ACC INHIBITORS FOR USE IN TREATING MYCOBACTERIAL DISEASES

Abstract

Compounds which are suitable for treating mycobacterial diseases and pharmaceutical compositions containing such compounds are provided. Also encompassed are such compounds for use in medicine. The disclosure further relates to a kit of parts comprising a pharmaceutical composition containing such compounds and at least one additional pharmaceutically active compound.

Claims

1. A method for treating a mycobacterial disease in macrophage host cells of a host organism comprising providing the host organism with a composition comprising a therapeutically effective amount of an inhibitor of the host acetyl-CoA-car-boxylase 2 (ACC2), at least one additional pharmaceutically active compound selected from an antibiotic, antifungal and/or anti-HIV compound, and optionally a pharmaceutically acceptable carrier, diluent, or excipient, wherein the administering step does not affect the viability of the host macrophages and does not decrease the replication rate of M. tuberculosis in the host organism, wherein the mycobacterial disease is tuberculosis caused by bacteria of Mycobacterium tuberculosis, and wherein said antibiotic is selected from the group consisting of Isoniazid, Rifampicin, Ethambutol, Pyrazinamide, Rifapentine, Rifabutin, Animoglycosides including Kanamycin and/or Amikacin, Polypetides including Capreomycin, Viomycin and/or Streptomycin, fluoroquinolones including Moxifloxacin, Levofloxacin, Ofloxacin and/or Gatifloxacin, thioamides including Ethionamide and/or Protionamide, Cycloserine, Terizidone, Thioacetone, p-Aminosalicylic acid, Clofazimine, Linezolid, Amoxicillin, Clavulanate, Thioacetazone, Imipenem, Cilastatin, Clarithromycin, Delamanid, and Bedaquiline.

2. The method of claim 1, wherein said composition comprises an ACC2 inhibitor having a structure according to formula I ##STR00007## or pharmaceutically acceptable salts of said inhibitor; wherein A-B is N—CH or CH—N; K is (CH.sub.2)r wherein r is 2, 3 or 4; m and n are each independently 1, 2 or 3 when A-B is N—CH or m and n are each independently 2 or 3 when A-B is CH—N; the dashed line represents the presence of an optional double bond; D is carbonyl or sulfonyl; G is carbonyl, sulfonyl or CR.sup.7R.sup.8; wherein R.sup.7 and R.sup.8 are each independently H, (C.sub.1-C.sub.6)alkyl, (C.sub.2-C6)alkenyl or (C.sub.2-C.sub.6)alkynyl or a five to seven membered partially saturated, fully saturated or fully unsaturated ring optionally having one heteroatom selected from oxygen, sulfur and nitrogen; E is a tricyclic ring, linked through the middle ring, consisting of two fused fully unsaturated six membered rings, taken independently each of said rings optionally having a nitrogen heteroatom, said two fused rings fused to a third partially saturated or fully unsaturated six membered ring, said third ring optionally having one nitrogen heteroatom; wherein said E ring is optionally mono-, di- or tri-substituted independently on each ring used to form the tri-cyclic or teraryl ring with halo, hydroxy, (C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.4)alkylthio, or mono-N- or diN, N-(C.sub.1-C.sub.6)alkylamino wherein said (C.sub.1-C.sub.6)alkyl and (C.sub.1C.sub.6)alkoxy substituents are also optionally mono-, di- or tri-substituted independently with halo, hydroxy or from one to nine fluorines; and J is NR.sup.2R.sup.3, wherein R.sup.2 and R.sup.3 can be taken together with the nitrogen atom to which they are attached to form a partially saturated or fully saturated five to six membered ring optionally having one additional heteroatom selected independently from oxygen and nitrogen; wherein said NR.sup.2R.sup.3 ring is optionally mono-, di-, tri- or tetra-substituted independently with halo, hydroxy, amino, oxo, (C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkoxy wherein said (C.sub.1-C.sub.6)alkyl substituent is optionally mono-, di- or tri-substituted independently with chloro, hydroxy, oxo, (C.sub.1-C.sub.6)alkoxy and said (C.sub.1-C.sub.6)alkyl substituent is also optionally substituted with from one to nine fluorines; or wherein R.sup.2 and R.sup.3 are each independently H, Q, or (C.sub.1-C.sub.6)alkyl, wherein said (C.sub.1-C.sub.6)alkyl is optionally mono-, di- or tri-substituted independently with halo, hydroxy, (C.sub.1-C.sub.4)alkylthio, (C.sub.1-C.sub.6)alkyloxycarbonyl, or mono-N- or di-N,N-(C.sub.1-C.sub.6)alkylamino or Q.sup.1; wherein Q and Q.sup.1 are each independently partially saturated, fully saturated or fully unsaturated three to seven membered ring optionally having one heteroatom selected independently from oxygen and nitrogen; wherein said Q and Q.sup.1 ring are each independently optionally mono-, di- or tri-substituted independently with halo, hydroxy, oxo, (C.sub.1-C.sub.6)alkyl or (C.sub.1-C.sub.6)alkoxy wherein said (C.sub.1-C.sub.6)alkyl substituent is also optionally substituted with from one to nine fluorines or a pharmaceutically acceptable salt thereof.

3. The method of claim 1, wherein said composition comprises an ACC2 inhibitor having a structure according to formula II ##STR00008## wherein R.sub.10 is selected from the group consisting of hydrogen, cycloalkyl, alkyl and haloalkyl; Y is selected form the group consisting of —(CR.sub.4aR.sub.4b).sub.p, —C(O)—, —O—, —N(H)—, —N(alkyl)- and —S—; wherein p is 1, 2 or 3; each of R.sub.4a, R.sub.4b, at each occurrence, is independently selected from the group consisting of hydrogen, alkyl, hydroxyalkyl, and haloalkyl when p is 1, 2 or 3; alternatively, R.sub.4a and R.sub.4b together with the carbon to which they are attached form a monocyclic cycloalkyl or heterocycle ring when p is 1; Ar.sub.3 is ##STR00009## A.sub.1, B.sub.1, E.sub.1, and D.sub.1 are —C(R)—; or one of A.sub.1, B.sub.1, E.sub.1 and D.sub.1 is N and the others are —C(R)—; wherein R is selected from the group consisting of hydrogen, —I, —Br, —Cl, and —F; Ar.sub.1 is selected from the group consisting of phenyl, pyridinyl, thienyl, furanyl, thiazolyl, and 1, 3, 4-thiadiazolyl; each of which is independently unsubstituted or substituted with one substituent selected form the group consisting of —I, —Br, —Cl, and —F; Ar.sub.2 is selected from the group consisting of thienyl, thiazolyl, isoxazolyl, 1,2,4-thiadiazolyl, and 1,2,4-oxadiazolyl; each of which is independently unsubstituted or substituted with one substituent selected from the group consisting of methyl and ethyl; R.sub.10 is selected form the group consisting of methyl and trifluoromethyl; Z is selected form the group consisting of —OR.sub.9a and —NR.sub.60R.sub.9b; wherein R.sub.9a is —S(O).sub.2(methyl), R.sub.50 is hydrogen, and R.sub.9b is selected from the group consisting of hydrogen, —C(O)NH.sub.2, —C(O)N(H)(methyl), —C(O)O(methyl), —S(O).sub.2(methyl), —CH.sub.2—C(O)O(methyl) and —C(O)R.sub.20 wherein R.sub.20 is methyl, ethyl, isopropyl or unsubstituted cyclopropyl; Y is —O—; and R.sub.50 is selected from the group consisting of methyl, ethyl, isopropyl, 2-methylpropyl, —R.sub.80, and-CH.sub.2-R.sub.80; wherein R.sub.80 at each occurrence is an unsubstituted ring selected from the group consisting of phenyl, cyclopropyl, cyclopentyl, cyclohexyl, tetrahydrofuranyl and tetrahydropyranyl, or a pharmaceutically acceptable salt, solvate or hydrate thereof.

4. The method of claim 2, wherein the composition comprises the ACC2 inhibitor ##STR00010##

5. The method of claim 3, wherein the composition comprises the ACC2 inhibitor ##STR00011##

6. The method of claim 1, wherein the host organism is a mammalian subject and wherein said composition is administered to the mammalian subject in an amount effective to treat said mycobacterial disease.

7. The method of claim 1, wherein said additional pharmaceutically active compound is an antibiotic selected from the group consisting of Isoniazid, Rifampicin, Ethambutol, Pyrazinamide, Rifapentine, Rifabutin, Animoglycosides including Kanamycin and/or Amikacin, Polypetides including Capreomycin, Viomycin and/or Streptomycin, fluoroquinolones including Moxifloxacin, Levofloxacin, Ofloxacin and/or Gatifloxacin, thioamides including Ethionamide and/or Protionamide, Cycloserine, Terizidone, Thioacetone and/or p-Aminosalicylic acid,

8. The method of claim 1, wherein said additional pharmaceutically active compound is an antibiotic selected from the group consisting of Isoniazid, Rifampicin, Ethambutol, Pyrazinamide, Rifapentine, and Rifabutin.

9. The method of claim 1, wherein said additional pharmaceutically active compound is an antibiotic selected from the group consisting of Isoniazid, Rifampicin, Ethambutol, and Pyrazinamide.

10. The method of claim 1, wherein the mycobacterial disease is tuberculosis caused by single- or multi-drug resistant strains of Mycobacterium tuberculosis.

11. A pharmaceutical composition comprising the ACC2 inhibitor, the at least one additional pharmaceutically active compound, and the pharmaceutically acceptable carrier, diluent, or excipient, of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0107] FIG. 1: Acetyl-CoA Carboxylase 2 (ACC2) inhibitor ab142090 does not affect the growth of Mycobacterium tuberculosis (Mtb) in liquid culture. Green-fluorescence protein (GFP)-expressing Mtb bacteria were cultivated in liquid culture in the absence (Ctrl) or presence of the indicated concentrations of an ACC2 inhibitor (ab142090) or the antibiotic rifampicin. Growth was determined by measuring fluorescence signal intensity at 528 nm after excitation at 485 nm in a microplate reader at the indicated time points. Depicted is the mean +/−SEM of two independent experiments.

[0108] FIG. 2: Acetyl-CoA Carboxylase 2 (ACC2) inhibitor ab142090 dose-dependently reduces intracellular growth of Mycobacterium tuberculosis in primary human macrophages. Human monocyte-derived macrophages (hMDM) of healthy donors were infected with Mycobacterium tuberculosis (Mtb H37Rv, multiplicity of infection 1:1) for four hours. Subsequently, cells were washed and incubated in the absence (ctrl) or presence of dimethylsulfoxid (DMSO) or various concentrations (100 nM, 300 nM) of an ACC2 inhibitor (ab142090) for seven days. Additional culture medium-containing inhibitor was added at day three post infection. Four hours post infection (Uptake) or at day seven post infection, cells were lysed and intracellular bacteria were plated in serial dilutions on 7H10 agar plates to determine the Colony Forming Units (CFU). Data is depicted as mean +/−SEM of three independent experiments (donors) which either shows the CFU per Well (left site) or the percentage of CFU relative to DMSO-treated cells (% of ctrl, right site). For statistical analysis, raw data was log-transformed and a 1WAY-ANOVA with Bonferroni-correction as post hoc test was used to compare data from DMSO-treated macrophages with inhibitor-treated cells (*p≤0.05, **0.01).

[0109] FIG. 3: Acetyl-CoA Carboxylase 2 (ACC2) inhibitor ab142090 in combination with the first line TB-drugs rifampicin and isoniazid reduces intracellular growth of Mycobacterium tuberculosis in primary human macrophages. Human monocyte-derived macrophages (hMDM) of healthy donors were infected with Mycobacterium tuberculosis (Mtb H37Rv, multiplicity of infection 1:1) for four hours. Subsequently, cells were washed and incubated for seven days with different ACC2 inhibitor ab142090 concentrations (100 nM or 300nM) in the absence (no antibiotic) or presence of the antibiotics rifampicin or isoniazid in suboptimal concentrations as indicated. Additional culture medium-containing inhibitor or antibiotic was added at day three post infection. At day seven post infection, intracellular bacteria were plated in serial dilutions on 7H10 plates to determine the Colony Forming Units (CFU). Data was normalized to DMSO-treated cells and is depicted as the mean +/−SEM of three independent experiments (donors). For statistical analysis, CFU data was log-transformed and a 1WAY-ANOVA with Bonferroni-correction as post hoc test was used to compare DMSO-treated macrophages with inhibitor treated macrophages (no antibiotic, left side) or the indicated groups treated with antibiotics (*p0.05).

[0110] FIG. 4: Acetyl-CoA Carboxylase 2 (ACC2) inhibitor CP640.186 dose-dependently reduces intracellular growth of Mycobacterium tuberculosis in primary human macrophages. Human monocyte-derived macrophages (hMDM) of healthy donors were infected with Mycobacterium tuberculosis (Mtb H37Rv, multiplicity of infection 1:1) for four hours. Subsequently, cells were washed and incubated in the absence (ctrl) or presence of dimethylsulfoxid (DMSO) or various concentrations (300nM, 500 nM) of an ACC2 inhibitor (CP640.186) for seven days. Additional culture medium-containing inhibitor was added at day three post infection. Four hours post infection (Uptake) or at day seven post infection, cells were lysed and intracellular bacteria were plated in serial dilutions on 7H10 agar plates to determine the Colony Forming Units (CFU). Data is depicted as mean +/−SEM of one out of two independent experiments (donors) which shows the CFU per Well.

[0111] FIG. 5: Acetyl-CoA Carboxylase 2 (ACC2) inhibitor ab142090 does not affect the viability of primary human macrophages within the range of tested concentrations. Human monocyte-derived macrophages (hMDM) were seeded in E-Plates (ACEA) and were incubated in the absence (Medium, control) or presence of DMSO (0.1%; solvent control), the apoptosis inducing agent staurosporine (1 μg/ml) or the ACC2-Inhibitor ab142090. The viability of macrophages is reflected by changes in the cell index, which depends on the electrical impedance on the bottom of the plate (monitored over a period of five days (120h) by use of a xCELLigence Real-Time Cell Analyzer (RTCA, ACEA)). Depicted is one representative experiment out of two.

[0112] FIG. 6: Acetyl-CoA Carboxylase 2 (ACC2) inhibitor CP640.186 does not affect the viability of primary human macrophages within the range of tested concentrations. Human monocyte-derived macrophages (hMDM) were seeded in E-Plates (ACEA) and were incubated in the absence (Medium),or presence of DMSO (0.1%; solvent control), the apoptosis inducing agent staurosporine (1 μg/ml) or the ACC2-Inhibitor CP640.186 at the concentrations indicated. The viability of macrophages is reflected by changes in the cell index, which depends on the electrical impedance on the bottom of the plate (monitored over a period of five days (120 h) by use of a xCELLigence Real-Time Cell Analyzer (RTCA, ACEA)). Depicted is one representative experiment out of two.

[0113] FIGS. 7A-D: Restriction of intracellular Mycobacterium tuberculosis growth by pharmacological inhibition of macrophage ACC2. Human monocyte-derived macrophages were infected with M. tuberculosis H37Rv (multiplicity of infection 1:1, inoculum) for four hours (=Uptake), and subsequently incubated for seven days (d7) in the absence (solvent, (Dimethylsulfoxid)) or presence of different ACC2 inhibitors ((A) MK-40474, (B) ND-646, (C) Diclofop, (D) Haloxyfop) with the concentrations indicated. Intracellular bacteria were enumerated by determining Colony forming units after lysis of cells. Data from three donors are shown (n=3). Statistical analyses were carried out using One-Way ANOVA with a suitable post-hoc test for multiple comparison. *p≤0.05, **p≤0.01, ***p0.001. All data are depicted as mean +/−SEM.

[0114] FIG. 8: MK-4074 and ND-646 do not affect growth of Mycobacterium tuberculosis (Mtb) in liquid culture. Green fluorescent protein (GFP)-expressing Mtb bacteria were cultured in liquid culture in the absence (ctrl) or presence of the antibiotic rifampicin or various ACC2 inhibitors at the indicated concentrations. Growth was determined by measuring fluorescence signal intensity at 528 nm after excitation at 485 nm in a microplate reader on day 7 post infection. Depicted is the mean +/−SEM of three independent experiments.

[0115] FIG. 9: CP640186 does not affect growth of Mycobacterium tuberculosis (Mtb) in liquid culture. Green fluorescent protein (GFP)-expressing Mtb bacteria were cultured in liquid culture in the absence (ctrl) or presence of the antibiotic rifampicin or the ACC2 inhibitor CP640186 at the indicated concentrations. Growth was determined by measuring fluorescence signal intensity at 528 nm after excitation at 485 nm in a microplate reader on day 7 post infection. Depicted is the mean +/−SEM of two independent experiments.

[0116] The invention will be described by way of examples further without restricting the invention thereto.

EXAMPLES

Methods

[0117] 1) Measurement of Acetyl-CoA Carboxylase Inhibitory Activity and adaptation of ACC Inhibition High Throughput Screening Format using a radiochemical method: (Harwood H J Jr, et al. (2003), J Biol Chem 278:37099-37111.) The procedure for measuring ACC1 and ACC2 activity utilizes a radiochemical method that measures incorporation of [14C]bicarbonate into [14C]malonyl-CoA and separates product from unused substrate at the end of the reaction through acidification, which serves to both quench the reaction and remove residual radiolabeled substrate as 14CO2.
2) Test of ACC2 inhibition in tissue/Detection of Malonyl CoA: The activity of ACC2 inhibition leading to reduced Malonyl Co A levels in tissue samples has been described before (Glund et al. Diabetologia (2012) 55:2044-2053.

[0118] Malonyl-CoA Measurement

Animals were killed and liver and muscle samples were immediately snap frozen in liquid N2. Aliquots were homogenised in 0.5 mol/l perchloric acid and subsequently filtered using Vivaspin Centrifugal Concentrators with a 0.2 pmpolyethersulfon membrane (VWR, Darmstadt, Germany). The sample was cleaned online using a weak anion-exchange column (Oasis WAX, Waters, Eschborn, Germany) with 1 mmol/l acetic acid (pH 3.5) as loading buffer and 200 mmol/l NH3 (pH 11) for elution made up 1:9 with solvent A (5 mmol/l dibutylamine [DBA], 15 mmol/l NH4OAc pH 7) for transfer onto the reverse-phase (RP) precolumn. Ionpairing RP-HPLC separation was carried out at 40° C. using a precolumn and separating column Zorbax Eclipse XDB-018 (Agilent, Böblingen, Germany), solvent A and for a gradient solvent B (5 mmol/l DBA, 15 mmol/l NH4OAc pH 7, in 50% acetonitrile). At 9.5 min, HPLC eluent was directed to an electrospray ionisation MS (Agilent 1946D) and data acquisition was started. Malonyl-CoA was detected at 854 m/z.
3) ACC test using a ACC/FAS coupled assay: ACC inhibition has been described by (T. S. Hague et al./Bioorg. Med. Chem. Lett. 19 (2009) 5872-5876).The authors use a ACC/Fatty-Acid-Synthase (FAS)-Coupled Assay according to Seethala et al. (Anal. Biochem. 2006, 58, 257) with minimal modifications.
Briefly, the assay buffer (50 mM HEPES pH 7.5, 10 mM sodium citrate, 20 mM MgCl2, 6 mM NaHCO3) and substrate mixture (containing 2.4 microM [3H] acetyl-CoA (PerkinElmer, NET-290) and 47.6 microM acetyl-CoA, 100 microM NADPH and 0.125 mM ATP in Assay Buffer) were all made fresh on the day of assay from stock solutions. Human ACC1 and human ACC2 were recombinant enzymes expressed in and purified from a bacculovirus system (Protein Expr. Purif. 2007, 51, 11). To each well containing 0.5 microL of compound in DMSO or DMSO as control in a 384-well phospholipid FlashPlate_(PerkinElmer) was added 30 microL of a solution of ACC (2-4.5 nM) and FAS (1 microg/assay) enzymes in assay buffer. After a 10 min incubation, the reaction was started via addition of 20 microL of substrate mixture. The reaction was carried out for 30 min at room temperature. After incubation, the reaction was quenched with the addition of 10 microL of 200 mM EDTA (˜33 mM final concentration). The [3H]-palmitic acid produced was determined by counting in a TopCount instrument (Perki-nElmer). The IC50 for each compound was calculated using a logistic 4 parameter fit equation: y=A+((B−A)/(1+((C/×).sup.^D))) in an in house developed data processing program TOOLSET.
4) ACC test using purified recombinant human ACC2: Human ACC2 inhibition is measured using purified recombinant human ACC2 (hACC2) (J. W. Corbett et al./Bioorg. Med. Chem. Lett. 20 (2010) 2383-2388) using the Transcreener ADP detection FP assay kit (Bellbrook Labs, Madison, Wis.) using the manufacturers' conditions for a 50 microM ATP reaction.
Human ACC2 inhibition is measured using purified recombinant human ACC2 (hACC2). A full length Cytomax clone of hACC2 was purchased from Cambridge Bio-science Limited and was sequenced and subcloned into PCDNA5 FRT TOTOPO (Invitrogen, Carlsbad, Calif.). The hACC2 was expressed in CHO cells by tetracycline induction and harvested in 5 L of DMEM/F12 with glutamine, biotin, hygromycin and blasticidin with 1 microg/mL tetracycline. The conditioned medium containing hACC2 was then applied to a Softlink Soft Release Avidin column (Promega, Madison, Wis.) and eluted with 5 mM biotin. hACC2 (4 mg) was eluted at a concentration of 0.05 mg/mL with an estimated purity of 95%. The purified hACC2 was dialyzed in 50 mM Tris, 200 mM NaCl, 4 mM DTT, 2 mM EDTA, and 5% glycerol. The pooled protein was frozen and stored at _80_C, with no loss of activity upon thawing. For measurement of hACC2 activity and assessment of hACC2 inhibition, test compounds are dissolved in DMSO and added to the hACC2 enzyme as a 5×stock with a final DMSO concentration of 1%. rhACC2 was assayed in a Costar #3767 (Costar, Cambridge, Mass.) 384-well plate using the Transcreener ADP detection FP assay kit (Bellbrook Labs, Madison, Wis.) using the manufacturers' conditions for a 50 microM ATP reaction. The final conditions for the assay are 50 mM HEPES, pH 7.5, 5 mM MgCl2, 5 mM tripotassium citrate, 2 mM DTT, 0.5 mg/mL BSA, 30 microM acetyl-CoA, 50 microM ATP, and 8 mM KHCO3. Typically, a 10 microL reaction is run for 1 h at room temperature and 10 microL of Transcreener stop and detect buffer is added and incubated for an additional 1 h. The data is acquired on an Envision Fluorescence reader (Perkin Elmer) using a 620 excitation Cy5 FP general dual mirror, 620 excitation Cy5 FP filter, 688 emission (S) and a 688 (P) emission filter.
1) Mycobacterium tuberculosis Growth Analysis in Liquid Culture
GFP-expressing Mycobacterium tuberculosis H37Rv (Michelucci, A., et al., Proc Natl Acad Sci USA, 2013. 110(19): p. 7820-5) were generated using the plasmid 32362:pMN437 (Addgene), kindly provided by M. Niederweis (University of Alabama, Birmingham, Ala.) (Song, H., et al., Tuberculosis (Edinb), 2008. 88(6): p. 526-44). 1×10.sup.6 bacteria were cultured in 7H9 medium supplemented with oleic acid-albumin-dextrose-catalase (OADC) (10%), Tween 80 (0.05%), and glycerol (0.2%) in a total volume of 100 μl in a black 96 well plate with clear bottom (Corning Inc, Corning, N.Y.) sealed with an air-permeable membrane (Porvair Sciences, Dunn Labortechnik, Asbach, Germany). Growth was as measured as RLU (relative light units) at 528 nm after excitation at 485 nm in a fluorescence microplate reader (Synergy 2, Biotek, Winooski, Vt.) at indicated time points, see FIG. 1.
2) Analysis of M. tuberculosis Growth in Human Primary Macrophages Mononuclear cells were isolated from peripheral blood (PBMC) of healthy volunteers by density gradient centrifugation. Monocytes were separated (purity consistently >95%) by counterflow elutriation. Human monocyte-derived Macrophages (hMDM) were generated in the presence of 10 ng/ml recombinant human macrophage colony-stimulating factor (M-CSF) from highly purified monocytes as described (Reiling, N., et al., J Immunol, 2001. 167(6): p. 3339-45). M. tuberculosis growth in human macrophages was analyzed as described (Reiling, N., et al., MBio, 2013. 4(4)). In brief 2×10.sup.5 hMDMs were cultured in 500 μl RPMI 1640 with 10% FCS and 4 mM L-glutamine in 48-well flat-bottom microtiter plates (Nunc) at 37° C. in a humidified atmosphere containing 5% CO.sub.2. Macrophages were infected with M. tuberculosis strain H37Rv with a multiplicity of infection (MOI) of 1:1. Four hours post-infection, non-phagocytosed bacteria were removed by washing three times with 0.5 ml Hanks' balanced salt solution (HBSS; Invitrogen) at 37° C. After washing and after 3 days of cultivation, 0.5 ml media containing the indicated inhibitors or antibiotics was added to the macrophage culture. At day 7 supernatants were completely removed and macrophage cultures were lysed by adding Saponin (Sigma, final concentration 0.2%) in HBSS at 37° C. for 15 min. Lysates were serially diluted in sterile water containing 0.05% Tween 80 (Merck, Darmstadt, Germany) and plated twice on 7H10 agar containing 0.5% glycerol (Serva) and 10% heat-inactivated bovine calf serum (BioWest, France). After 3 weeks at 37° C. the colony forming units (CFUs) were counted, see FIGS. 2, 3, and 4.

3) Cell Viability Analysis by Impedance Measurements

[0119] Real-time viability assays (5×10.sup.4 human monocyte derived macrophages/well) were performed with the xCELLigence System (Acea Biosciences Inc.) (Otero-Gonzalez L et al., Environ Sci Technol. 2012; 46(18):10271-8). Impedance measurement was carried out using plates with incorporated sensor array (E-Plate) and the Real-Time Cell Analyzer (RTCA) SP instrument for 120 h. After equilibration of the cells in RPMI 1640 with 10% FCS and 4 mM L-glutamine for 3 h, cells incubated in the absence (medium) or the presence of the ACC2 inhibitor (30, 100 and 300 nM) and staurosporine (1 μg/ml). Data obtained was analyzed using the RTCA Software 2.0 (Acea Biosciences Inc.) see FIGS. 5 and 6.

[0120] In brief, to demonstrate the specificity of the used compounds in this respect, we have first studied a putative direct effect of the inhibitors on the growth of Mtb bacteria. In FIG. 1 it is clearly shown that the ACC2 inhibitor tested does not impair mycobacterial growth, whereas Rifampicin, representing a first-line anti-Tb antibiotic, does. Nevertheless, a clear a dose-dependent inhibition of Mtb replication in human macrophages is observed, using the same ACC2 inhibitor. Thus, it can be concluded that the compounds used do not target the Mtb enzymes, but inhibit the activity of human acetyl-CoA carboxylase.

[0121] As shown in FIG. 7, pharmacological inhibition of macrophage ACC2 restricts intracellular Mycobacterium tuberculosis growth. Human monocyte-derived macrophages were infected with M. tuberculosis H37Rv (multiplicity of infection 1:1, inoculum) for four hours (=Uptake), and subsequently incubated for seven days (d7) in the absence (solvent, (Dimethylsulfoxid)) or presence of different ACC2 inhibitors ((A) MK-40474, (B) ND-646, (C) Diclofop, (D) Haloxyfop) with the concentrations indicated. Intracellular bacteria were enumerated by determining Colony forming units after lysis of cells.

[0122] As shown in FIG. 8, MK-4074 and ND-646 do not affect growth of Mycobacterium tuberculosis (Mtb) in liquid culture. Green fluorescent protein (GFP)-expressing Mtb bacteria were cultured in liquid culture in the absence (ctrl) or presence of the antibiotic rifampicin or various ACC2 inhibitors at the indicated concentrations. Growth was determined by measuring fluorescence signal intensity at 528 nm after excitation at 485 nm in a microplate reader on day 7 post infection.

[0123] As shown in FIG. 9, CP640186 does not affect growth of Mycobacterium tuberculosis (Mtb) in liquid culture. Green fluorescent protein (GFP)-expressing Mtb bacteria were cultured in liquid culture in the absence (ctrl) or presence of the antibiotic rifampicin or the ACC2 inhibitor CP640186 at the indicated concentrations. Growth was determined by measuring fluorescence signal intensity at 528 nm after excitation at 485 nm in a microplate reader on day 7 post infection.