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Oxadiazoles Have Butyrate-Specific Conditional Activity against Mycobacterium tuberculosis Julie V. Early,a Allen Casey,a Maria Angeles Martinez-Grau,b Isabel C. Gonzalez Valcarcel,c Michal Vieth,c Juliane Ollinger,a Mai Ann Bailey,a Torey Alling,a Megan Files,a Yulia Ovechkina,a Tanya Parisha TB Discovery Research, Infectious Disease Research Institute, Seattle, Washington, USAa; Eli Lilly and Company, Alcobendas, Madrid, Spainb; Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, USAc

Mycobacterium tuberculosis is a global pathogen of huge importance which can adapt to several host niche environments in which carbon source availability is likely to vary. We developed and ran a phenotypic screen using butyrate as the sole carbon source to be more reflective of the host lung environment. We screened a library of ⬃87,000 small compounds and identified compounds which demonstrated good antitubercular activity against M. tuberculosis grown with butyrate but not with glucose as the carbon source. Among the hits, we identified an oxadiazole series (six compounds) which had specific activity against M. tuberculosis but which lacked cytotoxicity against mammalian cells.

A

n estimated one-third of the world’s population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (1). More than 1 million people die of tuberculosis each year, despite significant international efforts to diagnose and treat the disease (2). Current regimens against drug-sensitive strains require 6 months of treatment with multiple antibiotics, while treatment of infections caused by drug-resistant strains requires 12 to 24 months. There is therefore a critical need to develop new therapeutics for tuberculosis. Carbon metabolism in M. tuberculosis has been widely studied, but several aspects remain unclear (reviewed in reference 3). M. tuberculosis can utilize a wide variety of carbon sources and is able to metabolize more than one carbon source simultaneously (4). During infection, M. tuberculosis has access to multiple carbon sources, including sugars, lipids, and fatty acids. While it is unclear if cholesterol and glucose are metabolized by M. tuberculosis in the host, it seems likely that M. tuberculosis uses fatty acids in the host (5–12). M. tuberculosis replicates both extracellularly and intracellularly inside macrophages, and the protein expression profiles of M. tuberculosis under these different conditions are distinct (13, 14). Several genes with differential expression in macrophages have roles in the metabolism of fatty acids, are involved with restructuring of the cell wall, or respond to a variety of stresses (15–21). Isocitrate lyase (ICL), a key enzyme of the glyoxylate shunt which catalyzes the conversion of isocitrate to succinate and glyoxylate, is upregulated in macrophages (13, 14, 22, 23). Disruption of icl leads to attenuation in the mouse model of tuberculosis, but only during chronic infection (23). However, it is not clear if attenuation is due to the accumulation of toxic metabolites or loss of the ability to metabolize fatty acids (24). Several other genes involved in central carbon metabolism—such as the gene for FadA5, which converts two acetyl coenzyme A (CoA) intermediates to CoA and acetoacetyl-CoA; the gene for PckA, which uses GTP to convert oxaloacetate to phosphoenolpyruvate and CO2; and the gene for DlaT, which converts pyruvate to acetyl-CoA and CO2—are also important for virulence (25–27). Fatty acids are metabolized by enzymes in the ␤-oxidation pathway and the glyoxylate shunt to generate energy, but shortchain fatty acids can also be used as building blocks to synthesize

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long-chain fatty acids and lipids. The targeting of enzymes involved in fatty acid anabolism can be a powerful treatment strategy, as demonstrated by the effectiveness of isoniazid and its effect on mycolic acids (28–30). Current antitubercular drugs have a narrow range of targets, and many new potential drugs are being progressed on the basis of their inhibition of growth in standard laboratory medium containing glucose as the carbon source. Some efforts to identify inhibitors effective under other conditions have been made. For example, the long-chain fatty acid palmitate (hexadecanoic acid) has been used as a carbon source (31, 32). Modification of standard medium has been used; for example, Se-methyl selenocysteine was identified to be an effective inhibitor under conditions of carbon and nitrogen limitation (33). Screening of compounds under complex conditions more relevant to infection (low pH and low oxygen, butyrate, and nitrate concentrations) has been done with sets of small compounds (34), and the screening of compounds for their activities against intracellular bacteria has been performed using high content analysis (35). These types of screens under disease-relevant conditions may be important for identifying inhibitors that are active in vivo (36, 37). We developed a phenotypic screen using a short-chain fatty acid as the primary carbon source based on the observation that carbon source modification could be used to generate gene expression profiles similar to those in the host environment (38). Our hypothesis was that, by forcing M. tuberculosis to metabolize fatty acids, not only will genes involved in ␤-oxidation, the glyoxy-

Received 2 December 2015 Returned for modification 30 December 2015 Accepted 24 March 2016 Accepted manuscript posted online 4 April 2016 Citation Early JV, Casey A, Martinez-Grau MA, Gonzalez Valcarcel IC, Vieth M, Ollinger J, Bailey MA, Alling T, Files M, Ovechkina Y, Parish T. 2016. Oxadiazoles have butyrate-specific conditional activity against Mycobacterium tuberculosis. Antimicrob Agents Chemother 60:3608 –3616. doi:10.1128/AAC.02896-15. Address correspondence to Tanya Parish, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.02896-15. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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late shunt, and anabolism be expressed but so will other genes important for survival in the human host. We focused on identifying compounds with differential activity, i.e., compounds that were active in medium with fatty acids but inactive in medium with glucose. This would identify inhibitors active against novel targets that are not expressed when glucose is the primary carbon source. MATERIALS AND METHODS Large-scale culture of M. tuberculosis. M. tuberculosis was grown in several different media. (i) 7H9-Tw-OADC medium consisted of Middlebrook 7H9 medium supplemented with 0.05% (wt/vol) Tween 80 (Tw) and 10% (vol/vol) oleic acid-albumin-dextrose-catalase (OADC) supplement (Becton Dickinson). (ii) 7H9-Ty-10BT medium consisted of 7H9 medium supplemented with 5 g/liter bovine serum albumin (BSA) fraction V, 0.8 g/liter NaCl, 0.05% (vol/vol) tyloxapol (Ty), and 10 mM sodium butyrate. (iii) 7H9-Ty medium consisted of 7H9 medium supplemented with 5 g/liter BSA fraction V, 0.8 g/liter NaCl, and 0.05% (vol/vol) tyloxapol plus a carbon source: 22 mM (0.4%, wt/vol) glucose, 5 mM (0.0288%, vol/vol) acetic acid, 1.25 mM (0.0136%, vol/vol) isovaleric acid, 0.25 mM (0.0064%, wt/vol) palmitic acid, 2.5 mM (0.0186%, vol/ vol) butyric acid, or 2.5 to 10 mM (0.0275 to 0.11%, wt/vol) sodium butyrate. Hygromycin was added to 50 ␮g/ml where appropriate. Standing cultures were grown in 10 ml medium in 50-ml conical tubes; rolling cultures were grown in 100 ml medium in 450-cm2 roller bottles. M. tuberculosis H37Rv expressing a red fluorescent protein (RFP; mCherry or DsRed) under the control of a constitutively active promoter was used (39, 40). For growth curves, a rolling culture was grown to late log phase (optical density at 590 nm [OD590] ⫽ 0.6 to 0.9) and used to inoculate 5 ml of medium in glass tubes 16 mm in diameter by 125 mm in length with stir bars to a theoretical OD590 of 0.04. The tubes were incubated on a stirring apparatus at 37°C. For culture in plates, M. tuberculosis was cultured in 30 ␮l of medium in 384-well plates or 100 ␮l of medium in 96-well plates. Preparation of assay plates. Compounds were screened at a single concentration (20 ␮M). Compounds were diluted to 1 mM in 100% dimethyl sulfoxide (DMSO) and stored at room temperature until use for up to 6 months. Rifampin was diluted in 100% DMSO and stored in aliquots at ⫺20°C. 7H9-Ty-10BT medium and compounds were dispensed into sterile 384-well black plates with clear bottoms (Greiner) using sterile tips and a MiniTrak system contained in a laminar HEPA enclosure. Assay plates were prepared by dispensing 19.4 ␮l 7H9-Ty-10BT medium into all wells, dispensing 0.6 ␮l of the maximum inhibition control into column 1, 0.6 ␮l of the minimum inhibition control into column 2, 0.6 ␮l of the midpoint inhibition control into column 23, 0.6 ␮l of test compound into each of columns 3 to 22, and 10 ␮l of 7H9-Ty-10BT medium into column 24. The tips were washed 4 times with water and once with DMSO after their use to prepare each plate; the tips were changed after they had been used to prepare 6 plates. The final plate layout was as follows: column 1, maximum inhibition control (2 ␮M rifampin); column 2, minimum inhibition control (2% DMSO); columns 3 to 22, test compounds at 20 ␮M each; column 23, midpoint inhibition control (0.04 ␮M rifampin); column 24, contamination control (medium only). High-throughput screen (butyrate). A roller culture of M. tuberculosis in 7H9-Ty medium plus 5 mM sodium butyrate and hygromycin was inoculated to a theoretical OD590 of 0.03 and incubated for 7 days at 37°C with rolling at 100 rpm. The final OD590 was 0.6 to 0.8. Forty milliliters of M. tuberculosis culture was filtered through a 5-␮m-pore-size polyethersulfone filter and adjusted to an OD590 of 0.09 with 7H9-Ty-10BT medium. Columns 1 to 23 of the assay plates were inoculated with 10 ␮l culture using a Multidrop Combi dispenser (Thermo Scientific) fitted with a standard 8-channel cassette. The plates were put into individually sealed bags and incubated in a humidified water-jacketed incubator at 37°C for 6 days. The plates were sealed with silicon adhesive film, and the

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fluorescence was measured using a Synergy 4 microplate reader (BioTek) with excitation at 586 nm and emission at 614 nm or a Synergy H4 microplate reader with excitation at 560 nm and emission at 590 nm. The background signal (the average for the wells in column 24) was subtracted from the reading from each well. Growth inhibition for test wells was calculated as the percent growth compared to that in the control wells. Plates were subjected to quality control using the following criteria: the coefficient of variation (CV) of each control column was ⬍20%, and Z= for the controls was ⬎0.5 (41). Screen validation. Two plates containing the minimum signal (the maximum inhibition obtained with 100 ␮M rifampin), two plates containing the maximum signal (the minimum inhibition obtained with DMSO), and two plates containing the midpoint inhibition control (obtained with 2 ␮M rifampin) were inoculated, and growth was measured after 6 days. Three independent experiments were run on three different days. Separately, three randomly selected plates from the initial compound set were screened as described above, except that the test compounds were used at final concentrations of 5 ␮M, 10 ␮M, and 20 ␮M. High-throughput screen (glucose). Compounds were screened in glucose medium with the following modifications: the medium used at all stages was 7H9-Tw-OADC medium, the culture was filtered using cellulose acetate filters, the plates were inoculated to a theoretical starting OD590 of 0.02, the midpoint inhibition control was 2.5 nM rifampin, and the assay duration was 5 days. MIC. MICs were determined as described previously (42). Briefly, compounds were tested for activity against M. tuberculosis in 2-fold serial dilutions in 96-well plates, starting at 20 ␮M. For glucose medium, the plates were incubated for 5 days. For butyrate medium, the plates were incubated for 6 days. Growth was measured using the optical density and the fluorescence. Two-fold serial dilutions of rifampin were included on each plate as a control. The MIC was determined from the resulting Gompertz curves, or the concentration required to inhibit growth by 90% (IC90) was determined from the Levenberg-Marquardt least-squares plot (43). For the reference compounds and MICs comparing the effects of detergents, assay plates were prepared using a multichannel pipette, but the assays were otherwise run as described above. Cytotoxicity. The Vero cell line CCL81 was propagated in high-glucose Dulbecco’s modified Eagle medium with GlutaMax (Invitrogen), 10% fetal bovine serum, 50 units/ml penicillin, and 50 ␮g/ml streptomycin. Cells were seeded into 384-well plates at 1,200 cells per well. Compounds were solubilized in DMSO, and a 10-point 3-fold serial dilution series was prepared starting at 100 ␮M. Compounds were added to the cells at 24 h postseeding (final DMSO concentration, 1%). Cells were incubated for 2 days at 37°C in 5% CO2, CellTiter-Glo reagent (Promega) was added, and the number of relative luminescence units was measured. Inhibition curves were fitted using the Levenberg-Marquardt algorithm, and the compound concentration that produced 50% inhibition (IC50) was calculated (44, 45). Oxadiazoles. Compounds 1, 2, and 4 to 12 were acquired from third parties as part of standard screening cassettes. Compound 3 was prepared by using the synthetic route presented in Fig. S1 in the supplemental material. Reaction of furan-2-carbohydrazide (compound 13) with chloroacetyl chloride in the presence of N-methylmorpholine afforded the intermediate acylsemicarbazide (compound 14), which was cyclized by heating with phosphoryl chloride. Replacement of chloride (compound 15) by 2-chloro-6-fluoro-N-methyl-benzylamine under microwave irradiation provided compound 3 (see Fig. S1 in the supplemental material). All final compounds were characterized by 1H nuclear magnetic resonance and liquid chromatography-mass spectrometry and had purities of greater than 95%. Details regarding purity and synthesis are provided in the methods in the supplemental material.

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FIG 2 Growth of M. tuberculosis in sodium butyrate. M. tuberculosis was grown in 100 ml 7H9-Ty-BT medium in roller bottles with various concentrations of sodium butyrate.

FIG 1 Growth of M. tuberculosis with different carbon sources. M. tuberculosis was inoculated to a theoretical OD590 of 0.03 and growth was monitored. The medium used was 7H9 plus 5 g/liter BSA fraction V, 0.8 g/liter NaCl, 0.05% (vol/vol) tyloxapol, and the indicated carbon source: 4 g/liter glucose, 5 mM acetic acid, 2.5 mM butyric acid, 1.25 mM isovaleric acid, 0.25 mM palmitic acid, 2.5 mM sodium butyrate, or no carbon source.

RESULTS

Assay development. We were interested in developing and running a screen to identify compounds active under host-relevant conditions. We focused on developing a screen in which fatty acids support the growth of M. tuberculosis, since these are most likely a source of carbon during infection. We used M. tuberculosis expressing a red fluorescent protein (mCherry or DsRed) to enable the detection of growth by the detection of fluorescence as well as by determination of the OD590 (39, 40, 42). We needed a medium that supported the robust and reproducible growth of M. tuberculosis and that was amenable to robotic manipulation and dispensing. We tested the growth of M. tuberculosis H37Rv in medium with a variety of short-chain fatty acids (acetic acid, butyric acid, isovaleric acid, and palmitic acid) and compared the growth with that obtained in medium with glucose. All carbon sources supported the growth of M. tuberculosis, with the cultures reaching an OD590 of 1.3 in glucose after 7 days, OD590s of 0.95 and 0.94 in acetic acid and butyric acid, respectively, and OD590s of 0.92 and 0.90 in isovaleric acid and palmitic acid, respectively (Fig. 1). The growth rate was the lowest in the presence of isovaleric acid, in which growth had not reached stationary phase by day 7. We saw limited growth in the absence of a carbon source, presumably due to carryover of glucose from the inoculum. However, growth with the carbon sources was higher than that in the absence of a carbon source in all cases. We selected butyrate for use in the screen because it provided robust growth, it was easy and reliable to work with, and would rely on ␤-oxidation for metabolism. We tested both butyric acid and sodium butyrate as carbon sources, since preparation of the culture medium with the latter would be easier; sodium butyrate and butyric acid similarly supported growth (Fig. 1). We determined the optimum concentration of sodium butyrate needed to support growth. A range of concentrations was

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able to support growth, with the highest concentration (10 mM) resulting in the highest final density of the culture (Fig. 2). However, the higher concentration of butyrate resulted in a lower initial growth rate, and the cultures took longer to reach maximal growth and stationary phase (10 days rather than 7 days). We selected 5 mM sodium butyrate, which gave the best combination of growth rate and OD590 after 7 days of growth. We next determined growth kinetics in 96-well plates using butyrate as the carbon source (Fig. 3A to C). In 96-well plates, 2.5 mM sodium butyrate resulted in limited growth, presumably due to exhaustion of the carbon source (Fig. 3A); 5 mM sodium butyrate was also growth limiting over 7 days, with cultures reaching stationary phase during this time (Fig. 3B). In contrast, 10 mM sodium butyrate supported good growth, with higher numbers of relative fluorescent units (RFU) being reached and without the cultures reaching stationary phase within 7 days (Fig. 3C). We also varied the starting inoculum to find the optimum number of bacteria to seed in the assay plates. Inoculation at an OD590 of 0.03 resulted in a strong signal with minimal variability after 6 days of growth and was selected for use in the screen (Fig. 3C). We also determined if hygromycin was required to maintain plasmid and RFP expression. We compared the number of RFU in the presence and absence of hygromycin and found that the values were the same, confirming that the plasmid was stable in the absence of selection over the time of the assay (Fig. 3A to C). As noted before (42), 2% DMSO had a minimal effect on growth, although the growth was slightly diminished (Fig. 3A to C). We used the same parameters in 384-well plates and confirmed that incubation for 6 days and use of a starting inoculum with an OD590 of 0.03 was suitable for the screen: the signal-to-background ratio was ⬃165, the signal-to-noise ratio was ⬃215, and the bacteria had not entered stationary phase at day 6 (Fig. 3D). Screen validation. We conducted a plate uniformity and reproducibility test according to National Center for Advancing Translational Sciences and Eli Lilly guidelines (46). Three independent runs were conducted using duplicate control plates with maximum, minimum, and midpoint signals. The interday variation was negligible, with all runs reporting similar RFU values, and the percent CVs for the controls were well within the guidelines: the minimum inhibition range was 93 to 160 RFU, the midpoint inhibition range was 32 to 69 RFU, and the maximum inhibition range was 12 to 19 RFU (Fig. 4A). This confirmed that the assay was reproducible with acceptable intra- and interexperiment variation. In order to select the appropria concentration of compound

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Butyrate-Specific Oxadiazoles against M. tuberculosis

FIG 3 Growth of M. tuberculosis in 96-well and 384-well plates. M. tuberculosis was grown in 7H9-Ty-BT medium in a 96-well (A to C) or 348-well (D) plate. Inocula with a range of OD590 values (indicated next to the symbols) were tested with 2.5 mM sodium butyrate (A), 5 mM sodium butyrate (B), or 10 mM sodium butyrate (C) in 96-well plates or 10 mM sodium butyrate in 384-well plates (D). Data are the averages for all wells in a single plate. Hyg, hygromycin.

for use in the single-point screen, we selected three library plates (960 compounds) at random and screened the compounds at concentrations of 5 ␮M, 10 ␮M, and 20 ␮M. Using an arbitrary cutoff of 95% inhibition, 1 compound was active at 5 ␮M or 10 ␮M, while 12 compounds were active at 20 ␮M. We therefore tested the compounds at 20 ␮M to provide the most sensitive detection of active compounds (Fig. 4B). Once we had selected the key parameters for the high-throughput assay, we determined the MICs of several reference compounds in 7H9-Ty-10BT medium and 7H9-Tw-OADC medium. With the exception of rifampin and 3-nitropropionic acid, all other compounds (isoniazid, kanamycin, levofloxacin, D-cycloserine, linezolid) had similar activities in both media (Table 1). The MIC for 3-nitropropionic acid in 7H9-Ty-10BT medium was 9.9 ␮M, whereas it was 74 ␮M in 7H9-Tw-OADC medium. The increased activity obtained using butyrate as a carbon source was expected, since 3-nitropropionic acid inhibits isocitrate lyase, an enzyme involved in the glyoxylate shunt, which should be utilized under these conditions (47). Interestingly, the MIC of rifampin was also changed, with the rifampin MIC showing that it had more activity in 7H9-Tw-OADC medium (0.003 ␮M) than in 7H9-Ty10BT medium (0.09 ␮M). We confirmed that the difference in MICs for rifampin was partially due to a change in the surfactant in the medium, since the MIC of rifampin in medium with butyrate and Tween 80 was 9-fold lower than that in 7H9-Ty-10BT medium (0.01 ␮M), consistent with what has previously been reported (48). In contrast, detergent did not affect the MIC of 3-nitropropionic acid, where the MIC in 7H9-OADC medium with tyloxapol was 36 ␮M, i.e., within ⬃2-fold of the MIC in the presence of Tween 80.

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High-throughput screen. We screened ⬃87,000 compounds from a diversity set of compounds from the Eli Lilly small-molecule library for activity against M. tuberculosis in both 7H9-Ty10BT medium and 7H9-Tw-OADC medium. The selected diversity set is a general screening cassette of compounds representing a diverse number of chemotypes. The Z= values for the controls ranged from 0.62 to 0.91, with the average Z= value being 0.76 throughout the screen. Using ⬎80% inhibition in 7H9-Ty-10BT medium as our cutoff, we identified 826 active compounds with a mean calculated logarithm of the partition coefficient between n-octanol and water (clogP), i.e., lipophilicity, of 4.3 (clogP was predicted by the use of BioByte software) and a mean polar surface area (PSA) of 53. Application of the same criteria to 7H9-TwOADC medium resulted in 1,266 active compounds with a mean clogP value of 4.1 and a mean PSA of 56. The very similar clogP values of the active set for both media are consistent with little or no solubilizing effect in the butyrate medium. Compounds that simultaneously inhibited M. tuberculosis in 7H9-Ty-10BT medium and 7H9-Tw-OADC medium were discarded from this initial set. For the purpose of identifying butyrate-specific molecules, we then selected 369 compounds with ⬎80% inhibition in 7H9Ty-10BT medium and ⬍95% inhibition in 7H9-Tw-OADC medium (Fig. 5A), making a hit rate of 0.2%. The compounds were resupplied, and we determined the MICs in both 7H9-Ty-10BT medium and 7H9-Tw-OAD medium; 166 compounds had confirmed activity (MICs ⱕ 20 ␮M) in butyrate medium, but only 36 were active in glucose medium (Fig. 5B). Compounds were tested for cytotoxicity against mammalian cells to exclude nonspecific effects; 48 compounds that had good selectivity, defined as an MIC of ⱖ20 ␮M in 7H9-Tw-OADC me-

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FIG 4 High-throughput assay validation. (A) A plate uniformity study was performed. Three independent cultures were used to prepare duplicate assay plates for the maximum signal (obtained with 2% DMSO), the minimum signal (obtained with 2 ␮M rifampin), and the midpoint signal (obtained with 0.04 ␮M rifampin). The plates were incubated for 6 days at 37°C, and the numbers of RFU were read. Black diamonds, day 1; light gray circles, day 2; dark gray squares, day 3. (B) Three compound plates were selected at random. The compounds were screened at three concentrations in 384-well plates. The assay mixture was incubated for 6 days at 37°C, and the numbers of RFU were read.

dium, an MIC of ⱕ10 ␮M in 7H9-Ty-10BT medium, and an IC50 of ⬎35 ␮M for Vero cells, were considered for further evaluation. From this set, several compounds were identified to be members of the oxadiazole series (Table 2). Other compounds with the desired in vitro profile showed quite diverse structural features

TABLE 1 MICs of standard antibioticsa MIC (␮M) Antibiotic

7H9-Tw-OADC

7H9-Ty-10BT

Isoniazid Kanamycin Levofloxacin D-Cycloserine Linezolid Rifampin 3-Nitropropionic acid

0.4 ⫾ 0.2 1.7 ⫾ 0.5 0.7 ⫾ 0.4 ⬎20 3.4 ⫾ 2.2 0.003 ⫾ 0.005 74 ⫾ 6.8

0.2 ⫾ 0.1 2.0 ⫾ 0.7 1.1 ⫾ 0.2 ⬎20 2.8 ⫾ 0.1 0.09 ⫾ 0.008 9.9 ⫾ 2.4

a The MICs of several reference compounds were determined in 7H9-Tw-OADC medium and 7H9-Ty-10BT medium. The results are the average ⫾ standard deviation from a minimum of 3 experiments.

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FIG 5 High-throughput screen to select active compounds. (A) Compounds were screened in 7H9-Ty-10BT medium and 7H9-Tw-OADC medium for their activity against M. tuberculosis. Growth inhibition (in percent) was determined for each compound. (B) Compounds that were active in 7H9-Ty10BT medium but not active in 7H9-Tw-OADC medium were resupplied, and MICs were determined in both media.

significantly different from those of the compounds in the oxadiazole series and are currently under evaluation. The active oxadiazoles (compounds 1 to 6) had the following property attributes: low molecular weights (284 to 322), clogP values of 0.3 to 4.4, and PSAs of 39 to 120. Five compounds (compounds 7 to 11) containing an oxadiazole ring in the initial screening set were inactive (⬍30% inhibition at 20 ␮M) (Table 3). One compound (compound 12) did not show selectivity and had low activity (MIC ⫽ 20 ␮M). The oxadiazole set evaluated in this screen mostly included 1,3,4-oxadiazoles; the exceptions were compounds 6 and 9, which contained the 1,2,4-oxadiazole ring. An encouraging finding was no direct relationship between lipophilicity (clogP) and biological activity (Table 2). The functionalization of oxadiazole compounds 1 to 12 influenced the antimicrobial activity. Active compounds with electron donor aromatic rings, such as the furan in compounds 3 and 5 or the thiophene in compounds 2, 4, and 6, were potent and demonstrated some diversity in position 5. A linker of one to three atoms in length benefited activity (compounds 1 to 6), while shorter or longer linkers containing amide groups were inactive (compounds 7 to 9 and 11). The nature of the linker between the ox-

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TABLE 2 Activities of oxadiazole compoundsa MIC (␮M) 7H9-Ty-10BT

7H9-Tw-OADC

IC50 (␮M) for Vero cells

clogP

Selectivity index

1

6.4 ⫾ 1.9

⬎20

⬎100

0.3

⬎15

2

1.5 ⫾ 0.3

⬎20

⬎100

2.3

⬎66

3

0.8 ⫾ 0.3

⬎20

⬎100

2.5

⬎125

4

0.4 ⫾ 0.1

⬎20

⬎100

3.9

⬎250

5

3.3 ⫾ 0.3

⬎20

⬎100

3.6

⬎30

6

2.2 ⫾ 0.4

⬎20

89.3

4.4

40.6

Compound

Structure

a MICs were determined in 7H9-Tw-OADC medium and 7H9-Ty-10BT medium. The results are the average ⫾ standard deviation from at least 2 experiments. Cytotoxicity against Vero cells was measured and is given as the concentration required to inhibit growth by 50% (IC50). Predicted clogP values were calculated with BioByte software. The selectivity index is calculated as IC50/MIC in 7H9-Ty-10BT medium.

adiazole and the third aromatic ring showed flexibility, with an ether, tertiary amine, alkyl, and thioether being the preferred substituents (compounds 2 to 5, respectively). On the basis of the findings obtained with this small set of compounds, linkers of one or three atoms in length look to be optimal for activity, since compounds 3 and 4 were the most potent. At the same time, substituents in position 5 with increased steric hindrance, as in compounds 10 and 12, lowered the activity. DISCUSSION

We observed the robust growth of M. tuberculosis with a variety of carbon sources: glucose, acetic acid, butyric acid, isovaleric acid, and palmitic acid. We selected butyric acid because it would force metabolism via hydrolysis of the beta carbon (unlike acetic acid), provided reproducible and robust growth (unlike isovaleric acid), and was water soluble (unlike palmitic acid). We noted that higher concentrations of butyrate ultimately resulted in a higher final cell density, suggesting that the carbon source may be limiting at lower concentrations. However, higher concentrations prolonged the lag phase and lowered the growth rate. The lag phase is influenced by the inoculum size, the degree of heterogeneity of the culture, the physiological status of the cells, and the physiochemical properties of the medium (49). The increased lag phase was not due to adaptation to the carbon source, as this was also seen when bacteria were subcultured from butyrate medium. Although fatty acids can be used as sole carbon sources by mycobacteria, they also inhibit growth (50, 51). We hypothesize that the increased lag phase in cultures exposed to higher concentrations of butyrate results from growth inhibition by butyrate and that this growth inhibition is removed as the butyrate concentration in the medium decreases during

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its utilization. Interestingly, a delay in exponential phase was also seen with an accD6 deletion strain, and AccD6 could be important for growth in medium where butyrate is the primary carbon source because it is involved with converting short-chain fatty acids to mycolic acids (52). We compared the activities of the entire library of compounds in two media (7H9-Ty-10BT medium and 7H9-Tw-OADC medium). As expected, the majority of the compounds were not active in either medium. However, we did note a general shift toward greater inhibition with active compounds in butyrate medium. We hypothesize this is due to the slower growth of M. tuberculosis in butyrate medium, but it could also reflect changes in cell wall structure and/or permeability or a more susceptible physiological state. For example, previous screening campaigns identified glycerol-dependent compounds whose activity was potentiated by toxic metabolites produced during glycerol metabolism (53). We identified from this screen a promising series of oxadiazoles for further evaluation. A number of other molecules with good properties were also identified; however, these were given a lower priority for investigation, due to either their lower potency or their structural features. We are currently evaluating other series. Oxadiazole derivatives, especially the 1,3,4-isomer, are privileged structures with enormous potential in medicinal chemistry. They have been evaluated according to a broad spectrum of pharmacological activities, with special attention being given to their properties as antimicrobial agents (54). We continue to explore the compounds in this series to characterize their potential and are also identifying more compounds that are active in the presence of fatty acids, as shown here, as they have potential to lead to new drugs effective against M. tuberculosis.

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TABLE 3 Activities of oxadiazole compounds in primary screena % inhibition Compound

7H9-Ty-10BT

7H9-Tw-OADC

clogP

7

Structure

5

13

3.1

8

15

45

2.7

9

10

24

3.6

10

24

22

3.8

11

4

10

2.9

12

82

78

1.2

a Inhibition of M. tuberculosis growth was determined by use of each compound at a fixed concentration of 20 ␮M in 7H9-Tw-OADC medium and 7H9-Ty-10BT medium. The results are expressed as the percent inhibition. For a percent inhibition value of ⬍30, compounds are normally considered inactive. The predicted clogP values, calculated using BioByte software, are given.

Conclusion. This work describes the rationale for a phenotypic screening that uses butyrate as a carbon source and the optimized screening conditions that allowed us to identify a set of 48 noncytotoxic compounds with condition-dependent activity. A set of six compounds containing an oxadiazole ring showed significant carbon source-dependent activity against M. tuberculosis when the bacteria were metabolizing butyrate but not glucose. ACKNOWLEDGMENTS We thank Aaron Korkegian, Alfredo Blakely Ruiz, Bjorn Sunde, Lindsay Flint, and Lena Anoshchenko for technical assistance. We thank Helena Boshoff for helpful discussions on suitable growth media and Joshua Odingo, Thierry Masquelin, and Philip Hipskind for insightful chemistry discussions. The work at the Infectious Disease Research Institute was funded in part by Eli Lilly and Company in support of the mission of the Lilly TB Drug Discovery Initiative and with funding from the Bill and Melinda Gates Foundation under grant OPP1024038.

FUNDING INFORMATION This work, including the efforts of Julie V. Early, Allen Casey, Juliane Ollinger, Mai Ann Bailey, Torey Alling, Megan Files, Yulia Ovechkina, and Tanya Parish, was funded by Lilly TB Drug Discovery Initiative. This work, including the efforts of Julie V. Early, Allen Casey, Juliane Ollinger, Mai Ann Bailey, Torey Alling, Megan Files, Yulia Ovechkina, and Tanya Parish, was funded by Bill and Melinda Gates Foundation (OPP1024038). The work at IDRI was funded in part by Eli Lilly and Company in support of the mission of the Lilly TB Drug Discovery Initiative and

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with funding from the Bill and Melinda Gates Foundation under grant no. OPP1024038.

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