Drugs and bacteria.

The series of quinoxalinic-derived chalcones (Fig 1) was synthesized and characterized essentially according to Mielcke et al. (2017) [13]. Rifampicin (RIF), ethambutol (EMB), and moxifloxacin (MFX) were purchased from Sigma-Aldrich, and isoniazid (INH) was purchased from ACROS Organics. M. tuberculosis strains were cultured as previously described [14].

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Fig 1. Chemical structures of the chalcones evaluated against the laboratorial M. tuberculosis H37Rv strain.

The non-methoxylated structures are highlighted in yellow, the monomethoxylated molecules are in green, the di-methoxylated in pink, and the structures containing tri-methoxy radicals on the A-ring are in orange.

https://doi.org/10.1371/journal.pone.0202568.g001

Determination of minimum inhibitory concentrations (MICs).

Individual MICs for each tested compound were determined in M. tuberculosis H37Rv strain as previously reported [15]. Briefly, the compounds were first solubilized in DMSO at a concentration of 4 mg/mL, except for compounds N3 and N7 (1 mg/mL), and then diluted in Middlebrook 7H9 broth to reach a concentration of 50 μg/mL, or 10 and 20 μg/mL for compounds N6 and N23, respectively. Serial two-fold dilutions were performed in 96-well U-bottom polystyrene microplates at concentration ranges of 25–0.05 μg/mL for all compounds, except for N6 (5–0.01 μg/mL) and N23 (10–0.02 μg/mL). Mycobacterial suspensions were thawed and diluted in 7H9 medium at a theoretic optical density (OD_600nm) of 0.006, and 100 μL were added to each well. Following 7 days of incubation at 37°C, 60 μL of a resazurin solution (0.01%) were added to the plates and the results were evaluated after 48 hours. MICs were determined by using the resazurin reduction microplate assay (REMA) as a growth indicator, and were considered as the lowest drug concentration that prevented a color change from blue (resazurin) to pink (resorufin) [16]. The values reported here were observed in two independent experiments, or were the highest value found among three independent tests. INH was used as a positive control drug. Compounds N9, N15 and N23 were subsequently tested against several resistant strains of M. tuberculosis: three drug-resistant clinical isolates (CDCT-10, CDCT-16, CDCT-27), one pan-susceptible strain (CDCT-28) and two laboratorial INH-resistant strains [pNIP::InhA(S94A) and pNIP::KatG(S315T)] (Table 1). All three clinical isolates carry the most common mutation found in the katG (Rv1908c) gene (S315T), CDCT-10 and CDCT-16 hold an additional mutation in the rpoB gene (H526D and D516V, respectively), responsible for causing resistance to RIF, and CDCT-16 also carries a mutation [C(-15)T] in the promoter sequence of the inhA (Rv1484) gene [17]. The pNIP::InhA(S94A) strain holds an additional copy of the inhA gene with a mutation that changed the naturally occurring Ser94 residue to an alanine, while the pNIP::KatG(S315T) strain had its wild katG gene knocked out and received a mutated copy of this gene, which changed the Ser315 to a threonine residue.

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Table 1. Activity of chalcones against M. tuberculosis clinical isolates and laboratorial strains.

https://doi.org/10.1371/journal.pone.0202568.t001

Combination of compound N9 with clinically used anti-TB drugs.

The effects of the combination of N9 with the clinically used anti-TB drugs INH, RIF, EMB, and MFX was assessed by a checkerboard assay in a two-drug association scheme, together with the REMA colorimetric method as a growth indicator, as previously reported [15]. Briefly, all compounds were solubilized in DMSO at concentrations of 1 mg/mL (INH, RIF, EMB, MFX) or 4 mg/mL (N9). Then, the compounds were diluted in 7H9 broth to obtain a concentration range in microplates of 10–0.039 μg/mL for INH and EMB; 0.3–0.001 μg/mL for RIF and MFX; and 6.25–0.098 μg/mL for compound N9. Anti-TB drugs were diluted horizontally (columns 2 to 10), while the N9 compound was diluted vertically (rows B to H). The mycobacteria inoculum, the incubation conditions of the microplates and the readout of results were carried out as described before for MICs determination. Values of fractional inhibitory concentration index (FICI) below 0.5 indicate a synergism between the compounds, in between 0.5 and 4 indicate an indifferent influence, and above 4 suggest an antagonistic effect [15]. Three independent experiments were performed.

Chemical and reagents.

The probe substrates phenacetin, acetaminophen, diclofenac, chlorzoxazone and nifedipine were purchased from Sigma-Aldrich (St. Louis, MO, USA); its metabolites 4′-hydroxydiclofenac, S-mephenytoin, 4′-hydroxymephenytoin, bufuralol, 1′-hydroxybufuralol, 6-hydroxychlorzoxazone and dehydronifedipine were acquired from Toronto Research Chemicals (Toronto, Canada); midazolam and 1′-hydroxymidazolam were obtained from Cayman Chemicals (Ann Arbor, MI, USA). Diazepam and tolbutamide, used as internal standards (IS), were purchased from Sigma-Aldrich (St. Louis, MO, USA). The following solutions were prepared: 962 μmol L^-1 phenacetin, 8000 μmol L^-1 S-mephenytoin, 432 μmol L^-1 bufuralol, 11672 μmol L^-1 chlorzoxazone, 560 μmol L^-1 nifedipine, 433 μmol L^-1 midazolam and 3956 μmol L^-1 diclofenac. Except for diclofenac, which was prepared in methanol, all dilutions were made in acetonitrile. The IS diazepam and tolbutamide were prepared at a concentration of 0.2 μmol L^-1 in methanol. All the solutions were stored in amber tubes at −20°C.

Human liver microsomes (HLMs), pooled from 150 donors, were purchased from Corning Life Science (Phoenix, AZ, USA). The constituents of the NADPH regeneration system, D-glucose-6-phosphate sodium salt, glucose-6-phosphate dehydrogenase, and β-nicotinamide adenine dinucleotide phosphate hydrate (NADP⁺), and macrogol glycerol ricinoleate (Cremophor EL) were acquired from Sigma-Aldrich (St. Louis, MO, USA). The solutions of the NADPH regeneration system were prepared in Tris-KCl buffer pH 7.4 (tris(hydroxymethyl)aminomethane 0.05 mol L^-1 and KCl 0.15 mol L^-1): 50 mmol L^-1 D-glucose-6-phosphate, 8.0 U mL^-1 glucose-6-phosphate dehydrogenase and 2.5 mmol L^-1 β-nicotinamide adenine dinucleotide phosphate hydrate. These solutions were stored at −20°C.

HPLC grade solvents, such as methanol, ethyl acetate and chloroform were purchased from Panreac (Castellar Del Vallès, Spain). Analytical grade reagents, such as hydrochloric acid, sodium phosphate monobasic and sodium phosphate dibasic were acquired from Synth (Diadema, Brazil), while potassium chloride was obtained from Mallinckrodt Baker (Xalostoc, Mexico), and tris(hydroxymethyl)aminomethane from JT Baker (Phillipsburg, NJ, USA).

Analysis conditions.

The analysis was performed using a Thermo Scientific LC system equipped with a pump (Accela 600 pump) and an autosampler coupled with Thermo Scientific TSQ Quantum Access Max and an electrospray triple quadrupole mass spectrometer. The chromatographic separation was carried out on an Ascentis Express Fused Core C18 column (100 x 4.6 mm ID, 2.7 μm particle size) connected to an Ascentis Express C18 guard column (3.0 mm x 4.6 mm ID, 2.7 μm particle size) purchased from Supelco (Bellefonte, PA, USA). The mobile phase comprised methanol: ultrapure water (90:10, v/v) that contained 0.1% of formic acid (v/v) for analysis of probes 1A2, 2C9, 2D6, 2E1 and 3A4/5, or 0.1% of ammonium hydroxide (v/v) for analysis of the CYP2C19 probe. Xcalibur software 2.0 (Thermo Fisher Scientific, Waltham, MA, USA) was used to control the instrument and to process data.

To optimize working conditions and to carry out the tandem mass spectrometry experiments, stock standard MeOH: ultrapure water (1:1, v/v) solution of each compound at concentration of 1 μg mL^-1 was infused at a flow-rate of 50 μL min^-1 using direct infusion via a syringe pump integrated into the TSQ instrument to mix the standard solution with the mobile phase (450 μL min^-1; MeOH:H₂O). Two selective reaction monitoring (SRM) transitions were used for each compound, one for quantification and one for confirmation (S1 Table).

Mass spectrometer conditions, such as cone voltage, capillary temperature, vaporizer temperature, sheath gas, ion sweep gas, and auxiliary gas, are summarized at S2 Table. Nitrogen was used as a sheath gas, ion sweep gas and auxiliary gas, and argon was used as a collision-induced-dissociation gas at 1.8 mTorr.

CYP450 inhibition experiments.

To evaluate the potential of N9 to inhibit CYP450 enzymes, human liver microsomes were used to assess the rate of metabolism of the probe substrate of each CYP450 isoform, evaluated in the absence or the presence of N9. The concentration of N9 that caused 50% reduction of the activity of each CYP450 isoform (IC₅₀) was determined. The enzyme inhibition assays were performed using a probe substrate for each CYP450 isoform. The probe substrate concentrations are equal to their K_M values, as summarized in the S3 Table. The compound N9 was evaluated at seven different concentrations: 10.0 nmol L^-1, 46.4 nmol L^-1, 215.4 nmol L^-1, 1.0 μmol L^-1, 4.6 μmol L^-1, 21.5 μmol L^-1, and 100 μmol L^-1. Control samples were also tested in the absence of N9. The experiments were performed in triplicate.

The microsomal medium was composed by N9 solution (5 μL), probe substrate solution (5 μL), HLMs (90 μL), NADPH regeneration system solution (100 μL), and sodium phosphate buffer pH 7.4 0.1 mol L^-1+ 0.34% (m/v) Cremophor EL (200 μL). The incubation conditions were previously determined by Habenschus et al. (2017) [20], and are described in S3 Table. After 5 min prewarmed at 37°C in a Dubnoff metabolic shaking incubator SL157 (Solab, Piracicaba, Brazil), the reactions were initiated by the addition of 100 μL of NADPH regeneration system solution. After the incubation period, the reaction was stopped by adding the extraction solvent (1.0 mL) and modifier, when necessary (S3 Table). The IS solution was then added (50 μL), and the samples were shaken in a Vibrax VXR agitator system (IKA. Staufen, Germany) for 15 min at 1000 rpm, and then centrifuged at 4°C and 1800 × g for 10 min (Hitachi CF16RXII, Tokyo, Japan). The extractor phase was collected (600 μL) and dried using a Concentrator Plus Speed Vacuum (Eppendorf, Hamburg. Germany). Then, the residue from each sample was solubilized in the mobile phase (200 μL) and analyzed by LC-MS/MS under the conditions described above. IC₅₀ values were determined by a nonlinear regression of the percentage of remaining enzyme activity versus the logarithm concentration of N9. The GraphPad Prism Software 5.0 (San Diego, CA, USA) was used to analyze these results.

Salmonella/microsome mutagenicity test.

Salmonella typhimurium strains TA98, TA97a, TA100, TA1535, and TA102 were provided by MOLTOX^® (Molecular Toxicology Inc. USA). Mutagenicity was assayed according to the pre-incubation procedure following the recommendation described in Mortelmans and Zieger (2000) [21]. The S9 metabolic activation mixture (S9 mix) was prepared according to Maron and Ames (1983) [22]. Chalcone N9 was tested with the highest concentration limited by its solubility in DMSO, reaching 400 μg/plate. Briefly, 100 μL of test bacterial cultures (1–2 x 10⁹ cells/mL) were incubated at 37 ^oC with different concentrations of N9 (10–400 μg/plate) in the presence or absence of S9 mix, for 20 min, without shaking. Subsequently, 2 mL of soft agar (agar 0.6%, NaCl 0.5%, histidine 50 μM, biotin 50 μM, pH 7.4, 42 ^oC) were added to the test tube and poured immediately onto a plate of minimal agar (1.5% agar, Vogel-Bonner E medium, containing glucose 2%). 2-aminoanthracene (2-AA, 5 μg/plate) was used as positive control for all strains in the presence of metabolic activation (with S9 mix). In the absence of metabolic activation, 4-nitroquinoline-oxide (4-NQO, 0.5 μg/plate) was used for TA98, TA97a, and TA102 strains, and sodium azide (NaN₃, 1 μg/plate) was used for TA100 and TA1535 strains. Plates were incubated in the dark at 37 ^oC for 48 h before counting the revertant colonies. Assays were repeated twice and the plating for each concentration was conducted in triplicate. Data were evaluated by one-way analysis of variance (ANOVA), followed by Dunnett's post-test, using GraphPad Prism 5.0 (GraphPad, San Diego, CA, USA). Differences were considered significant at the 95% level of confidence.

Genotoxicity investigation.

The alkaline comet assay was performed to evaluate possible genotoxic effects of N9. Human hepatocellular carcinoma (HepG2) cells were plated in a 6-well culture microtiter plate (5 x 10⁵ cells per well) in DMEM and allowed to adhere overnight prior to incubation for 24 h with N9 (2.5, 5, or 10 μg/mL). Methyl methanesulfonate (MMS; Sigma-Aldrich), a known mutagenic alkylating agent, was incubated for 1 h (100 μM), as positive control [23]. N9 solutions were prepared in DMSO, and the final concentration of DMSO in the assays was 0.5%. Sixty μL of HepG2 cell suspension was mixed with 180 μL of low-melting point agarose (Invitrogen) and scattered on two pre-coated microscope slides with normal agarose (Invitrogen). Cells were then lysed in freshly prepared ice-cold solution (10 mM Tris, 2.5 M NaCl, 100 mM EDTA with 10% DMSO and 1% Triton X-100), for 24 h. The slides were then incubated for 20 min at 4°C with fresh buffer (300 mM NaOH e 1 mM EDTA) to induce DNA unwinding and to expose the alkali-labile sites, with light protection. Electrophoresis was conducted for 20 min at 25 V and 300 mA. Slides were fixed and silver stained after neutralization. One hundred cells were scored according to the size and amount of DNA in the tail. Individually, the cells were given an arbitrary value of 0 (undamaged) to 4 (maximally damage) [24]. The statistical analysis was performed as described above for Salmonella mutagenicity test.