial fatty acid synthetic enzymes may account for the ability of GFZ to inhibit fatty acid synthesis in certain bacteria, without affecting the viability of mammalian cells.

[0261] ¹⁴C-acetate Incorporation into Whole L. pneumophila.

[0262] To test whether GFZ inhibited fatty acid synthesis, ¹⁴C-acetate was added to the medium of log phase L. pneumophilain AYE broth in the presence of increasing concentrations of GFZ (FIG. 21). Cerulenin, a known inhibitor of bacterial and mammalian fatty acid synthesis, was used as a control. Concentrations of GFZ as low as 10 μg/ml (40 μM) inhibited of ¹⁴C-acetate incorporation into wild type L. pneumophilawithin 15 minutes after the addition to the medium. Fifty percent inhibition relative to the control was achieved with a GFZ concentration of 25 mg/ml (100 mM) within 15 minutes of the drugs addition to the medium. However, inhibition of ¹⁴C-acetate incorporation into F4b, the L. pneumophiladerived mutant with moderate resistance to GFZ (MIC₉₉=50 μg compared to 10 μg/ml for wild type L. pneumophila) required 100 μg/ml (400 μM) GFZ to inhibit ¹⁴C-acetate incorporation by 50% (FIG. 22).

[0263] To confirm that incorporation of ¹⁴C-acetate into TCA precipitable material in whole bacteria accurately reflected ¹⁴C-acetate utilization for fatty acid synthesis, I analyzed ¹⁴C acetate incorporation into chloroform/methanol extractable material from L. pneumophilagrown in the presence or absence of GFZ. L. pneumophilawas incubated for one hour in medium containing ¹⁴C-acetate and increasing concentrations of GFZ. The bacteria were then pelleted, extracted with chloroform/methanol, and the extracts were analyzed by thin later chromatography (TLC). Assessment of the amounts of ¹⁴C radiolabel recovered in the chloroform/methanol extracts and autoradiography of the TLC plates (FIG. 23), confirmed that GFZ inhibited ¹⁴C-acetate incorporation into fatty acids in a dose dependent manner. TLC analysis showed that the decrease in ¹⁴C-acetate incorporation was equally distributed among the various fatty acid containing moieties. We draw two conclusions from thus experiment. First, GFZ inhibits fatty acid synthesis in L. pneumophila. Second, it does so by blocking an early step in fatty acid synthesis, since ¹⁴C-acetate incorporation into all fatty acid containing lipids was inhibited equally.

[0264] ¹⁴C-acetate Incorporation into L. pneuaophilaLysates.

[0265] To determine whether GFZ inhibited ¹⁴C-acetate incorporation into lipids in cell lysates lysates were prepared from log phase L. pneumophilaand incubated for one hour at 37° C. in 50 mM TrisHCl buffer (pH 7.6) containing ATP, Mg⁺⁺, CoA, ¹⁴C-acetate, and GFZ. As observed with intact bacteria, GFZ inhibited ¹⁴C-acetate incorporation into TCA-precipitable material in these lysates. Further analysis of chloroform/methanol extracts of these lysates confirmed that the ¹⁴C-acetate was largely incorporated into lipids (FIG. 24).

[0266] While GFZ inhibited ¹⁴C-acetate incorporation into fatty acids in the lysate, it was not as effective an inhibitor in these lysates as it was in whole cells. 0.4 mM GFZ only inhibited ¹⁴C-acetate incorporation into TCA precipitable material in a lysate by 15%, while 0.4 mM GFZ inhibited ¹⁴C-acetate incorporation into TCA precipitable material in whole cells by greater than 90% (FIG. 21). Similarly, cerulenin was a less effective inhibitor of ¹⁴C-acetate incorporation into lysates than whole cells (compare FIGS. 21 and 24).

[0267] Control experiments showed that lysates that had been boiled prior to ¹⁴C-acetate addition, or, had been pre-incubated with 10 mM EDTA, did not incorporate ¹⁴C-acetate into TCA precipitable material (FIG. 25). EDTA inhibits fatty acid synthesis by chelating Mg⁺⁺ which is a required cofactor for ATP-dependent enzymes. In the presence of EDTA, CoA synthase is unable to form acetyl-CoA so malonyl-ACP is not formed and elongation does not occur.

[0268] The effect of a second fibric acid, bezafibrate (BZF) on ¹⁴C-acetate incorporation in L. pneumophilalysates was compared with that of GFZ and cerulenin. Surprisingly, BZF was a better inhibitor than GFZ in a lysate (FIG. 25).

[0269] Effect of GFZ Analogs on ¹⁴C-acetate Incorporation.

[0270] Structural analogs of GFZ were also tested for inhibition of fatty acid synthesis in whole cells (FIG. 26). L. pneumophilacultures were incubated in medium containing ¹⁴C-acetate and each of seven GFZ analogs (FIGS. 27 A-B) (a generous gift from TEVA pharmaceuticals) at a 0.4 mM concentration. Analogs C and D were found to be better inhibitors at 0.4 mM than GFZ at this concentration. Analog B was as effective as GFZ. Dose response experiments were performed for analogs C and D (FIGS. 28A-B). Analogs C and D at a concentration of 0.4 mM, inhibited ¹⁴C-acetate incorporation into L. pneumophilaby 50%. In contrast, a concentration of 0.1 mM (25 μg/ml) GFZ was required to effect a 50% inhibition of ¹⁴C-acetate incorporation (FIG. 21). Additional commercially available compounds with structural similarity to GFZ (FIG. 26) were examined at concentrations of 0.5 mM. Of those tested, salicyclic acid, clofibric acid, and p-aminosalicyclic acid demonstrated some efficacy, although not as great as that found with GFZ at 0.4 mM (FIG. 28B).

[0271] The finding that GFZ inhibited ¹⁴C-acetate incorporation into lipids, as measured by TCA precipitation and by chloroform/methanol extraction of L. pneumophilacultures and lysates, was consistent with the hypothesis that GFZ inhibited fatty acid synthesis. The dose-response studies demonstrating that ¹⁴C-acetate incorporation is inversely and proportionately related to GFZ concentration, suggested that GFZ had a direct effect on fatty acid synthesis. The argument that GFZ has a direct effect on fatty acid synthesis is supported by the GFZ-mediated inhibition of ¹⁴C-acetate incorporation into lysates.

[0272] Cerulenin, an inhibitor of the β-ketoacyl synthase reaction in bacteria and eukaryotes [25,115] with MIC values ranging from 1 to 100 μg/ml }[116], was used as a positive control for the inhibition ¹⁴C-acetate into fatty acids [117]. The inhibitory action of cerulenin on β-ketoacyl synthase is irreversible; 1 mol of cerulenin binds to 1 mol of β-ketoacyl synthase when inhibition approaches 100% [115]. Cerulenin is also a potent inhibitor of the mammalian fatty acid synthetase enzyme (FAS) [25], which contains a β-ketoacyl synthase domain, and for this reason is not useful as an antibiotic in humans. However, it is being pursued as an anticancer drug since certain types of tumors (e.g. ovarian, endometrial, breast, colorectal, and prostate) overexpress FAS [118,119].

[0273] Similar to GFZ, cerulenin had an immediate and sustained inhibitory effect on the incorporation of ¹⁴C-acetate into L. pneumophilalipids. Cerulenin at 100 μg/ml (0.45 mM) was a slightly better inhibitor of ¹⁴C-acetate incorporation than GFZ at 100 μg/ml, 0.4 mM, in whole L. pneumophila. The observation that GFZ is nearly as potent as cerulenin in whole L. pneumnophilaindicates that it may also have a one to one (drug to target) relationship. The observation that GFZ is not as potent as cerulenin in lysates, indicates that GFZ may need to be metabolized to an active form.

[0274] In our studies, GFZ caused a similar dose-dependent decrease in ¹⁴C-acetate incorporation into TCA precipitable material and into chloroform/methanol extractable material. Chloroform/methanol extraction is reported to selectively extract up to 99% of the total lipids from a sample [120,121]. Since bacteria do not synthesize sterols, the majority of the incorporated ¹⁴C-acetate in a chloroform/methanol extract will be found in phospholipids, free fatty acids bound to ACP or CoA, lipid A of the lipoploysaccharaide (LPS), the lipoproteins of the outer membrane, and polyhydroxyalkanoate. Other minor lipid species comprise less than 1% of total bacterial cell lipids. These include ubiquinone [5,24,122-124] and the lipid containing coenzymes biotin and lipoic acid.

[0275] Chloroform/methanol extractions were performed using the method of Bligh and Dyer as this method required smaller volumes of chloroform and methanol, and had been used successfully in L. pneumophilaand other bacteria [41,125]. Although TCA precipitation is not selective for lipids, it is often utilized to assess ¹⁴C-acetate incorporation into bacterial lipids [40,113,126-128]. Tesh et al [113] examined the incorporation of ¹⁴C-acetate into the TCA soluble and the chloroform/methanol soluble fractions of L. pneumophilaover a 12-15 hour time period. They demonstrated that 72% of the total radioactivity was recovered in the chloroform/methanol soluble fraction, while 91% was recovered in the TCA precipitate [113].

[0276] Acetyl-CoA is utilized by many bacteria as a primer for PHB synthesis. Further, increased pools of acetyl CoA due to inhibition of the TCA cycle in the PHB producing bacteria Azotobacter vinelandiiand R. eutropha, resulted in an increased flux of acetyl-CoA into PHB synthesis [129,130]. If L. pneumophilautilizes acetyl-CoA as a precursor for PHB synthesis, then the greater recovery of radiolabel in the TCA precipitable fraction may be due to a greater recovery of PHB in the TCA precipitate.

[0277] Due to our use of log phase L. pneumophila, which do not accumulate PHB, and the length of our incubations (i.e. 15-60 minutes of ¹⁴C-acetate exposure), it is unlikely that significant amounts of ¹⁴C-acetate were incorporated into PHB. However, a small percentage of the ¹⁴C label recovered in our TCA precipitable fractions, could be at least partially due to the incorporation of ¹⁴C-acetate into PHB. Incorporation of ¹⁴C-acetate into PHB would be expected to increase with time as precursors and intermediates accumulated, and the enzymes responsible for synthesizing PHB were induced. An experiment comparing ¹⁴C-acetate incorporation into PHB, in L. pneumophilaincubated in the absence and presence of GFZ over a longer time period would address the hypothesis that inhibition of fatty acid synthesis by GFZ shunts fatty acid precursors/intermediates into PHB synthesis.

[0278] The lysate experiments were performed to confirm the results obtained from the whole cell experiments in which GFZ inhibited the incorporation of ¹⁴C-acetate into lipids. In addition to confirming these results, the lysate experiments revealed that BZF, a related fibric acid used to treat hypertriglyceridemia, also inhibits lipid synthesis in L. pneumophila. Since BZF does not inhibit growth of intact L. pneumophila, it is likely that BZF does not penetrate the membrane, or, is a substrate for an efflux transporter in the L. pneumophilamembrane. Experiments to assess permeability and active transport using radioactive BZF might resolve these issues.

[0279] As a first step in determining what aspects of the GFZ molecule are important for the growth inhibitory effects of GFZ, the ability of other structurally similar compounds to inhibit lipid synthesis in whole L. pneumoiphilawas examined. In general, a free carboxylate moiety linked to a hydrophobic moiety, such as an aliphatic chain, appeared most important. Hydrophobicity is likely to be important for membrane permeability. Substitutions on the aromatic ring that increased the hydrophobicity of TEVA analogs C and D, may have increased the membrane permeability thereby accounting for their increased antibacterial activity against L. pneumophila.

[0280] The presence of the carboxylate group is likely to be important for the activation of GFZ by L. pneumophila. GFZ, like free fatty acids contains a carboxylate group linked to analiphatic chain. Therefore, GFZ, like long chain fatty acids (e.g. C₁₂-C₁₈fatty acids), may be converted to its CoA derivative concommitant with transport into bacteria [131-135]. Alternatively, GFZ may be converted to its ACP derivative, similar to short chain fatty acids (e.g. C₆-C₁₁fatty acids), and a minor proportion (2%) of long chain fatty acids in E. coli[136]. Analog B, which contains an amide instead of a carboxylate, but is still capable of inhibiting fatty acid synthesis, may have a different mechanism of inhibiting fatty acid synthesis. Alternatively, it may be converted to a carboxylate by L. pneumophila. Experiments to test this possibility may yield insights into the mechanisms by which TEVA analog B acts to block ¹⁴C-acetate incorporation into lipids in L. pneumophila.

[0281] In conclusion, GFZ was found to be an inhibitor of lipid synthesis in L. pneumophila. This finding was consistent with reports that GFZ and other fibrates inhibit fatty acid elongation in mammalian systems [67,68].

[0282] ¹⁴C-acetate Incorporation into Whole L. pneumophila.

[0283] L. pneumophila were grown to log phase (OD=0.6-1.0) in AYE broth at 37° C. 0.5-1.0 ml aliquots were pelleted and resuspended in an equivalent volume of fresh AYE medium. GFZ and other inhibitors were added to the cultures. ¹⁴C-acetate (specific activity 48.9 mCi/mmol) (Sigma) was added immediately thereafter (final concentration 5 μCi/ml) and the cultures were incubated at 37° C. At various time points, 100 μl aliquots of the culture were added to 600 μl 12% TCA on ice; final concentration 10% TCA. TCA precipitates were pipetted onto a Whatman GF/F glass microfibre filter and washed with 30 ml of cold 10% TCA. The filters were dried and radioactivity was assessed by scintillation counting [40,113126,127,139]. Alternatively, at various time points the bacteria were placed on ice, pelleted at 4° C., washed three times with 1 ml of ice cold AYE , and the pellet was extracted with 400 μl of chloroform/methanol (1:1 vol:vol). The extract was washed with 180 ml of water according to the method of Bligh and Dyer [113,120]. 5 μl of the chloroform layer was combined with 5 mls of CytoScint and counted in a scintillation counter. GFZ, salicylate, paraaminosalicylate, isoniazid, clofibrate, 4-hydroxypropionate were obatined from Sigma; 3,4-hydroxypropionate was obtained from Aldrich.

[0284] Thin Layer Chromatography.

[0285] 50 μl of each chloroform/methanol extract was spotted onto EM Science™ Silica Gel 60 F254 plates (Fisher) and developed in chloroform:methanol:acetic acid (65:25:2) until the solvent front was halfway up the plate. The TLC plate was dried, and then developed in chloroform:methanol:sodium acetate pH 3.4 until the solvent from was 1 cm from the top of the plates. The plates were air dried, exposed to iodine vapors to visualize lipid bands. The presence of ¹⁴C-acetate labeled lipids was assessed by exposing the TLC plate to Fuji™ RX film for five days at −80° C.

[0286] ¹⁴C-acetate incorporation into Lysates.

[0287] One liter of log phase L. pneumophila, grown in AYE broth at 37° C., was pelleted, frozen, thawed, resuspended in 5 mls lysis buffer (5 mM Tris HCl pH 7.6, lysozyme 1 mg/ml, EDTA 1 mM, and one Complete™ protease inhibitor tablet) and sonicated at 4° C. The lysate was centrifuged for 10 minutes at 8,000×g at 4° C. to remove intact cells. 50 ul of lysate were combined with 50 ul of a cocktail consisting of 50 mM Tris HCl (pH 7.6), 5 mM MgCl₂, 5 mM ATP, 1 mM CoA, 1 mM DTT, 1 mM NADH, 1 mM NADPH, and ¹⁴C-acetate (10 μCi/ml) in 1.5 ml eppendorf tubes on ice. Boiled lysates were prepared by heating a 50 μl aliquot of the lysate for 10 minutes in a boiling water bath prior to the addition of the above cocktail. GFZ, BZE, CER, were added to the lysates to a final concentration of 2 mM. EDTA was added to the lysates to a final concentration of 10 mM. The reaction mixtures were incubated for 10-30 minutes at 37° C. At the times indicated, the 100 μl reaction were placed on ice and precipitated by the addition of 600 μl of ice cold 12% TCA. The precipitates were pelleted in a microfuge at 4° C., and washed 3× with 1 ml of 10% TCA in each wash. The washed TCA precipitated material was extracted with 200 μl of chloroform/methanol (1:1), and the extract was washed with 100 ul of water. The aqueous layer was removed, washed with 100 μl of chloroform/methanol (1:1), and the two organic extracts were combined. The entire organic extract was added to a scintillation tube, evaporated overnight and counted in 5 mls of CytoScint with a scintillation counter.

[0288] The finding that L. pneumophilaaccumulated polyhydroxybutyrate (PHB) in response to GFZ led us to hypothesize that GFZ inhibited enzyme(s) involved in fatty acid synthesis/elongation. Support for this hypothesis was obtained by studies of the ability of GFZ to inhibit ¹⁴C-acetate incorporation into L. pneumophilalipids. Since GFZ inhibited lipid synthesis in L. pneumophila, we reasoned that the most likely target would be a regulatory enzyme in fatty acid synthesis.

[0289] Putative targets included the β-ketoacyl synthase, the target of the fatty acid synthesis inhibitors cerulenin [115] and thiolactomycin [28]; b-hydroxydecenoyl dehydrase, the target for the unsaturated fatty acid synthesis inhibitor N-decenoyl-N-acetylcysteamine [33]; and enoyl reductase, the target for diazoborines [140], triclosan [40], and isoniazid [1] (FIG. 29).

[0290] Since the FabI enoyl reductase of E. coliwas reported to be an essential enzyme involved in regulating the rate of fatty acid synthesis, we chose to examine this protein for sensitivity to GFZ. A second reason for the selection of enoyl reductase, was the observation that inhibition of the FabI enoyl reductase in E. colistimulated the accumulation of 3-hydroxybutyryl-ACP (3-HB-ACP) [17]. Under normal conditions, crotonoyl-ACP, the substrate for E. coliFabI, is unstable and is present at {fraction (1/10)} the level of 3-HB-ACP [14]. Therefore, inhibition of FabI in E. colistimulates the accumulation of 3-HB-ACP rather than crotonoyl-ACP. If L. pneumophilacontains a PhaG homolog capable of converting 3-HB-ACP to 3-HB-CoA, then GFZ-mediated enoyl reductase inhibition might result in the accumulation of a precursor for PHB synthesis.

[0291] 1. Banerjee, A., E. Dubnau, A. Quemard, V. Balasubramanian, K. S. Um, T. Wilson, D. Collins, G. de Lisle and W. R. Jacobs Jr. 1994. inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science. 263:227-230.

[0292] 2. Rozwarski, D. A., G. A. Grant, D. H. R. Barton, W. R. Jacobs, Jr. and J. C. Sacchettini. 1998. Modification of the NADH of the isoniazid target (InhA) from Mycobacterium tuberculosis. Science. 279:98-102.

[0293] 3. Quemard, A., J. C. Sacchettini, A. Dessen, C. Vilcheze, R. Bittman, W. R. Jacobs and J. S. Blanchard. 1995. Enzymatic characterization of the target for isoniazid in Mycobacterium tuberculosis. Biochemistry. 34:8235-8241.

[0294] 4. Smith, S. 1994. The animal fatty acid synthase: one gene, one polypeptide, seven enzymes. FASEB J. 8:1248-1259.

[0295] 5. Cronan, J., J. E. and C. O. Rock. 1996. Biosynthesis of membrane lipids, 612-636. In F. C. Neidhardt (ed), Escherichia coliand Salmonella Cellular and Molecular Biology. ASM Press, Washington, D.C.

[0296] 6. Barker, J., P. A. Lambert and M. R. Brown. 1993. Influence of intra-amoebic and other growth conditions on the surface properties of Legionella pneumophila. Infect Immun. 61:3503-10.

[0297] 7. Moll, H., A. Sonesson, E. Jantzen, R. Marre and U. Zahringer. 1992. Identification of 27-oxo-octacosanoic acid and heptacosane-1,27-dioic acid in Legionella pneumophila. FEMS Microbiol. Lett. 97:1-6.

[0298] 8. Dreher, P. K., K. Poralla and W. A. Kong. 1976. Synthesis of w-alicyclic fatty acids from cyclic precursors in Bacillus subtilis. J. Bacteriol. 127:1136-1140.

[0299] 9. Clark, D. P., D. DeMendoza, M. L. Polacco and J. E. Cronan, Jr. 1983. Beta-hydroxydecanoyl thio ester dehydrase does not catalyze a rate-limiting step in Escherichia coli unsaturated fatty acid synthesis. Biochemistry. 22:5897-902.

[0300] 10. Garwin, J. L., A. L. Klages and J. Cronan, J. E. 1980. β-ketcacyl-acyl carrier protein synthase II of Escherichia coli. Evidence for function in the thermal regulation of fatty acid synthesis. J. Biol. Chem. 255:3263-3265.

[0301] 11. Jackowski, S. and C. O. Rock. 1983. Ratio of active to inactive forms of acyl carrier protein in Escherichia coli. J Biol Chem. 258:15186-91.

[0302] 12. Oku, H., N. Yagi, J. Nagata and I. Chinen. 1994. Precursor role of branched-chain amino acids in the biosynthesis of iso and anteiso fatty acids in rat skin. Biochim Biophys Acta. 1214:279-87.

[0303] 13. Kaneda, T. 1991. Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance. Microbiol. Rev. 55:288-302.

[0304] 14. Heath, R. J. and C. O. Rock. 1995. Enoyl-acyl carrier protein reductase (fabI) plays a determinant role in competing cycles of fatty acid elongation in Escherichia coli. J. Biol. Chem. 270:26538-26542.

[0305] 15. Heath, R. J. and C. O. Rock. 1996. Roles of the FabA and FabZ beta-hydroxyacyl-acyl carrier protein dehydratases in Escherichia colifatty acid biosynthesis. J Biol Chem. 271:27795-801.

[0306] 16. Heath, R. J., S. Jackowski and C. O. Rock. 1994. Guanosine tetraphosphate inhibition of fatty acid and phospholipid synthesis in Escherichia coliis relieved by overexpression of glycerol-3-phosphate acyltransferase (plsB). J Biol Chem. 269:26584-90.

[0307] 17. Heath, R. J. and C. O. Rock. 1996. Regulation of fatty acid elongation and initiation by acyl—acyl carrier protein in Escherichia coli. J Biol Chem. 271:1833-6.

[0308] 18. Magnuson, K., S. Jackowski, C. O. Rock and J. E. Cronan, Jr. 1993. Regulation of fatty acid biosynthesis in Escherichia coli. Microbiol Rev. 57:522-42.

[0309] 19. Sinesky, M. 1971. Temperature control of phospholipid biosynthesis in Escherichia coli. J. Bact. 106:449-455.

[0310] 20. Cronan, J. E., Jr. 1975. Thermal regulation of the membrane lipid composition of Escherichia coli. J. Biol. Chem. 250:7074-7077.

[0311] 21. Black, P. N., C. C. DiRusso, A. K. Metzger and T. L. Heimert. 1992. Cloning, sequencing, and expression of the fadD gene of Escherichia coliencoding acyl coenzyme A synthetase. J Biol Chem. 267:25513-20.

[0312] 22. DiRusso, C. C., T. L. Heimert and A. K. Metzger. 1992. Characterization of FadR, a global transcriptional regulator of fatty acid metabolism in Escherichia coli. Interaction with the fadb promoter is prevented by long chain fatty acyl coenzyme A [published erratum appears in J Biol Chem 1992 Nov 5;267(31):22693]. J Biol Chem. 267:8685-91.

[0313] 23. DiRusso, C. C., A. K. Metzger and T. L. Heimert. 1993. Regulation of transcription of genes required for fatty acid transport and unsaturated fatty acid biosynthesis in Escherichia coilby FadR. Mol Microbiol. 7:311-22.

[0314] 24. DiRusso, C. C. and T. Nystrom. 1998. The fats of Escherichia coliduring infancy and old age: regulation by global regulators, alarmones and lipid intermediates. Mol Microbiol. 27:1-8.

[0315] 25. Vance, D., I. Goldberg, O. Mitsuhashi and K. Bloch. 1972. Inhibition of fatty acid synthetases by the antibiotic cerulenin. Biochem Biophys Res Commun. 48:649-56.

[0316] 26. Pizer, E. S., F. D. Wood, H. H. S., F. E. Romantsev, G. R. Pasternack and F. P. Kuhajda. 1996. Inhibition of fatty acid synthesis delays disease progression in a xenograft model of ovarian cancer. Cancer Res. 56:1189-1193.

[0317] 27. Pizer, E. S., C. Jackisch, F. D. Wood, G. R. Pasternack, N. E. Davidson and F. P. Kuhajda. 1996. Inhibition of fatty acid synthesis induces programmed cell death in human breast cancer cells. Cancer Res. 56:2745-2747.

[0318] 28. Jackowski, S., C. M. Murphy, J. E. Cronan, Jr. and C. O. Rock. 1989. Acetoacetyl-acyl carrier protein synthase. A target for the antibiotic thiolactomycin. J Biol Chem. 264:7624-9.

[0319] 29. Noto, T., S. Miyakawa, H. Oishi, H. Endo and H. Okazaki. 1982. Thiolactomycin, a new antibiotic. III. In vitro antibacterial activity. J Antibiot (Tokyo). 35:401-10.

[0320] 30. Lomovskaya, O., K. Lewis and A. Matin. 1995. EmrR is a negative regulator of the Escherichia colimultidrug resistance pump EmrAB. J Bacteriol. 177:2328-34.

[0321] 31. Furukawa, H., J. T. Tsay, S. Jackowski, Y. Takamura and C. O. Rock. 1993. Thiolactomycin resistance in Escherichia coliis associated with the multidrug resistance efflux pump encoded by emrAB. J Bacteriol. 175:3723-9.

[0322] 32. Kass, L. R. 1968. The antibacterial activity of 3-decynoyl-N-acetylcysteamine. J. Biol. Chem. 243:3223-3228.

[0323] 33. Helmkamp, G. M., Jr., D. J. Brock and K. Bloch. 1968. Beta-hydroxydecanoly thioester dehydrase. Specificity of substrates and acetylenic inhibitors. J Biol Chem. 243:3229-31.

[0324] 34. Mandell, G. L. and M. A. Sande. 1990. Drugs used in the chemotherapy of tuberculosis and leprosy, In A. G. Gilman, T. W. Rail, A. S. Nies and P. Taylor (ed), The pharmacalogical basis of therapeutics. Pergamon Press, New York.

[0325] 35. Dessen, A., A. Quemard, W. R. Blanchard and J. C. Sacchettini. 1995. Crystal structure and function of the isoniazid target of Mycobacterium tuberculosis. Science. 267:1638-1641.

[0326] 36. Miesel, L., T. R. Weisbrod, J. A. Marcinkeviciene, R. Bittman and W. R. Jacobs, Jr. 1998. NADH dehydrogenase defects confer isoniazid resistance and conditional lethality in Mycobacterium smegmatis. J Bacteriol. 180:2459-67.

[0327] 37. iszhang, Y., T. Garbe and D. Young. 1993. Transformation with katG restores isoniazid-sensitivity in Mycobacterium tuberculosisisolates resistant to a range of drug concentrations. Mol Microbiol. 8:521-4.

[0328] 38. Grassberger, M. A., F. Turnowsky and J. Hildebrandt. 1984. Preparation and antibacterial activities of new 1,2,3-diazaborine derivatives and analogues. J Med Chem. 27:947-53.

[0329] 39. Baldock, C., J. B. Rafferty, S. E. Sedelnikova, P. J. Baker, A. R. Stuitje, A. R. Slabas, T. R. Hawkes and D. W. Rice. 1996. A mechanism of drug action revealed by stuctural studies of enoyl reductase. Science. 274:2107-2110.

[0330] 40. McMurry, L. M., M. Oethinger and S. B. Levy. 1998. Triclosan targets lipid synthesis [letter]. Nature. 394:531-2.

[0331] 41. Heath, R. J., Y. T. Yu, M. A. Shapiro, E. Olson and C. O. Rock. 1998. Broad spectrum antimicrobial biocides target the FabI component of fatty acid synthesis. J Biol Chem. 273:30316-20.

[0332] 42. Turnowsky, F., K. Fuchs, C. Jeschek and G. Hogenauer. 1989. envM Genes of Salmonella typhimuriumand Escherlchia coli. J. Bacteriol. 171:6555-6565.

[0333] 43. Cao, C., S. C. Silverstein, H. C. Neu and T. Steinberg. 1992. J774 Macrophages secrete antibiotics via organic anion transporters. J. Infect. Dis. 165:322-328.

[0334] 44. Cao, C. X., H. C. Neu and S. C. Silverstein. 1991. Gemfibrozil inhibits organic anion secretion and enhances norfloxacin accumulation in J774 macrophage-like cells. J Cell Biol. 115:476a (Abstr.).

[0335] 45. Cao, C. X. 1991. Organic anion transporters in white blood cells. Ph.D. thesis. Dept. Physiology. 119.

[0336] 46. Rudin, D. E., P. X. Gao, C. X. Cao, H. C. Neu and S. C. Silverstein. u1992. Gemfibrozil enhances the Listeriacidal effects of fluoroquinolone antibiotics in J774 macrophages. J. Exp. Med. 176:1439-1447.

[0337] 47. Stout, J. E. and V. L. Yu. 1997. Legionellosis. New Eng. J. Med. 337:682-687.

[0338] 48. Horwitz, M. A. 1984. Phagocytosis of the Legionnaires' disease bacterium (Legionella pneumophila) occurs by a novel mechanism: engulfment within a pseudopod coil. Cell. 36:27-33.

[0339] 49. Swanson, M. S. and R. R. Isberg. 1995. Association of Legionella pneumophilawith the macrophage endoplasmic reticulum. Infect Immun. 63:3609-20.

[0340] 50. Horwitz, M. A. 1983. Formation of a novel phagosome by the Legionnaires' disease bacterium (Legionella pneumophila) in human monocytes. J Exp Med. 158:1319-31.

[0341] 51. Horwitz, M. A. 1983. The Legionnaires' disease bacterium (Legionelia pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J Exp Med. 158:2108-26.

[0342] 52. Horwitz, M. A. and F. R. Maxfield. 1984. Legionella pneumophilainhibits acidification of its phagosome in human monocytes. J Cell Biol. 99:1936-43.

[0343] 53. Marra, A., M. A. Horwitz and H. A. Shuman. 1990. The HL-60 model for the interaction of human macrophages with the Legionnaires' disease bacterium. J. Immunol. 144:2738-2744.

[0344] 54. Horwitz, M. A. and S. C. Silverstein. 1980.

[0345] Legionnaire's disease bacterium (Legionella pneumophila) multiplies Intracellularly in human monocytes. J. Clin. Invest. 66:441-450.

[0346] 55. Muller, A., J. Hacker and B. C. Brand. 1996. Evidence for apoptosis of human macrophage-like HL-60 cells by Legionelia pneumophila. Infect. Immun. 64:4900-4906.

[0347] 56. Peck, R₁₉₈₅. A one-plate assay for macrophage bactericidal activity. J Immunol Methods. 82:131-40.

[0348] 57. Denizot, F. and R. Lang. 1986. Rapid calorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods. 89:271-7.

[0349] 58. Sadosky, A. B., L. A. Wiater and H. A. Shuman. 1993. Identification of Legionella pneumophila genes required for growth within and killing of human macrophages. Infect. Immun. 61:5361-5373.

[0350] 59. Jin, F. Y., V. S. Kamanna, M. Y. Chuang, K. Morgan and M. L. Kashyap. 1996. Gemfibrozil stimulates apolipoprotein A-I synthesis and secretion by stabilization of mRNA transcripts in human hepatoblastoma cell line (Hep G2). Arterioscler. Thromb. Vasc. Biol. 16:1052-1062.

[0351] 60. Hemingway, C. J., K. K. Tey and M. R. Munday. 1995. Short-term inhibition of fatty acid and cholesterol biosynthesis by the lipid-lowering drug gemfibrozil in primary rat hepatocyte cultures and rat liver in vivo. Biochem Soc Trans. 23:496S.

[0352] 61. Kahri, J., T. Sane, A. van Tol and M. R. Taskinen. 1995. Effect of gemfibrozil on the regulation of HDL subfractlons in hypertriglyceridaemic patients. J Intern Med. 238:429-36.

[0353] 62. Latruffe, N. and J. Vamecq. 1997. Peroxisome proliferators and peroxisome proliferator activated receptors (PPARs) as regulators of lipid metabolism. Biochimie. 79:81-94.

[0354] 63. Schoonjans, K., B. Staels and J. Auwerx. 1996. The peroxisome proliferator activated receptors (PPAPS) and their effects on lipid metabolism and adipocyte differentiation. Biochim Biophys Acta. 1302:93-109.

[0355] 64. Miller, M., P. S. Bachorik, B. W. McCrindle and P. O. Kwiterovich, Jr. 1993. Effect of gemfibrozil in men with primary isolated low high-density lipoprotein cholesterol: a randomized, double-blind, placebo-controlled, crossover study. Am J Med. 94:7-12.

[0356] 65. Baldo, A., A. D. Sniderman and K. Cianflone. 1994. Increase in intracellular triglyceride synthesis induced by gemfibrozil. Metabolism. 43:257-262.

[0357] 66. Schoonjans, K., B. Staels, P. Grimaldi and J. Auwerx. 1993. Acyl-CoA synthetase mRNA expression is controlled by fibric acid derivatives, feeding and liver proliferation. Eur. J. Biochem. 216:615-622.

[0358] 67. Sánchez, R. M., M. Viñals, M. Alegret, M. Vazquez, T. Adzet, M. Merlos and J. C. Laguna. 1992. Inhibition of rat liver microsomal fatty acid chain elongation by gemfibrozil in vitro. FEBS. 300:89-92.

[0359] 68. Sánchez, R. M., M. Viñals, M. Alegret, M. Vázauez, A. Adzet, M. Merlos and J. C. Laguna. 1993. Fibrates modify rat hepatic fatty acid chain elongation and desaturation in vitro. Biochem. Pharm. 46:1791-1796.

[0360] 69. Tam, S. P., L. Dory and D. Rubinstein. 1981. Fate of apolipoproteins C-1, C-iii, and E during lipolysis of human very low density lipoproteins in vitro. J Lipid Res. 22:641-51.

[0361] 70. Havlichek, D., L. Saravolatz and D. Pohold. 1987. Effect of quinolones and other antimicrobial agents on cell-associated Legionella pneumophila. Antimicrob. Agents. Chemother. 31:1529-1534.

[0362] 71. Horwitz, M. A. and S. C. Silverstein. 1983. Intracellular multiplication of Legionnaires' disease bacteria (Legionella pneumophila) in human monocytes is reversibly inhibited by erythromycin and rifampin. J Clin Invest. 71:15-26.

[0363] 72. Barker, J. E. and I. D. Farrell. 1990. The effects of single and combined antibiotics on the growth of Legionella pneumophilausing time-kill studies. J. Antimicrob. Chemother. 26:45-53.

[0364] 73. Vilde, J. L., E. Dournon and P. Rajagopalan. 1986. Inhibition of Legionella pneumophilamultiplication within human macrophages by antimicrobial agents. Antimicrob. Agents Chemother. 30:743-748.

[0365] 74. Silverstein, S. C. and C. A. Kabbash. 1994. Penetration, retention, intracellular localization, and antimicrobial activity of antibiotics within phagocytes. Current Ooinion in Hematology, Functions and Disorders of Leukocytes, 1993, 1(1):85-92.

[0366] 75. Feeley, J. C., R. J. Gibson, G. W. Gorman, N. C. Langford, J. K. Rasheed, D. C. Mackel and W. B. Baine. 1979. Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J Clin Microbiol. 10:437-41.

[0367] 76. Liebers, D. M., A. L. Baltech, R. P. Smith, M. C. Hammer and J. V. Conroy. 1989. Susceptibility of Legionella pneumophilato eight antimicrobial agents including four macrolides under different assay conditions. J. Antimicrob. Chemother. 23:37-41.

[0368] 77. Barker, J., H. Scaife and M. R. Brown. 1995. intraphagocytic growth induces an antibiotic-resistant phenotype of Legionella pneumophila. Antimicrob. Agents Chemother. 39:2684-2688.

[0369] 78. FDA Summary Basis of Approval, N.-W. -. L. C., p.1-9. FDA Summary Basis of Approval, NDA 18 422. Warner-Lambert Co., p.1-9.

[0370] 79. Pharmacologist Review of NDA 1-422, 0. S. S. t. F. b. W.-L. C., Mar. 12, 1981, p1-18. Pharmacologist Review of NDA 1-422, Original Summary. Submitted to FDA by Warner-Lambert Co., Mar. 12, 1981, p1-18.

[0371] 80. Brown, M. S. and J. L. Goldstein. 1990. Drugs used in the treatment of hyperlipoproteinemias, 874-896. In A. G. Gilman, T. W. Rall, A. S. Nies and P. Taylor (ed), The pharmalogical basis of therapeutics. Pergamon Press, New York.

[0372] 81. Nakagawa, A., A. Shigeta, H. Iwabuchi, M. Horiguchi, K. Nakamura and H. Takahagi. 1991. Simultaneous determination of gemfibrozil and its metabolites in plasma and urine by a fully automated high performance liquid chromatographic system. Biomed Chromatogr. 5:68-73.

[0373] 82. Cayen, M. N. 1985. DisDosition, metabolism, and pharmacokinetics of antihyperlipidemic agents in laboratory animals and man. Pharmac. Ther. 29:157-204.

[0374] 83. Horwitz, M. A. 1983. Symbiotic interactions between Legionella pneumophilaand human leukocytes. Int Rev Cytol Suppl. 14:307-28.

[0375] 84. W. H. O. Ed. 1996. The World Health Report.

[0376] 85. McCray, E., C. M. Weinbaum, C. R. Braden and I. M. Onorato. 1997. The epidemiology of tuberculosis in the United States. Clin Chest Med. 18:99-113.

[0377] 86. Raviglione, M. C., D. E. Snider, Jr. and A. Kochi. 1995. Global epidemiology of tuberculosis. Morbidity and mortality of a worldwide epidemic [see comments]. Jama. 273:220-6.

[0378] 87. Antonucci, G., E. Girardi, M. C. Raviglione and G. Ippolito. 1995. Risk factors for tuberculosis in HIV-infected persons. A prospective cohort study. The Gruppo Italiano di Studio Tubercolosi e AIDS (GISTA). Jama. 274:143-8.

[0379] 88. Markowitz, N., N. I. Hansen, P. C. Hopewell, J. Glassroth, P. A. Kvale, B. T. Mangura, T. C. Wilcosky, J. M. Wallace, M. J. Rosen and L. B. Reichman. 1997. Incidence of tuberculosis in the United States among HIV-infected persons. The Pulmonary Complications of HIV Infection Study Group. Ann Intern Med. 126:123-32.

[0380] 89. Bifani, P. J., B. B. Plikaytis, V. Kapur, K. Stockbauer, X. Pan, M. L. Lutfey, S. L. Moghazeh, W. Eisner, T. M. Daniel, M. H. Kaplan, J. T. Crawford, J. M. Musser and B. N. Kreiswirth. 1996. Origin and interstate spread of a New York City multidrug-resistant Mycobacterium tuberculosisclone family [see comments]. Jama. 275:452-7.

[0381] 90. Awaya, J., T. Ohno, H. Ohno and S. Omura. 1975. Substitution of cellular fatty acids in yeast cells by the antibiotic cerulenin and exogenous fatty acids. Biochim Biophys Acta. 409:267-73.

[0382] 91. Greenspan, M. D. and R. C. Mackow. 1977. The effect of cerulenin on sterol biosynthesis in Saccharomyces cerevisiae. Lipids. 12:729-40.

[0383] 92. Hayashi, T., O. Yamamoto, H. Sasaki, H. Okazaki and A. Kawaguchi. 1984. Inhibition of fatty acid synthesis by the antibiotic thiolactomycin. J Antibiot (Tokyo). 37:1456-61.

[0384] 93. Cole, S. T., R. Brosch, J. Parkhill, T. Garnier, C. Churcher, D. Harris, S. V. Gordon, K. Eiglmeier, S. Gas, C. E. Barry, 3rd, F. Tekaia, K. Badcock, D. Basham, D. Brown, T. Chillingworth, R. Connor, R. Davies, K. Devlin, T. Feltwell, S. Gentles, N. Hamlin, S. Holroyd, T. Hornsby, K. Jagels, B. G. Barrell and et al. 1998. Deciphering the biology of Mycobacterium tuberculosisfrom the complete genome sequence [see comments] [published erratum appears in Nature 1998 Nov 12;396(6707):190]. Nature. 393:537-44.

[0385] 94. Chandler, F. W., R. M. Cole, M. D. Hicklin, J. A. Blackmon and B. S. Callaway. 1979. Ultrastructure of the Legionnaire's disease bacterium. Ann. Int. Med. 90:642-647.

[0386] 95. Rodgers, F. G. and M. R. Davey. 1982. Ultrastructure of the cell envelope layers and surface details of Legionella pneumophila. J. Gen. Microbiol. 128:1547-1557.

[0387] 96. Mauchline, W. S. and K. C. W. 1991. Development of the BIOLOG substrate utilization system for identification of Legionella spp. Appl. Environ. Microbiol. 57:3345-3349.

[0388] 97. Moffie, B. G. and R. P. Mouton. 1988. Sensitivity and resistance of Legionella pneumophilato some antibiotics and combinations of antibiotics. J Antimicrob Chemother. 22:457-62.

[0389] 98. Ostle, A. G. and J. G. Holt. 1982. Nile Blue: A as a fluorescent stain for poly-β hydroxybutyrate. Appl. Environ. Microbiol. 44:238-241.

[0390] 99. de Smet, M. J., G. Eggink, B. Witholt, J. Kingma and H. Wynberg. 1983. Characterization of intracellular inclusions formed by Pseudomonas oleovoransduring growth on octane. J Bacteriol. 154:870-8.

[0391] 100. Findlay, R. H. and D. C. White. 1983. Polymericβ-hydroxyalkanoates from environmental samples and Bacillus megaterium. Appl. Environ. Microbiol. 45:71-78.

[0392] 101. Riis, W. and W. Mai. 1988. Gas chromatographic determination of poly-8-hydroxybutyric acid in microbial biomass after hydrochloric acid propanolysis. J. Chromatog. 445:285-289.

[0393] 102. Anderson, A. J. and E. A. Dawes. 1990. Occurrence, metabolism, metabolic rate, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54:450-472.

[0394] 103. Madison, L. L. and G. W. Huisman. 1999. Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Biol Rev. 63:21-53.

[0395] 104. West, A. A., J. Rogers, J. V. Lee and C. W. Keevil. 1993. Lack of dormancy in Legionella pneumophila?, 201-203. In J. M. Barbaree, R. F. Breiman and A. P. Dufour (ed), Legionella: current status and emerging perspectives. American Society for Microbiology, Washington D.C.

[0396] 105. James, B. W., W. S. Mauchline, P. J. Dennis, C. W. Keevil and R. Wait. 1999. Poly-3-hydroxybutyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments. Appl. Environ. Microbiol. 65:822-827.

[0397] 106. Hrabak, O. 1992. Industrial production of poly-b-hydroxybutyrate. FEMS Microbiol Rev. 103:251-256.

[0398] 107. Wallace, K. K., S. Lobo, L. Han, H. A. McArthur and K. A. Reynolds. 1997. In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus. J Bacteriol. 179:3884-91.

[0399] 108. Kaneda, T. and E. J. Smith. 1980. Relationship of primer specificity of fatty acid de novo synthetase to fatty acid composition in 10 species of bacteria and yeasts. Can J Microbiol. 26:893-8.

[0400] 109. Doi, Y., A. Tamaki, M. Kunioka and K. Soga. 1988. Production of copolyesters of 3-hydroxybutyrate and 3-hydroxyvalerate by Alcaligenes eutrophusfrom butyric and pentanoic acids. Appl. Microbiol. Biotechnol. 28:330-334.

[0401] 110. Rehm, B. H. A., N. Kruger and A. Steinbuchel. 1998. A new metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid synthesis. J Biol Chem. 273:24044-24051.

[0402] 111. Fukui, T., N. Shiomi and Y. Doi. 1998. Expression and characterization of (R)-specific enoyl coenzyme A hydratase involved in polyhydroxyalkanoate biosynthesis by Aeromonas caviae. J Bacteriol. 180:667-73.

[0403] 112. Clark, D. P. and J. E. Cronan, Jr. 1996. Two-carbon compounds and fatty acids as carbon sources, 343-357. In F. C. Neidhardt (ed), Escherichia coliand Salmonella Cellular and Molecular Biology. ASM Press, Washington, D.C.

[0404] 113. Tesh, M. J., S. A. Morse and R. D. Miller. 1983. Intermediary metabolism in Legionella pneumophila: utilization of amino acids and other compounds as energy sources. J. Bacteriol. 154:1104-1109.

[0405] 114. Cronan, J. E., Jr. and S. Subrahmanyam. 1998. FadR, transcriptional co-ordination of metabolic expediency. Mol Microbiol. 29:937-43.

[0406] 115. D'Agnolo, G., I. S. Rosenfeld, A. J., S. Omura and P. R. Vagelos. 1973. Inhibition of fatty acid synthesis by the antibiotic cerulenin. Specific inactivation of β-ketoacyl-acyl carrier protein synthetase. Biochim. Biophys. Acta. 326:155-166.

[0407] 116. Omura, S. 1981. Cerulenin. Methods Enzymol. 72:520-32.

[0408] 117. Nomura, S., T. Horiuchi, S. Omura and T. Hata. 1972. J. Antibiot. 25:365-368.

[0409] 118. Kuhajda, F. P., K. Jenner, F. D. Wood, R. A. Hennigar, L. B. Jacobs, J. D. Dick and G. R. Pasternack. 1994. Fatty acid synthesis: a potential selective target for antineoplastic therapy. Proc Natl Acad Sci U S A. 91:6379-83.

[0410] 119. Pizer, E. S., F. D. Wood, G. R. Pasternack and F. P. Kuhajda. 1996. Fatty acid synthase (FAS): a target for cytotoxic antitetabolites in HL60 promyelocytic leukemia cells. Cancer Res. 56:745-51.

[0411] 120. Bligh, E. G. and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 37:911-917.

[0412] 121. Folch, J., M. Lees and G. H. Stanley. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226:497-509.

[0413] 122. Helander, I. M., B. Lindner, U. Seydel and M. Vaara. 1993. Defective biosynthesis of the lipid A component of temperature-sensitive firA (omsA) mutant of Escherichia coli. Eur J Biochem. 212:363-9.

[0414] 123. Lambert, M. A. and W. Moss. 1989. Cellular fatty acid compositions and isoprenoid quinone contents of 23 Legionella species. J. Clin. Microbiol. 27:465-473.

[0415] 124. Henry, A., C. M. Allen and P. W. Stacpoole. 1992. Fibric acid derivatives: effects on the synthesis of isoprenoid lipids in cultured human lymphocytes. Biochim. Biophys. Acta. 1127(2): 168-173.

[0416] 125. Mindich, L. 1972. Control of fatty acid synthesis in bacteria. J Bacteriol. 110:96-102.

[0417] 126. Letts, V., P. Shaw, L. Shapiro and S. Henry. 1982. Synthesis and utilization of fatty acids by wild-type and fatty acid auxotrophs of Caulobacter crescentus. J Bacteriol. 151:1269-78.

[0418] 127. Harder, M. E., R. C. Ladenson, S. D. Schimmel and D. F. Silbert. 1974. Mutants of Escherichia coliwith temperature-sensitive malonyl coenzyme A-acyl carrier protein transacylase. J. Biol. Chem. 249:7468-7475.

[0419] 128. Weiss, E., M. G. Peacock and J. C. Williams. 1980. Glucose and glutamate metabolism of Legionella pneumophila. Curr Microbiol. 4:1-6.

[0420] 129. Manchak, J. and W. J. Page. 1994. Control of polyhydroxyalkanoate synthesis in Azobacteri vinelandiistrain UWD. Microbiology. 140:953-963.

[0421] 130. Park, J. S. and Y. H. Lee. 1996. Metabolic characteristics of the isocitrate dehydrogenase leaky mutant of Alcaligenes eutrophusand its utilization for poly-β-hydroxybutyrate production. J. Ferment. Bioeng. 81:197-205.

[0422] 131. Overath, P., G. Pauli and H. U. Schairer. 1969. Fatty acid degradation in Escherichia coli. An inducible acyl-CoA synthetase, the mapping of old-mutations, and the isolation of regulatory mutants. Eur J Biochem. 7:559-74.

[0423] 132. Klein, K., R. Steinberg, B. Fiethen and P. Overath. 1971. Fatty acid degradation in Escherichia coli. An inducible system for the uptake of fatty acids and further characterization of old mutants. Eur J Biochem. 19:442-50.

[0424] 133. Cronan, J. E., Jr. 1997. In vivo evidence that acyl coenzyme A regulates DNA binding by the Escherichia coliFadR global transcription factor. J Bacteriol. 179:1819-23.

[0425] 134. Elovson, J. and P. R. Vagelos. 1968. Acyl carrier protein. X. Acyl carrier protein synthetase. J Biol Chem. 243:3603-11.

[0426] 135. Rock, C. O. and S. Jackowski. 1982. Regulation of phospholipid synthesis in Escherichia coli. Composition of the acyl-acyl carrier protein pool in vivo. J Biol Chem. 257:10759-65.

[0427] 136. Rock, C. O. and S. Jackowski. 1985. Pathways for the incorporation of exogenous fatty acids into phosphatidylethanolamine in Escherichia coli. J Biol Chem. 260:12720-4.

[0428] 137. Horsmans, Y., J. P. Desager and C. Harvengt. 1993. Effects of gemfibrozil on the activities of plasma lipolytic enzymes in normolipidemic subjects. Clinca Chimica Acta. 218:223-228.

[0429] 138. Gnasso, A., B. Lehner, W. Haberbosch, O. Leiss, K. von Bergmann and J. Augustin. 1986. Effect of gemfibrozil on lipids, apoproteins, and postheparin lipolytic activities in normolipidemic subjects. Metabolism. 35:387-93.

[0430] 139. Heath, R. J. and C. O. Rock. 1996. Inhibition of beta-ketoacyl-acyl carrier protein synthase III (FabH) by acyl—acyl carrier protein in Escherichia coli. J Biol Chem. 271:10996-1000.

[0431] 140. Bergler, H., P. Wallner, A. Ebeling, B. Leitinger, S. Fuchsbichler, H. Aschauer, G. Kollenz, G. Högenauer and F. Turnowsky. 1994. Protein EnvM is the NADH-dependent enoyl-ACP reductase (FabI) of Escherichia coli. J. Biol. Chem. 269:5493-5496.

[0432] 141. Bergler, H., S. Fuchsbichler, G. Högenauer and F. Turnowsky. 1996. The enoyl-[acyl-carrier-protein] reductase (FabI) of Escherichia coli, which catalyzes a key regulatory step in fatty acid biosynthesis, accepts NADH and NADPH as cofactors and is inhibited by palmitoyl-CoA. Eur. J. Biochem. 242:689-694.

[0433] 142. Baldock, C., J. B. Rafferty, A. R. Stuitje, A. R. Slabas and D. W. Rice. 1998. The X-ray structure of Escherichia colienoyl reductase with bound NAD+ at 2.1 A resolution. J Mol Biol. 284:1529-46.

[0434] 143. Gibson, T., D. Higgins and J. Thompson. 1996. Clusta1W.

[0435] 144. Maloy, S. R., C. L. Ginsburgh, R. W. Simons and W. D. Nunn. 1981. Transport of long and medium chain fatty acids by Escherichia coliK12. J Biol Chem. 256:3735-42.

[0436] 145. Ginsburgh, C. L., P. N. Black and W. D. Nunn. 1984. Transport of long chain fatty acids in Escherichia coli. Identification of a membrane protein associated with the fadl gene. J Biol Chem. 259:8437-43.

[0437] 146. Kameda, K. 1986. Partial purification and characterization of fatty acid binding protein(s) in Escherichia colimembranes and reconstitution of fatty acid transport system. Biochem Int. 13:343-50.

[0438] 147. McClain, M. S., M. C. Hurley, J. K. Brieland and E. N. C. 1996. The Legionella pneumophilahel locus encodes intracellularly induced homologs of heavy-metal ion transporters of Alcaligenes spp. Infect. Immun. 64:1532-1540.

[0439] 148. Arroyo, J., M. C. Hurley, M. Wolf, M. S. McClain, B. I. Eisenstein and N. C. Engleberg. 1994. Shuttle mutagenesis of Legionella pneumophila: Identification of a gene associated with host cell cytopathocity. Infect. Immun. 62:4075-4080.

[0440] 149. Goldman, P. and R. P. Vagelos. 1961. J Biol Chem. 236:2620.

[0441] 150. Fernandes, N. D. and P. E. Kolattukudy. 1998. A newly identified methyl-branched chain fatty acid synthesizing enzyme from Mycobacterium tuberculosisvar. bovis BCG. J Biol Chem. 273:2823-8.

[0442] 151. Cornick, N. A., M. Silva and S. L. Gorbach. 1990. In vitro antibacterial activity of bismuth subsalicylate. Rev. Inf. Dis. 12[Suppl 1]:S9-S10.

[0443] 152. Manhart, M. D. 1990. In vitro antimicrobial activity of bismuth subsalicylate and other bismuth salts. Rev. Inf. Dis. 12[Suppl 1]:S11-S15.

[0444] 153. Levy, C. W., A. Roujeinikova, S. Sedelnikova, P. J. Baker, A. R. Stuitje, A. R. Slabas, D. W. Rice and J. B. Rafferty. 1999. Molecular basis of triclosan activity [letter]. Nature. 398:383-4.

[0445] 154. Black, P. N. and C. C. DiRusso. 1994. Molecular and biochemical analyses of fatty acid transport, metabolism, and gene regulation in Escherichia coli. Biochim Biophys Acta. 1210:123-45.

[0446] 155. Cleland, W. W. 1979. Statistical analysis of enzyme kinetic data. Methods Enzymol. 63:103-38.

[0447] 156. Martinez-Blanco, H., A. Reglero, L. B. Rodriguez-Aparicio and J. M. Luengo. 1990. Purification and biochemical characterization of phenylacetyl-CoA ligase from Pseudomonas putida. A specific enzyme for the catabolism of phenylacetic acid. J Biol Chem. 265:7084-90.