Nitazoxanide Analogs Require Nitroreduction for Antimicrobial Activity

Article pubs.acs.org/jmc

Nitazoxanide Analogs Require Nitroreduction for Antimicrobial Activity in Mycobacterium smegmatis Maria V. Buchieri,*,† Mena Cimino,† Sonia Rebollo-Ramirez,‡ Claire Beauvineau,§ Alessandro Cascioferro,∥ Sandrine Favre-Rochex,† Olivier Helynck,⊥ Delphine Naud-Martin,§ Gerald Larrouy-Maumus,‡ Hélène Munier-Lehmann,*,⊥ and Brigitte Gicquel† †

Unité de Génétique Mycobactérienne, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France MRC Centre for Molecular Bacteriology & Infection, Imperial College London, London SW7 2AZ, United Kingdom § PSL Research University,CNRS, INSERM, Chemical Library, Institut Curie UMR9187/U1196, UMR3666/U1143, 91405 Orsay Cedex, France ∥ Unité de Pathogénomique Mycobactérienne Intégrée, Institut Pasteur, 75724 Paris Cedex 15, France ⊥ Unité de Chimie et Biocatalyse, Département de Biologie Structurale et Chimie, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France ‡

S Supporting Information *

ABSTRACT: In this study, we aimed to decipher the natural resistance mechanisms of mycobacteria against novel compounds isolated by whole-cell-based high-throughput screening (HTS). We identified active compounds using Mycobacterium aurum. Further analyses were performed to determine the resistance mechanism of M. smegmatis against one hit, 3bromo-N-(5-nitrothiazol-2-yl)-4-propoxybenzamide (3), which turned out to be an analog of the drug nitazoxanide (1). We found that the repression of the gene nf nB coding for the nitroreductase NfnB was responsible for the natural resistance of M. smegmatis against 3. The overexpression of nf nB resulted in sensitivity of M. smegmatis to 3. This compound must be metabolized into hydroxylamine intermediate for exhibiting antibacterial activity. Thus, we describe, for the first time, the activity of a mycobacterial nitroreductase against 1 analogs, highlighting the differences in the metabolism of nitro compounds among mycobacterial species and emphasizing the potential of nitro drugs as antibacterials in various bacterial species.

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INTRODUCTION Among diseases caused by mycobacterial species, tuberculosis is one of the major causes of death due to infectious diseases worldwide, with 1.4 million deaths and 10.4 million new cases reported each year. It is estimated that one-third of the world’s population is infected with Mycobacterium tuberculosis (latent tuberculosis), with a 10% lifetime probability of developing the disease. The probability of developing disease is higher in immunocompromised individuals.1 This is also true for infections produced by nontuberculous mycobacterial species (NTM).2 Furthermore, specific host conditions such as cytokine pathway defects,3−5 systemic illness,6,7 and the genetic disease cystic fibrosis8,9 enhance susceptibility to NTM infection. NTMs are associated with various clinical manifestations,10 with M. avium complex (MAC) the most common etiological agent of NTM lung infections.10 Because of their high resistance to antibiotics, including standard treatment for drug susceptible tuberculosis, NTM infections are difficult to treat.10−13 Treatment for NTM infections consists of multidrug therapy. For example, treatment with a combination of © 2017 American Chemical Society

azithromycin, ethambutol, and rifampin for 3 months is recommended for MAC lung disease.10 The difficulties associated with NTM treatment coupled with the emergence of multidrug-resistant (MDR) and extremely drug-resistant (XDR) tuberculosis have made the discovery of new antimycobacterial drugs and the improvement of known drugs a major priority for research groups worldwide. Recently, several drug candidates have been identified and several are currently in human clinical trials, among them pretomanid (previously known as PA-824), a nitroimidazole in late stage trials.14,15 Pretomanid is a prodrug, which is activated by the deazaflavin-dependent nitroreductase (Ddn), giving rise to the metabolites responsible for its killing activity in M. tuberculosis.16 Prodrugs have the advantage that they can be devoid of undesirable drug properties.17,18 For example, prodrugs are only active in bacteria after transformation and are not toxic to the host cells because of their inert nature until activation. This Received: May 17, 2017 Published: August 28, 2017 7425

DOI: 10.1021/acs.jmedchem.7b00726 J. Med. Chem. 2017, 60, 7425−7433

Journal of Medicinal Chemistry

Article

characteristic has been exploited in cancer chemotherapy using nitro prodrugs, which are activated by bacterial nitroreductases conjugated with cell-specific antibodies to direct them to the tumor.19,20 Nitro groups can follow a partial or total enzymatic reduction pathway yielding highly toxic or inactive products or both, depending on the nitroreductase and the structure of the compound.21 Hydroxylamino products are highly toxic; they can interact with DNA and other biomolecules, causing cytotoxic and mutagenic effects.22 In contrast, amino products are associated with drug inactivation.23 It has been proposed that Giardia lamblia nitroreductase GLNRI carries out the activation of the antibacterial and parasitic drugs nitazoxanide (1, Alinia Romark Laboratories) and metronidazole (MTZ); conversely, nitroreductase GLNRII inactivates MTZ.24 Nitroreductases are widespread throughout nature,25 and thus they are involved in the mechanisms of action and resistance to nitro drugs in various organisms. Altogether, these properties make nitroreductase/nitrodrug combination an attractive research subject for drug development laboratories. In the present study, we screened chemical compounds for antibacterial activity against a series of mycobacterial species and investigated the natural resistance of some. We used high throughput screening (HTS), physiological, and genetic approaches to analyze the resistance mechanism of M. smegmatis against one hit, which turned out to be an analog of 1. We describe, for the first time, the activity of a mycobacterial nitroreductase against analogs of 1. We also identified the intermediate responsible for its toxicity in M. smegmatis using LC−MS-based approaches. This study highlights the potential of nitro drugs as growth inhibitors of various bacterial species, emphasizing the differences in the metabolism of these types of molecules between M. tuberculosis and environmental mycobacteria, such as M. smegmatis, as a proofof-principle.

Scheme 1. Chemical Structure of Nitozoxanide (1) and Analogs 2,4-Dichloro-N-(5-nitrothiazol-2-yl)benzamide (2), 3-Bromo-N-(5-nitrothiazol-2-yl)-4-propoxybenzamide (3), and N-(5-Nitrothiazol-2-yl)-2-(trifluoromethoxy)benzamide (4)a

a

Structures were drawn using ChemDraw 16.0.

potency as 2 and 3 and was thus selected for further studies. The MICs against the various bacterial species tested are shown in Tables 2 and 3. These compounds had little or no antibacterial activity against M. smegmatis and M. tuberculosis complex strains. Hits 2, 3, and 4 Are Nitazoxanide Analogs. On the basis of their chemical structures, compounds 2, 3, and 4 are analogs of the antibacterial and antiparasitic thiazolide nitazoxanide (1, Scheme 1). 1 is a prodrug that is deacetylated at low pH into its active metabolite tyzaxonide (TIZ). 1 and TIZ inhibit the pyruvate:ferredoxin oxidoreductase (PFOR) of H. pylori, G. lamblia, and other anaerobic or microaerophilic microorganisms.27 However, 1 is moderately active against M. tuberculosis (MIC of 0.016 mg/mL),28 a species that has no PFOR homologs. Furthermore, the action of 1 on M. tuberculosis has been associated with the disruption of the membrane potential and intracellular pH homeostasis, leading to a multitarget mechanism of action.29 Hits 2, 3, and 4 are position and functional group isomers of 1. Indeed, the three compounds of the NTB series have different modifications of the functional group in the benzene ring moiety of the tail region of molecule 1 (Scheme 1). These modifications are responsible for their inactivity in M. tuberculosis. Moreover, 2, 3, and 4 have a limited spectrum of activity in mycobacteria, suggesting a more specific mechanism of action than the disruption of the membrane potential and intracellular pH homeostasis observed for 1 in M. tuberculosis. On the basis of these observations, it is possible that the antimicrobial effect of 2, 3, and 4 in mycobacterial species is due to the reduction of their nitro group. Several studies have analyzed the behavior of 1 analogs in which the nitro or tail groups have been deleted or substituted30−32 to decipher their mode of action in the various species analyzed. Indeed, denitro1 analogs were 2−3 times more active against M. tuberculosis than 1, showing that the nitro group is not essential for the activity of the compound in this bacterial species.31 However, analogs of 1 for which the nitro moiety was replaced or deleted were not active against anaerobic microorganisms,33 supporting

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RESULTS AND DISCUSSION Identification of Active Antimycobacterial Compounds by Whole-Cell-Based HTS Using M. aurum. We performed whole-cell-based HTS with the aim of identifying compounds with antimycobacterial activity. A chemical library of 35 860 compounds was screened against the growth of M. aurum (see Experimental Section and Figure S1 in Supporting Information for details), which was quantified using the resazurin colorimetric assay.26 Here, we report only the data pertaining to the “Institute Curie chemical library”, which is part of the French “Chimiothèque Nationale” (see Experimental Section). This library was screened at 0.15 μg/mL (average concentration of 0.4 μM). We identified 20 compounds showing greater than 40% growth inhibition: eight were eliminated from further consideration, as they were known to be cytotoxic or genotoxic compounds. Among the remaining hits, eight belonged to the same chemical family nitrothiazolylbenzamide, namely, the NTB series: those presenting MICs lower than 10 μM were retained. We then tested the validated hits for their antibacterial activity against various mycobacterial species, including M. tuberculosis and other bacterial species. We identified compounds active against only NTM. These compounds were the focus of this study. From this group, two compounds, 2 and 3 (Scheme 1), showed potent growth inhibition of M. aurum and M. marinum and no toxicity on Vero cells (IC50 > 100 μM). We tested other analogs available at the Institut Curie from the NTB chemical series: among them, compound 4 (Scheme 1) had the same 7426



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our hypothesis. Moreover, 1 can interact with enzymes other than PFOR, leading to an alternative hypothesis about its mechanism of action. For example, G. lamblia nitroreductase GLNR1 has been proposed to be an activator of 1, as its overexpression in the parasite increases susceptibility to the drug34 and its expression is down-regulated in resistant strains.24 Expression of MSMEG_6503 Affects Resistance of M. smegmatis to Compound 3. In addition to acquired drug resistance produced by spontaneous chromosomal mutations, mycobacterial species present inherent resistance to a variety of antibiotics, including macrolides35,36 and β-lactams.37 This is in part due to the presence of enzymes which modify or degrade the antibiotic and/or its target,38−40 efflux mechanisms,41 and cell wall impermeability.42 Various strategies have been tested to bypass or overcome these mechanisms, such as combining βlactamase inhibitors and β-lactams for the potential treatment of MDR and XDR tuberculosis.43,44 Given the importance of these mechanisms, we analyzed the origin of resistance of M. smegmatis against compound 3 (MIC > 129 μM). Such information could also provide clues about the mechanism of action of the molecules. Recently, HTS, using transposon libraries of M. smegmatis, has been successfully applied to the isolation of isoniazid hypersensitive mutants, thus revealing novel insights on the mechanism of action of the drug.45 Similarly, we performed HTS screening using a Tn611 transposon mutant collection of M. smegmatis and identified hypersensitive mutants to 3 that failed to grow at a low concentration of the compound (51.6 μM). Growth inhibition was visualized using the resazurin reduction test. We determined the MIC for a total of 81 positive mutants obtained in the first round of the assay and identified one hypersensitive mutant, called 126E6, with a MIC of 10.1 μM. This mutant was also more sensitive to the analogs 2 and 4 with a MIC of 18.6 μM and 19.6 μM, respectively. We determined the genomic position of the transposon insertion by performing ligation-mediated PCR (LM-PCR).46 We then sequenced the genome fragments flanking the transposon and found that the insertion site was inside the ORF MSMEG_6503. The length of the insertion obtained during Tn611 transposition is 20.7 kb, thus preventing efficient amplification by PCR of the MSMEG_6503 gene with specific primers (data not shown). We validated this result by complementing the 126E6 mutant strain with the wild type MSMEG_6503 gene using the plasmid pAL64::MSMEG_6503. After transformation, the mutant recovered resistance to 3, thus confirming that the hypersensitivity observed in the mutant was due to the interruption of the MSMEG_6503 gene (Figure 1). MSMEG_6503 encodes a transcriptional regulator of the TetR family, which has been demonstrated to be a repressor of the MSMEG_6505 gene.23 This gene is named nf nB and encodes the nitroreductase NfnB. We analyzed the levels of nf nB gene expression in wild type M. smegmatis and the 126E6 mutant strain by RT-qPCR to confirm the overexpression of NfnB in the mutant strain. Chart 1 shows the relative quantification (RQ) or fold change in the expression of nfnB in mutant 126E6 over that in the wild type strain. M. smegmatis 126E6 had a RQ of 6000, thus confirming overexpression of the nfnB gene in this strain. NfnB activity has been associated with benzothiazinone (BTZ) resistance through inactivation of the drug in M. smegmatis and M. tuberculosis.23 This process is due to the complete reduction of the nitro group of BTZ into an amino group catalyzed by

Figure 1. M. smegmatis 126E6 recovers resistance against 3 after complementation with the MSMEG_6503 gene. (1) Western blot showing overexpression of the MSMEG_6503 gene in M. smegmatis 126E6 pAL64::6503 (a) and its absence in the control strain M. smegmatis 126E6 pAL64 (b). (2) MIC determination using the resazurin microdilution assay with strains a (rows E−H) and b (rows A−D). The first six wells in rows A and E are growth controls (medium without antibiotic), and the next six wells in the same rows are negative controls (medium without bacteria). The test drugs (ofloxacin and 3) were arranged in 2-fold dilutions from left to right. Duplicates were performed for 3. Ofloxacin test wells are in rows B and F; 3 test wells are in rows C, D, G, and H. Bacterial growth was read as the change of color from blue to pink.

Chart 1. Study of the Expression of nf nB in the Mutant M. smegmatis 126E6 by RT-qPCRa

a

The bar chart shows the fold difference of nfnB gene expression (RQ) in M. smegmatis 126E6 relative to that of the WT M. smegmatis calibrator (RQ = 1). Gene expression was normalized using sigA as an invariant transcript. The mean values + SE are given for triplicates.

NfnB.23 BTZs target the DrpE1 subunit of the mycobacterial enzyme decaprenylphosphoryl-β-D-ribose 2′-epimerase,47 a key component for the synthesis of arabinogalactan and lipoarabinomanan,48,49 both essential polysaccharides of mycobacterial and related Actynomicetales cell walls. Makarov et al. demonstrated that BTZ hydroxylamine and amino derivatives were less active than the original molecule, thus highlighting the importance of a nonreduced nitro group in BTZ molecule for its antimycobacterial activity.47 The case of 3 contrasts with that of BTZ; NfnB apparently activates 3 instead of inactivating it. We can affirm that the sensitivity of M. smegmatis to 3 is associated with the expression of NfnB, based on the MIC of 3 against wild type M. smegmatis, mutant 126E6, and strain pAL64::MSMEG_6503, and the expression profiles of nf nB in M. smegmatis and mutant 126E6 and is possibly due to the production of active radicals after reduction of the molecule via its nitro group. Nitro-compound toxicity is associated with their interaction with specific targets47,50 and/or the reactive metabolites generated during reduction of the nitro group.51 Nitroreductases play a central role in this process. The enzymatic reduction of nitro compounds yields reactive metabolites, and its complete reduction to an amino group 7427



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Scheme 2. Empirical Reduction Pathway of 3a

a

Electrons involved in each reduction step are represented by e−. Structures were drawn with ChemDraw 16.0.

requires six electrons. This reaction generally produces a hydroxylamine, an amine, or both,21 which can interact with cellular macromolecules by direct or indirect covalent binding, producing genotoxic and cytotoxic effects.22 NfnB Reduces Compound 3 and Generates a Hydroxylamine Radical. We attempted to identify the chemical-related species present in cultures of M. smegmatis 126E6 with 3 to determine whether this hit was effectively reduced in the strain. Empirically, we know that NTB 3 can follow a reduction route with the formation of various radicals (Scheme 2), for which the exact m/z can be calculated. Thus, we performed LC−MS to explore this hypothesis by examining all ions corresponding to 3 degradation products in culture filtrates from M. smegmatis WT and the 126E6 mutant grown in the presence of 3 (Supporting Information Figure 2). Cultures grown in the absence of 3 were used as negative controls. The only ion we could detect in the positive ion mode in filtrates of WT M. smegmatis was at m/z 385.9805 [M + H]+, corresponding to 3. We conclude that 3 was not degraded in the wild type strain, as none of the other degradation products could be detected. In addition to the peak at m/z 385.9805, we detected a peak in filtrates of the 126E6 mutant at m/z 372.0012 in the positive ion mode, corresponding to the degradation of 3. This peak corresponds to the molecular mass of the hydroxylamine intermediate generated by the reduction of 3. The abundance of the detected peaks for both strains is shown in Chart 2. This result suggests that the compound is reduced to a hydroxylamine intermediate in the 126E6 mutant in association with NfnB expression and that this radical is responsible for 3 toxicity. According to the literature, hydroxylamine intermediates are highly toxic.22 Indeed, the nitroreductase RdxA activates the nitro drug MTZ in Helicobacter pylori resulting in DNA damage and mutagenesis.52−55 Similarly, in Trichomonas, this compound is toxic and mutagenic due to its reduction to a hydroxylamine intermediate.56

Chart 2. Abundance of 3 and Its Hydroxylamine Intermediate in Cultures of M. smegmatis and M. smegmatis 126E6 Determined by Targeted Metabolomics Analysisa

a The bar chart shows the abundance of 3 at m/z 385.9805 (black bars) and the hydroxylamine intermediate at m/z 372.0012 (gray bar) in the positive ion mode expressed as ion counts per milliliter of culture medium of M. smegmatis or M. smegmatis 126E6.

The mechanism of activation of 3 is likely due to NfnB expression because 3 had no activity in M. tuberculosis, which does not have NfnB analogs.23 Only M. marinum was sensitive to 3 and the other two molecules analyzed, 2 and 4. M. marinum carries several genes coding for nitroreductases. According to Marinolist (http://mycobrowser.epfl.ch/ marinolist.html), a total of six genes are annotated as nitroreductases: MMAR_0119, MMAR_0959, MMAR_1160, MMAR_1739, MMAR_2938, and MMAR_4721. M. marinum and M. tuberculosis have a common environmental ancestor, as they share a common genetic core.57 However, the genome size and the gene order in M. marinum are similar to that of M. smegmatis.57 During its evolution, M. marinum has become specialized to an intracellular lifestyle and can infect macrophages. However, it has retained genes from its ancestor, which has allowed it to survive in changing environmental conditions.57 This may be associated with the presence of a large number of genes that confer metabolic plasticity to the bacterium and, as a consequence, several genes encoding 7428



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resolution mass spectrometry (ESI-MS) was performed by the I.C.S.N. (Institut de Chimie des Substances Naturelles, Gif-sur-Yvette). Materials. 2,4-Dichloro-N-(5-nitrothiazol-2-yl)benzamide (2) (C10H5Cl2N3O3S, MW = 318 1360 g·mol−1), 3-bromo-N-(5-nitrothiazol-2-yl)-4-propoxybenzamide (3) (C13H12BrN3O4S, MW = 386 2211 g·mol−1) (purity >95% by liquid chromatography−mass spectrometry) came from the Institute Curie/CNRS chemical library. N-(5-Nitrothiazol-2-yl)-2-(trifluoromethoxy)benzamide (4). 5-Nitro-1,3-thiazol-2-amine 5 (131 mg, 0.9 mmol) was dissolved in dichloromethane (2 mL). 2-(Trifluoromethoxyl)benzoyl chloride 6 (0.17 mL, 1.1 mmol) dissolved in dichloromethane (1.5 mL) was added at 0 °C. The reaction was stirred at room temperature for 36 h. The organic layer was decanted, and the aqueous layer was extracted with CH2Cl2 (3 × 10 mL). The organic solutions were mixed and washed with a saturated solution of NaCl. The organic solution was dried over MgSO4, filtered, and evaporated. The crude product was purified by column chromatography on silica gel with dichloromethane as eluent. The chromatographic fractions were evaporated to yield 79 mg (26%) of a yellow powder. 1H NMR (300 MHz, CDCl3) δ = 10.36 (s, 1H, NH), 8.26 (s, 1H, H4′), 8.19 (dd, J = 7.8, 1.6 Hz, 1H, H6), 7.73 (td, J = 8.3, 1.7 Hz, 1H, H5), 7.55 (t, J = 7.75 Hz, 1H, H4), 7.44 ppm (d, J = 8.2 Hz, 1H, H3). 13C NMR (75, MHz, CDCl3) δ 162.52 (C2′), 161.01 (CO), 146.60 (C1), 144.01 (C5′), 140.45 (C4′), 135.07 (C5), 132.39 (C6), 128.02 (C4), 124.28 (C2), 122.04 (CF3, J = 260 Hz), 121.23 (C3). LRMS (ESI-MS) m/z = 334 [M + H]+. HRMS,18 m/z calculated for C11H6N3O4F3S + H+ [M + H]+: 334.0109. Found: 334.0098. Resazurin Microdilution Assay. The determination of the minimal inhibitory concentrations of antibiotics was performed in 96 wells.63 Briefly, 2-fold dilutions of each antibiotic were made in test wells with 100 μL of the appropriate culture medium (Table 1). A

nitroreductases, even if the specific physiological function of these enzymes is still unknown.58 Our findings could be translated to other environmental mycobacteria that affect human health, thus reflecting the high potential of these compounds in drug discovery research.

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CONCLUSIONS Our studies reflect the potential of exploring resistance mechanism as a tool to discover novel insights of the mechanism of drug action. This approach can be used as an alternative or complementary method to the traditional analysis of resistant mutants of normally sensitive bacteria. It has allowed us to identify the repression of the gene nf nB, coding for NfnB, as the factor responsible for the natural resistance of M. smegmatis to the hit 3. We report, for the first time, the identification of metabolic products of an analog of 1 in the genus Mycobacterium, as well as the reduction of analogs of 1 in mycobacteria. Our results suggest that NfnB reduces the compound 3 in a sensitive mutant strain of M. smegmatis, thus generating a hydroxylamine intermediate. This is associated with toxicity in this bacterial species. More definitive studies remain to be performed to define the mechanism of action of these compounds in sensitive mycobacterial species.

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EXPERIMENTAL SECTION

Bacterial Strains and Culture and Growth Conditions. M. tuberculosis H37Rv,59 M. smegmatis mc2155,60 M. marinum M,61 M. aurum,62 M. avium, and the derivatives M. smegmatis pAL64, M. smegmatis pAL64::MSMEG6503, and M. smegmatis 126E6 were cultured at 37 °C (or 32 °C for M. marinum) in Middlebrook 7H9 medium supplemented with ADC (0.5% bovine serum albumin, 0.2% dextrose, 0.085% NaCl, 0.0003% beef catalase; Difco), 0.5% glycerol, and 0.05% Tween 80 or on solid Middlebrook 7H11 medium supplemented with OADC (0.05% oleic acid, 0.5% bovine serum albumin, 0.2% dextrose, 0.085% NaCl, 0.0003% beef catalase; Difco). Clinical isolates of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter aerogenes, and Klebsiella pneumonia were cultured at 37 °C in Mueller−Hinton broth (Sigma-Aldrich). Streptococcus agalactiae NEM316 was grown in Todd−Hewitt broth (Sigma-Aldrich) at 37 °C. E. coli XL10-Gold Ultracompetent cells used for cloning procedures were grown according to supplier’s instructions (Agilent Technologies). When required, the antibiotics kanamycin (41 μM) or hygromycin B (180 μM for E. coli or 90 μM for mycobacteria) were added to the medium. General Procedures. All solvents and chemicals were used as purchased without further purification. The progress of all reactions was monitored on Merck precoated silica gel plates (with fluorescence indicator UV254) using dichloromethane/ethanol as the solvent system. Column chromatography was performed using Fluka silica gel 60 (230−400 mesh ASTM) with the solvent mixtures specified in the corresponding experiment. Spots were visualized by irradiation with ultraviolet light (254 nm). Proton (1H) and carbon (13C) NMR spectra were recorded on a Bruker Avance 300 (300 MHz for 1H; 75 MHz for 13C), using TMS as internal standard. Deuterated CDCl3 was purchased from SDS. Chemical shifts are given in parts per million (ppm) (δ relative to the residual solvent peak for 1H and 13C). The following abbreviations are used: singlet (s), doublet (d), triplet (t), and multiplet (m). The purity was determined by high performance liquid chromatography (HPLC). The purity of all final compounds was 95% or higher. The instrument used was an Alliance Waters system [Alliance Waters 2695 (pump) and Waters 2998 (photodiiode array detector)], and the column was a Waters XBridge C-18, 3.5 μm particle size (3.0 mm × 100 mm). Low-resolution mass spectrometry (ESI-MS) was recorded on a micromass ZQ 2000 (Waters). High-

Table 1. Medium and Incubation Conditions Used in Resazurin Microdilution Assay To Test the Antibiotic Susceptibility of Various Bacterial Species incubation temp (°C)

strain

medium

time

M. avium M. marinum M. tuberculosis H37Rv M. aurum M. smegmatis mc2155 E. coli S. aureus P. aeruginosa E. aerogenes K. pneumonia S. agalactiae

LBa Middlebrook 7H9 + ADCb + 0.5% glycerol

7 days 4 days 7 days overnight

30 32

MHc

6h

37

Todd−Hewitt broth

a

Luria−Bertani broth. b0.5% bovine serum albumin, 0.2% dextrose, 0.085% NaCl, 0.0003% beef catalase. cMueller−Hinton broth.

bacterial inoculum of approximately 1 × 103 UFC was added to each well. A growth control, without antibiotic, and a medium control,

Table 2. MICs of Compounds 2, 3, and 4 against Various Mycobacterial Speciesa compd

M. tuberculosis

M. aurum

M. marinum

M. smegmatis

M. avium

2 3 4

78.5 77.6 75.02

2.45 1 4.65

18.8 4.03 9

78.5 >129 75.02

46.63 77.68 11.70

a

MICs were determined using the resazurin microdilution assay. Test conditions were adjusted depending on the bacterial species under analysis. Values are expressed in μM units.

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genomic region of M. smegmatis mc2155 was amplified using the primers MSMEG_6503fw, 5′-TTGGCCGCTAGCGTGGACTACAAAGACGATGACGACAAGACCAAACCGCGCGGTCGTGGAG-3′, and MSMEG_6503Rv, 5′-TTCAGGGGATCCTTAGAGCCCGGCGGCGGTCAGCAG-3′, specifically designed for the addition of NheI/BamHI restriction sites and a FLAG epitope tag to the N-terminus of the protein to facilitate its detection on Western blots. The fragment was digested with NheI and BamHI and cloned in frame with the hsp60 promoter into the mycobacterial replicative vector pAL64.66 M. smegmatis was transformed following the protocol published by Jacobs and Hatfull.67 Empty pAL64 was used as a control. After selection, transformed clones were grown in 7H9 broth and hygromycin B until OD600 = 0.6 was reached. The cells were then collected by centrifugation and resuspended in 1 mL of protein lysis buffer (50 mM Tris-HCl, pH 7, 0.6 M NaCl, 10% glycerol, 0.1 mg/mL lysozyme, and Pierce protease inhibitor tablets; Thermo Fisher Scientific). Cell disruption was carried out using 2 mL of Lysing Matrix B tubes (MP Biomedicals) in a cell disruptor machine. Total protein concentration in the extracts was determined using the Pierce BCA protein assay kit. For Western blot experiments, approximately 10 μg of protein was separated on an SDS−polyacrylamide gel and transferred onto nitrocellulose membranes (iBlot Transfer Stacks) using a iBlot dry blotting system, according to the manufacturer’s recommendations (Invitrogen by Life Technologies). Membranes were incubated with mouse monoclonal ANTI-FLAG M2 antibody (Sigma-Aldrich) followed by HRP-coupled goat anti-mouse IgG (H+L) secondary antibody (Thermo Fisher Scientific) and visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Ligation-Mediated PCR (LM-PCR).46 The insertion site of Tn611 in the M. smegmatis genome was determined by LM-PCR. Briefly, a SalI-specific synthetic linker was constructed by annealing two nonphosphorylated oligonucleotides (Salgd, 5′-TAGCTTATTCCTCAAGGCACGAGC-3′, and Salpt, 5′-TCGAGCTCGTGC-3′). Genomic DNA extracted from transposon mutants was digested by SalI (New England Biolabs) and then ligated to the SalI linker using T4 DNA ligase (New England Biolabs). After ligase inactivation, the mix was redigested to cleave remaining restriction sites. This ligation mix was used as the PCR template in a reaction performed using AmpliTaq “Gold” DNA polymerase (Thermo Fischer Scientific), specific primers for Tn611 (oligoF, 5′-AAGAATTCATCGTTCCGTCCGTCCAATCTCC-3′, and oligoG, 5′-GAGCGACAGCCTACCTCTGACT3′), and a second specific primer for the SalI linker. The resulting fragments were purified and sequenced (Eurofins Genomics, Cochin sequencing platform). Quantification of nf nB Expression Levels by Quantitative Reverse Transcription PCR (RT-qPCR). Total RNA was isolated from M. smegmatis and M. smegmatis 126E6 as follows: 100 mL of cultures grown in Middlebrook 7H9 broth until OD600 = 0.35−0.45 was centrifuged at 5310g for 15 min. Pellets were resuspended in 1 mL of QIAzol lysis reagent (Qiagen), and cell disruption was carried out using 2 mL of Lysing Matrix B tubes (MP Biomedicals) in a cell disruptor machine. The tubes were centrifuged at 16 595g for 10 min at 4 °C and the supernatants collected. The extraction protocol was continued with PureLink RNA Mini Kit (Ambion), according to the manufacturer’s instructions. Resulting RNA samples were treated with TURBO DNA-free kit (Invitrogen) and subjected to control PCR to discard those contaminated with DNA. Total RNA (1 μg) was reverse transcribed using SuperScript II reverse transcriptase (Invitrogen) and random primers. qPCR was performed with samples corresponding to 20 ng RNA using SYBR Green PCR Master Mix in a StepOnePlus real-time PCR system (Applied Biosystems). The primer pairs used in the reactions were as follows: nfnBFw 5′-CAGGGCTACAACACCGAGA-3′ and NfnBRv 5′-CGTAAGAGCTCAACAGTGCC-3′ for nf nB (MSMEG_6505); sigFw 5′-GGTCGAGGTGATCAACAAGC-3′ and sigARv 5′-TCGCTGTCCTCGATGAAGTC-3′ for the internal calibrator sigA (MSMEG_2758). Expression levels of the nfnB gene were normalized to the expression of the sigA gene and analyzed using

Table 3. MICs of Compounds 2, 3, and 4 against Various Bacterial Speciesa compd

E. coli

S. agalactiae

S. aureus

P. aeruginosa

E. aerogenes

K. pnemonia

2 3 4

>93.05 >129 >93.62

>93.05 >129 >93.62

6.2 2.33 48.8

93.05 >129 93.63

>93.05 >129 >93.62

>93.05 >129 >93.62

a

MICs were determined using the resazurin microdilution assay. Test conditions were adjusted depending on the bacterial species under analysis. Values are express in μM.

without antibiotic or bacteria, were included in each assay. The reduction−oxidation dye resazurin (Sigma) was added to each well at a final concentration of 0.003%. Different incubation times and temperatures were used depending on the bacterial species (Table 1). When antibiotics do not inhibit bacterial growth, resazurin is reduced to resorufin, changing its color from blue to pink as an indicator of cell viability. The lowest concentration of drug that prevented the color change was defined as the minimum inhibitory concentration. Screening Procedure. The screening of a chemical library composed of 35 860 compounds from Prestwick Chemical (1120 compounds; www.prestwickchemical.com), CHEM-X-INFINITY (10 000 compounds; www.chem-x-infinity.com), and the French “Chimiothèque Nationale”64 (24 740 compounds obtained from Université de Lyon, Faculté de Pharmacie de Strasbourg, Centre d’Etude et de Recherche sur le Médicament de Normandie (CERMN), Institut Curie and Institut Pasteur) was performed on a Freedom EVO platform (Tecan). Compounds were transferred from mother plates into clear, flat bottom, bar-coded, cell-culture-treated 96-well plates with lids (Greiner Bio-One). Columns 1 and 12 were dedicated to controls: DMSO was used as a positive control to define growth of the bacteria without any compound (100% viability), and kanamycin was used as a negative control to kill all bacteria (0% viability). A 135 μL aliquot of a mixture containing M. aurum (20-fold dilution of a culture at an optical density (OD) of 600 nm of 0.2−0.3) in 7H9-S medium Middlebrook broth, 0.1% casitone, and 0.5% glycerol, supplemented with oleic acid, albumin, dextrose, catalase, and resazurin (0.002% final) was added to each well. Plates were incubated 18 h at 37 °C and assessed for color development by measuring the OD differences (ΔOD, corresponding to OD570 − OD604) at 570 and 604 nm on a Safire2 (Tecan). For each plate, the mean values and corresponding standard deviations were calculated for the positive and negative controls: ΔOD0 was the average ΔOD value of the negative control wells (in the presence of the antibiotic), and ΔOD100 was the average ΔOD value of the positive control wells (in the presence of DMSO alone). These values were used to determine the Z′-factor65 and the signal-to-background ratio (S/B = ΔOD100/ΔOD0). The average Z′-factor was calculated to be 0.87 ± 0.04 (no value below 0.5; see Figure S1), and S/B was >3 for all plates. The percent viability for each compound was calculated as follows: [(ΔODcmpd − ΔOD0)/ (ΔOD100 − ΔOD0)] × 100. Selection of Hypersensitive Mutants to 3 by HTS. For this experiment we used a transposon mutant collection of M. smegmatis previously constructed in our laboratory by Tn611 transposition.46 The collection was stocked in 96-well plates. HTS was performed as follows; 2 μL from each well of the collection plates was transferred into 96-well plates containing (i) Middlebrook 7H9 medium with ADC, 0.05% Tween 80, and kanamycin (growth control) or (ii) Middlebrook 7H9 medium with ADC, 0.05% Tween 80, kanamycin, and hit 3 (final concentration 20 μg/mL) (test plates). The antimicrobial mixture BBL MGIT PANTA was added to the media as a contaminant growth inhibitor. Plates were incubated for 48 h at 37 °C. At the midpoint of incubation (24 h), resazurin was added to a final concentration of 0.003% as a cell viability indicator. The clones that failed to grow in the presence of 3 (hypersensitive) were selected for further characterization. MSMEG_6503 Overexpression and Western Blot Analysis. For the overexpression of the MSMEG_6503 gene, the corresponding 7430



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the comparative CT method (also known as the 2−ΔΔCT method).68 qPCR data are presented as the fold change of nf nB gene expression in the mutant 126E6 over that in M. smegmatis WT. Liquid Chromatography−Mass Spectrometry (LC−MS): Lysate Preparation and Processing. WT M. smegmatis and mutant 126E6 were grown in Middlebrook 7H9 broth until OD600 = 0.6 at 37 °C. The cultures were then divided in two 5 mL aliquots; one was treated with 0.3 mg of 3 (limit of detection of compound in LC−MS), and the other was used as a control without antibiotic. The cultures were incubated for 3 h and then sonicated. The lysates were centrifuged and the resulting supernatants filtered using 0.45 μm Millex HA filter units (Merk Millipore) and treated as described below for LC−MS analysis. An amount of 100 μL of each sample was mixed with 100 μL of acetonitrile/methanol/water 40:40:20 v/v/v, followed by the addition of 200 μL of 0.2% acetonitrile in acetic acid, and centrifuged at 17 000g for 10 min at 4 °C; supernatants were analyzed by LC−MS. Aqueous normal phase liquid chromatography was performed using an Agilent 1290 Infinity II LC system equipped with a binary pump, temperaturecontrolled autosampler (set at 4 °C), and temperature-controlled column compartment (set at 25 °C), containing a Cogent diamond hydride type C silica column (150 mm × 2.1 mm; dead volume 315 μL). A flow-rate of 0.4 mL/min was used. Elution of polar metabolites was carried out using solvent A, consisting of 0.2% acetic acid in deionized water (resistivity of ∼18 MΩ cm), and solvent B consisting of 0.2% acetic acid in acetonitrile. The gradient was as follows: 0 min 85% B; 0−2 min 85% B; 3−5 min to 80% B; 6−7 min 75% B; 8−9 min 70% B; 10−11 min 50% B; 11.1−14 min 20% B; 14.1−25 min hold at 20% B, followed by a 5 min re-equilibration period in 85% B at a flow-rate of 0.4 mL/min. Accurate mass spectrometry was carried out using an Agilent Accurate Mass 6545 QTOF apparatus. Dynamic mass axis calibration was achieved by continuous postchromatography infusion of a reference mass solution using an isocratic pump connected to an ESI dual jet stream ionization source, operated in the positive- and negative-ion mode. Nozzle voltage and fragmentor voltages were set at 2000 and 100 V, respectively. The nebulizer pressure was set at 50 psig, and the nitrogen drying gas flow rate was set at 5 L/min. The drying gas temperature was maintained at 300 °C. The MS acquisition rate was 1.5 spectra/s, and m/z data ranging from 50 to 1200 were stored. This instrument routinely enabled accurate mass spectral measurements with an error of less than 5 parts-permillion (ppm), mass resolution ranging from 10 000 to 25 000 over the m/z range of 121−955 atomic mass units, and a 100 000-fold dynamic range with picomolar sensitivity. Detected m/z data were considered to be identified metabolites based on unique accurate massretention time identifiers for masses exhibiting the expected distribution of accompanying isotopomers. Typical variation in the abundance of most metabolites was between 5% and 10% under these experimental conditions.

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ORCID

Maria V. Buchieri: 0000-0001-5957-5210 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is part of the European Seventh Framework Program Nanotherapeutics against Resistant Emerging Bacterial Pathogens (NAREB Project 604237). This work was also supported by the “Programme de Recherche sur les Nouvelles Molécules Intervenant dans la Lutte Contre la Tuberculose” of the Gabonese Republic, the Department of Life Sciences and the Faculty of Natural Sciences kick-start funding award from Imperial College London, U.K., and Agilent Technologies, U.K., concerning all LC−MS experiments performed on the Agilent 6545 QToF. We thank Yves Janin for fruitful discussions and technical support.

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ABBREVIATIONS USED BTZ, benzothiazinone; kb, kilobase; LM-PCR, ligationmediated PCR; MAC, M. avium complex; MTZ, metronidazole; μM, micromolar; NTM, nontuberculous mycobacterial species; qPCR, quantitative PCR; RQ, relative quantification; RT- qPCR, quantitative reverse transcription PCR; TIZ, tyzaxonide; V, volt; XDR, extremely drug-resistant

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00726. Figure 1 showing Z′ factor value plotted for each 96-well plate; Figure 2 showing targeted metabolomics analysis of 3 and its intermediates in WT M. smegmatis and M. smegmatis 126E6 (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*M.V.B.: phone, (+33) 1 45 68 88 40; fax, (+33) 1 45 68 88 43; e-mail, [email protected]. *H.M.-L.: phone, (+33) 1 45 68 83 81; e-mail, [email protected]. 7431



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