ABSTRACT
8-Nitro-benzothiazinones (BTZs), such as BTZ043 and PBTZ169, inhibit decaprenylphosphoryl-β-d-ribose 2′-oxidase (DprE1) and display nanomolar bactericidal activity against Mycobacterium tuberculosis in vitro. Structure-activity relationship (SAR) studies revealed the 8-nitro group of the BTZ scaffold to be crucial for the mechanism of action, which involves formation of a semimercaptal bond with Cys387 in the active site of DprE1. To date, substitution of the 8-nitro group has led to extensive loss of antimycobacterial activity. Here, we report the synthesis and characterization of the pyrrole-benzothiazinones PyrBTZ01 and PyrBTZ02, non-nitro-benzothiazinones that retain significant antimycobacterial activity, with MICs of 0.16 μg/ml against M. tuberculosis. These compounds inhibit DprE1 with 50% inhibitory concentration (IC50) values of <8 μM and present favorable in vitro absorption-distribution-metabolism-excretion/toxicity (ADME/T) and in vivo pharmacokinetic profiles. The most promising compound, PyrBTZ01, did not show efficacy in a mouse model of acute tuberculosis, suggesting that BTZ-mediated killing through DprE1 inhibition requires a combination of both covalent bond formation and compound potency.
INTRODUCTION
The discovery of the 8-nitro-benzothiazinone (BTZ) lead compound BTZ043 [2-[(2S)-2-methyl-1,4-dioxa-8-azaspiro[4,5]dec-8-yl]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one] (Fig. 1) and the identification of the enzyme decaprenylphosphoryl-β-d-ribose 2′-oxidase (DprE1) as its target provided one of the most potent agents against tuberculosis (TB) known to date (MIC of 1 ng/ml) and one of the most attractive targets for antituberculosis drug discovery, respectively (1, 2). More recently, the preclinical candidate PBTZ169 [2-[4-(cyclohexylmethyl)piperazin-1-yl]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one] arose from a lead optimization campaign and is currently on track to enter clinical trials (3). PBTZ169 has an MIC against Mycobacterium tuberculosis of 0.3 ng/ml.
Structures of BTZ043, PBTZ169, and PyrBTZ derivatives and their respective MICs against M. tuberculosis H37Rv.
DprE1, together with its partner enzyme DprE2, catalyzes the conversion of decaprenylphosphoryl-β-d-ribose (DPR) to its arabinose counterpart decaprenylphosphoryl-β-d-arabinose (DPA), which is the sole donor of arabinose sugars that are essential for cell wall biosynthesis in M. tuberculosis and other Corynebacterineae species (4). The drug susceptibility of DprE1 is illustrated by recent publications reporting not only covalent inhibitors such as BTZ (5–10) but also a multitude of chemical scaffolds that inhibit this target noncovalently, with some showing in vivo efficacy.
The BTZs and other reported nitroaromatic DprE1 inhibitors are suicide inhibitors of DprE1. The nitro group of these compounds is reduced specifically by DprE1 to a nitroso group, which then reacts with a key active site cysteine residue (Cys387 in M. tuberculosis) to form an irreversible covalent adduct. The nitro group of the BTZs is essential for this mechanism of inhibition, as earlier structure-activity relationship (SAR) studies demonstrated that its replacement by groups such as H, Br, CN, CF3, NH2, N,N-diacetyl, NCHN-dimethyl, NCH-phenyl, furyl, COOR, and COH led to MIC increases of >500-fold with respect to BTZ043 and greatly reduced bactericidal activity (1, 11). These substitutions were introduced because of concerns about potential mutagenicity associated with the nitro group, although the concerns have proved groundless. Here, we report the first non-nitro BTZ analogues that retain significant antimycobacterial activity through inhibition of DprE1, with a pyrrole ring replacing the nitro group.
MATERIALS AND METHODS
Chemistry.The four pyrrole-BTZs (PyrBTZs) were synthesized starting from their nitro analogues (1, 3), as described in the supplemental material.
Bacterial strains and culture conditions.All mycobacterial strains, BTZ-resistant mycobacterial mutants, and M. tuberculosis strain H37Rv were grown at 37°C, with shaking, in Middlebrook 7H9 broth (Difco) supplemented with 10% albumin-dextrose-catalase (ADC) enrichment, 0.2% glycerol, and 0.05% Tween 80. M. tuberculosis strain 18b was grown in the same medium supplemented with 50 μg/ml streptomycin.
Drug susceptibility testing.The in vitro activities against all mycobacterial strains were measured with the resazurin reduction microplate assay (REMA), by 2-fold serial dilution of the compounds in the working bacterial culture in 96-well plates (final volume of 100 μl). For M. tuberculosis and Mycobacterium bovis BCG, the plates were incubated for 1 week at 37°C; for Mycobacterium smegmatis strains, the incubation time was 24 h. Bacterial viability was determined by adding sterile resazurin (10 μl, 0.025% [wt/vol]), incubating the mixture, and measuring resazurin turnover by fluorescence (excitation wavelength, 560 nm; emission wavelength, 590 nm), using a Tecan Infinite M200 microplate reader.
DprE1 assays.The C387G and C387S mutant DprE1 proteins were generated using the pET28a-M. tuberculosis DprE1 plasmid (3) and the QuikChange site-directed mutagenesis kit (Agilent), with the primers 5′-GGCTGGAACATCGGCGTCGACTTCCCC-3′ and 3′-CCGACCTTGTAGCCGCAGCTGAAGGGG-5′ (C387G) and 5′-GGCTGGAACATCAGCGTCGACTTCCCC-3′ and 3′-CCGACCTTGTAGTCGCAGCTGAAGGGG-5′ (G387S) (mutated bases are underlined). Wild-type M. tuberculosis DprE1 and the mutant enzymes were expressed and purified as described elsewhere (3).
The 50% inhibitory concentrations (IC50s) for DprE1 were determined as described previously (12), using a coupled Amplex Red/horseradish peroxidase assay, with farnesyl-phosphoryl-β-d-ribofuranose (FPR) as the substrate. The conversion of Amplex Red to resorufin was followed by fluorescence measurements (excitation wavelength, 560 nm; emission wavelength, 590 nm) on a Tecan M200 reader, in kinetic mode, at 30°C. A negative-control sample with no inhibitor was used, and the background rate (no added FPR) was subtracted from measured rates. IC50s were determined using Prism (GraphPad Software) by fitting the inhibitor concentration (log[I]) and normalized response (V) to the equation V = 100/{10[(logIC50 − log[I])h]}, where h is the Hill coefficient for DprE1.
Decaprenylphosphoryl-β-d-ribose epimerization by M. tuberculosis H37Ra cells.Aliquots of 6 ml of M. tuberculosis H37Ra culture grown to an optical density at 600 nm (OD600) of 1.31 were harvested and washed with buffer A (50 mM MOPS [morpholinepropanesulfonic acid] [pH 7.9], 10 mM MgCl2, 5 mM 2-mercaptoethanol). The cells (∼30 mg) were incubated for 15 min on ice with 50 μl of buffer A, 16 nmol NADH, and 16 μg of PyrBTZ01 or PyrBTZ02 or 2 μg of BTZ043 in 5 μl dimethyl sulfoxide (DMSO), in a final volume of 80 μl. The reactions were started with the addition of 15,000 dpm of 5-phospho-[14C]ribose 1-diphosphate (P[14C]RPP) prepared from [14C]glucose (specific activity, 290 mCi/mmol; American Radiolabeled Chemicals, Inc.), as described elsewhere (13). After 2 h of incubation at 37°C, the reactions were stopped with 1.5 ml CHCl3/CH3OH (2:1) and subjected to a biphasic Folch wash (14). The organic phase was dried and dissolved in 40 μl of CHCl3/CH3OH/H2O/NH4OH (65:25:3.6:0.5 [vol/vol]); 25% of the sample was separated by thin-layer chromatography on silica gel plates (Merck) in CHCl3/CH3OH/NH4OH/1 M ammonium acetate/H2O (180:140:9:9:23 [vol/vol]) and visualized by autoradiography (Biomax MR-1 film; Kodak). The intensity of the bands was quantified using ImageJ software (NIH).
Molecular modeling.Docking of PyrBTZ01 and PyrBTZ02 to DprE1 was performed using the induced-fit docking module of Schrödinger-Maestro v9.1, with standard precision settings (15). The M. tuberculosis DprE1 protein structure (PDB accession no. 4NCR) was used as a template for docking. The sulfur atom of Cys387 was chosen as the centroid of the box, and the box size was generated automatically. The ligands were placed in the same pocket as PBTZ169 by superposition of the benzothiazinone core. Initial glide docking for each ligand was carried out. Side chains were trimmed automatically based on B-factors, with receptor and ligand van der Waals scaling of 0.70 and 0.50, respectively, and 20 poses were generated. Prime side chain prediction and minimization were carried out for residues within 5.0 Å of ligand poses, and side chains were optimized. This led to ligand structures and conformations that were induced to fit each pose of the receptor structure. Finally, Glide SP redocking was carried out for structures with scores within 30.0 kcal/mol of the best structure and within the top 9 structures overall.
Cytotoxicity studies.The cytotoxicity of the compounds was measured as described previously (8), against two human hepatic cell lines (HepG2 and Huh7), a human lung epithelial cell line (A549), and a human monocytic cell line (THP-1). Briefly, cells were incubated (4,000 cells/well) with serial dilutions of compounds (2-fold dilutions; 100 to 0.1 μg/ml) in a 96-well microplate. Following incubation for 3 days at 37°C, cell viability was determined by adding resazurin for 4 h at 37°C and measuring the fluorescence of the resorufin metabolite (excitation wavelength, 560 nm; emission wavelength, 590 nm) using a Tecan Infinite M200 microplate reader. Data were corrected for background (no-cell control) and expressed as a percentage of the value for untreated cells (cells only).
Metabolic stability in vitro.The intrinsic clearance (CLint) of compounds was determined using both mouse and human liver microsomes. Briefly, 100 μg of mouse (CD-1) or human liver microsomes (both from Invitrogen) were mixed in 0.1 M phosphate buffer (pH 7.4) containing 1 μl of compound dissolved in DMSO at 100 μg/ml, in a final volume of 50 μl. In parallel, an NADPH-regenerating system (Promega) was prepared in 0.1 M phosphate buffer (pH 7.4). The solutions were preincubated at 37°C for 10 min before the intrinsic clearance assessment was initiated by mixing the two solutions (50 μl of each; final compound concentration, 1 μg/ml) at 37°C. After 0, 5, 10, 15, 30, and 60 min, the reactions were terminated by transferring 100 μl of the reaction mixture into 100 μl of acetonitrile and placing the mixture on ice for 30 min, for full protein precipitation. Samples were then centrifuged at 12,000 × g for 10 min, and the supernatant was injected onto a high-performance liquid chromatography (HPLC) column (Dionex) to quantify the amount of parent compound remaining over time. Carbamazepine (1 μg/ml) was used as a control for low intrinsic clearance.
Pharmacokinetic studies in mice.PyrBTZ01 (50 mg/kg) was administered by gavage as a solution (5 mg/ml) in 0.5% carboxymethyl cellulose (CMC) in water, using three BALB/c mice per time point. At each time point (0, 15, 30, 60, 120, 240, and 480 min), mice were sacrificed by cardiac puncture and blood was collected. Plasma was separated by centrifugation for 10 min at 2,200 × g at 4°C and was flash frozen in liquid nitrogen for storage at −80°C for subsequent mass spectrometric (MS) analysis. Quantification was performed using an external calibration curve of PyrBTZ01 at different concentrations in mouse serum. Ultraperformance liquid chromatography (UPLC) separation was performed with an Agilent 1290 Infinity liquid chromatography (LC) system, including a 1290 Infinity LC system binary pump with an integrated degasser, a high-performance autosampler, and a thermostatted column compartment. Samples (2 μl) were injected onto a Zorbax Extend-C18 analytical column (2.1 by 50 mm; particle size, 1.8 μm; Agilent Technologies) operated at 40°C, using H2O/0.1% HCOOH as mobile phase A and CH3CN/0.1% HCOOH as mobile phase B, with a gradient of 2 to 100% mobile phase B in 5 min, at a flow rate of 0.4 ml/min. All samples were analyzed in duplicate. The UPLC system was interfaced with a 6530 accurate-mass quadrupole-time of flight (Q-TOF) LC/MS system (Agilent Technologies). Electrospray ionization MS data were acquired in the positive ionization mode in the mass range of m/z 100 to 1,000, at a rate of 2 spectra/s. Experimental parameters were set as follows: fragmentor voltage, 190 V; capillary voltage, 3,500 V; gas temperature, 300°C; sheath gas temperature, 350°C. External calibration of the instrument was carried out with ESI-L solution (Agilent). Data were processed using MassHunter quantitative analysis compliance software. Extracted ion chromatograms (EICs) of ions at m/z 432.0835 (eluted at 4.24 min) were integrated, and quantification was performed using the external calibration curve for PyrBTZ01 in mouse serum.
Efficacy studies in vivo.Male BALB/c mice (age, 5 to 6 weeks; Central Research Institute of Tuberculosis, Russian Academy of Medical Sciences, Moscow, Russia) were infected through intravenous injection of 5 × 106 CFU of M. tuberculosis H37Rv in the lateral tail vein (10 mice per study group). Treatment began on day 8 after infection, and PyrBTZ01 (50 mg/kg) or isoniazid (25 mg/kg) was administered by gavage 5 days/week for 4 weeks. A group of untreated mice was used as a control group. The mice were euthanized, lungs were excised and homogenized, and dilutions were plated on Dubos agar. When the mice died before the end of the experiment, lung CFU counts were performed immediately. Plates were incubated for 24 days at 37°C, and the number of CFU in the lungs was determined. Experiments were approved by the State Veterinary Service of the Russian Federation (authorization no. 250 0219882).
RESULTS
Pyrrole-BTZ analogues are potent antimycobacterial agents.Following our discovery of BTZ043, we performed extensive SAR studies of this scaffold. Significant modifications were tolerated at position 2 of the BTZ ring system, and this led to the development of the preclinical candidate PBTZ169 from the lead compound BTZ043 (3). We also explored several functional groups as replacements for the 8-nitro group, which invariably led to greatly reduced antimycobacterial activity, i.e., 500- to 5,000-fold increases in MICs (1, 2). An interesting exception was the 8-pyrrole substitution (yielding PyrBTZ compounds) (Fig. 1), which was introduced into the BTZ or PyrBTZ scaffold by using the respective nitro-BTZs as starting materials, as shown in Fig. 2.
Synthetic routes for the synthesis of pyrrole-BTZs. Step a, NaHS, NH4Cl, ethanol; step b, (i) 2,5-dimethoxytetrahydrofuran, acetic acid, reflux; (ii) NaOH; step c, 2,5-hexanedione, p-toluenesulfonic acid, toluene, reflux.
Compounds PyrBTZ01 [2-[(2S)-2-methyl-1,4-dioxa-8-azaspiro[4,5]dec-8-yl]-8-(1H-pyrrol-1-yl)-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one] and PyrBTZ02 [2-[4-(cyclohexylmethyl)piperazin-1-yl]-8-(1H-pyrrol-1-yl)-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one], the pyrrole analogues of BTZ043 and PBTZ169, respectively, both displayed MICs of 0.16 μg/ml against M. tuberculosis H37Rv. PyrBTZ03 [2-(4-butylpiperazin-1-yl)-8-(1H-pyrrol-1-yl)-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one], a PyrBTZ analogue bearing a n-butyl chain on the piperazine ring, presented a higher MIC of 0.62 μg/ml (Fig. 1). Previous literature reports described the oxidation of pyrroles, particularly dimethyl-substituted pyrroles, in the presence of nucleophiles (16). Therefore, we synthesized PyrBTZ04 [2-[4-(cyclohexylmethyl)piperazin-1-yl]-8-(2,5-dimethyl-1H-pyrrol-1-yl)-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one], a dimethylpyrrole analogue of PBTZ169, to evaluate whether this group could be oxidized by flavin-containing DprE1 and whether the resulting product could form a covalent adduct with Cys387, making this compound an irreversible inhibitor of the enzyme. However, because the MIC of PyrBTZ04 (2.5 μg/ml) was significantly higher than those observed for the unsubstituted pyrrole compounds PyrBTZ01, PyrBTZ02, and PyrBTZ03 (Fig. 1), PyrBTZ04 was not explored further.
PyrBTZs kill M. tuberculosis through inhibition of DprE1.The two most potent PyrBTZ analogues, PyrBTZ01 and PyrBTZ02, were characterized in depth to confirm their mechanisms of action. Because the PyrBTZs were expected to target DprE1, we first determined their MICs against various mycobacterial strains (Table 1) that were fully susceptible or resistant to BTZ due to mutations in the key active site Cys residue (Cys387 in M. tuberculosis and M. bovis and Cys394 in M. smegmatis). Both compounds displayed >600-fold increases in MICs against the BTZ-resistant strains versus wild-type M. tuberculosis, thus confirming DprE1 as their target. Similar results were obtained with matched BTZ-susceptible strains and resistant mutants of M. bovis BCG and M. smegmatis (Table 1). Like other BTZ derivatives, and cell wall inhibitors generally, PyrBTZ01 and PyrBTZ02 were inactive against the streptomycin-starved 18b strain (ss18b), a model for nonreplicating M. tuberculosis (17).
MIC99 values for PyrBTZ01 and PyrBTZ02 against wild-type and BTZ-resistant mycobacterial strains
Genetic analysis.To assess whether additional mutations in the essential flavoenzyme DprE1 could cause resistance to PyrBTZs, we generated PyrBTZ01-resistant mutants of M. tuberculosis. The mutants arose at a frequency of 10−8 and displayed MICs of >100 μg/ml for both PyrBTZ01 and PyrBTZ02. Surprisingly, Sanger sequencing of the dprE1 gene in four distinct mutants revealed single nucleotide changes in the Cys387 codon, which translated to a Cys→Ser mutation, as seen in the BTZ043-resistant NTB1 strain (Table 1) (1).
The cross-resistance to the PyrBTZs displayed by the NTB1 strain and the newly generated PyrBTZ01-resistant mutants, due to the Cys387Ser mutation, was unexpected, since Cys387 is required for the binding of covalent nitroaromatic inhibitors, including nitro-BTZs, but not noncovalent inhibitors (18). The PyrBTZs with nonsubstituted pyrroles were not expected to form covalent adducts with Cys387. To investigate this, we incubated M. tuberculosis DprE1 with its substrate farnesyl-phospho-ribose in the presence of PyrBTZ01 or PyrBTZ02, followed by mass spectrometric analysis, as described previously (3). In contrast to the situation observed for BTZ043 or PBTZ169, for which covalent adducts with DprE1 were clearly identified (3, 19), no covalent adducts were observed following incubation with PyrBTZ01 or PyrBTZ02.
Enzymatic analysis.PyrBTZ01 and PyrBTZ02 were then tested in the Amplex Red peroxidase-coupled enzymatic assay with recombinant wild-type M. tuberculosis DprE1 and were found to inhibit the enzyme with IC50 values of 1.61 μM (PyrBTZ01) and 7.34 μM (PyrBTZ02) (Table 2). The IC50 values were higher than expected based on the relatively low MICs in M. tuberculosis and in comparison with values reported elsewhere for other noncovalent inhibitors such as the best aminoquinolones (IC50 of 0.02 μM and MIC of 0.39 μM) (20) and pyrazolopyridones (IC50 of 0.01 μM and MIC of 0.1 μM) (21). The favorable MICs observed for the PyrBTZs could be indicative of better uptake, easier access to DprE1, and accumulation in the bacterium, or even a secondary target in M. tuberculosis. The inhibition of BTZ-resistant DprE1 followed the trend observed in the MIC measurements, with the C387G mutant being more resistant to inhibition by PyrBTZ01, PyrBTZ02, and BTZ043 (∼7- to 9-fold increases in IC50) than the C387S mutant (∼2.5- to 4-fold increases in IC50).
IC50 values measured for PyrBTZ01, PyrBTZ02, and BTZ043 against wild-type and BTZ-resistant M. tuberculosis DprE1
Much lower sensitivity of the DprE1 enzyme to PyrBTZ01 and PyrBTZ02, compared to BTZ043, was confirmed in a radioactive assay that monitored the incorporation of P[14C]RPP into [14C]DPA and its precursors by M. tuberculosis H37Ra cells. We observed partial inhibition of DPA synthesis when 200 μg/ml PyrBTZ01 and PyrBTZ02 were added (Fig. 3), whereas complete inhibition was obtained in the presence of only 25 μg/ml BTZ043.
Comparative inhibition of DPA synthesis. Decaprenylphosphoryl-β-d-ribose epimerization by M. tuberculosis H37Ra cells was inhibited by PyrBTZ01 (200 μg/ml), PyrBTZ02 (200 μg/ml), and BTZ043 (25 μg/ml). The ratios of the intensities of the DPA and DPR bands (quantified in ImageJ) are also shown.
Structural studies of PyrBTZ binding to DprE1.To understand the mode of binding of PyrBTZ01 and PyrBTZ02 to DprE1 and the intriguing role of Cys387, we have performed extensive cocrystallization studies of these compounds with DprE1 from M. tuberculosis and M. smegmatis, but no PyrBTZ has been found in the crystals to date. Previous reports described the formation of aromatic-thiol hydrogen bonds in proteins, involving cysteine residues (22, 23); in theory, this could explain the role of Cys387 in binding PyrBTZ compounds. To test this hypothesis, we modeled PyrBTZ01 and PyrBTZ02 in the active site of M. tuberculosis DprE1, using the cocrystal structure containing PBTZ169 (PDB accession no. 4NCR) that was disclosed recently (3). The modeling work assessed the possibility that the active site of DprE1 could accommodate PyrBTZ01 or PyrBTZ02 by placing the pyrrole rings in the vicinity of Cys387 and maintaining the overall binding mode of the BTZ ring system that was observed for BTZ043 and PBTZ169 (3, 19). Results of the induced-fit docking studies produced similar poses for both pyrrole derivatives, but these were slightly shifted in comparison with the positions of BTZ043 and PBTZ169 (Fig. 4A and B). Indeed, the pyrrole ring is placed just below Cys387, leading to PyrBTZ01 and PyrBTZ02 being less embedded (Fig. 4C and D). Conformational changes in a few neighboring residues (for example, Lys418 or Trp230) would be required to stabilize the pyrrole compounds in the pocket in the same manner as PBTZ169 in the crystal structure of DprE1 (3).
Molecular docking of pyrrole-BTZs in the DprE1 active site. Superposition of the M. tuberculosis DprE1 crystal structure with BTZ043 (A) or PBTZ169 (B) and the modeled structures of docked PyrBTZ01 (blue carbons) (C) and docked PyrBTZ02 (red carbons) (D) show an induced-fit complex. Cocrystallized DprE1 with PBTZ169 (PDB accession no. 4NCR) (yellow carbons) was used as the template. Residues important for the interactions are shown as sticks. FAD, flavin adenine dinucleotide.
PyrBTZs and BTZ043 present similar absorption-distribution-metabolism-excretion/toxicity (ADME/T) profiles.PyrBTZ01 and PyrBTZ02 were tested in parallel with BTZ043 for their cytotoxicity (50% toxic dose [TD50]) against four human cell lines, namely, liver carcinoma HepG2, hepatoma Huh7, lung epithelial A549, and monocytic THP-1 cell lines (Table 3). Both compounds were less cytotoxic than BTZ043, with PyrBTZ02 being the least cytotoxic. BTZ043 presented the highest selectivity index (SI) due to its extremely low MIC, followed by PyrBTZ02 and PyrBTZ01 (Table 3). As reported previously for BTZ043 and PBTZ169 (1, 3), neither PyrBTZ01 nor PyrBTZ02 was mutagenic when tested in the SOS chromotest (24).
Cytotoxicity (TD50) and selectivity index values
Next, we evaluated, using both mouse and human liver microsomes, the in vitro metabolic stabilities (intrinsic clearance [CLint]) of PyrBTZ01 and PyrBTZ02 in parallel with BTZ043, PBTZ169, and carbamazepine (a control compound for low CLint). PyrBTZ01 and PyrBTZ02 presented intermediate CLint values in the presence of mouse and human microsomes (Table 4), and these values were similar to those observed for BTZ043 and PBTZ169, thus suggesting reasonable compound exposure in mice.
Metabolic stability of PyrBTZ01 and PyrBTZ02 in the presence of mouse and human microsomes
To confirm this, we performed an in vivo pharmacokinetic (PK) study of PyrBTZ01 in BALB/c mice, following oral administration of a single 50-mg/kg dose. Comparison of the PK profiles of PyrBTZ01 and its analogue BTZ043 at a dose of 25 mg/kg (Fig. 4) showed that PyrBTZ01 and BTZ043 had similar half-lives of ∼100 min. The higher maximum concentration (Cmax) observed for PyrBTZ01 (approximately 3,500 ng/ml) versus BTZ043 (1,923 ng/ml) is most likely due to the higher dose used for the former compound and not improved absorption.
PyrBTZ01 is not active in a mouse model of acute TB.Given the encouraging ADME/T and PK data obtained for PyrBTZ01, we performed an in vivo efficacy study using a mouse model of acute TB. Mice were infected by intravenous injection of M. tuberculosis H37Rv and then treated with PyrBTZ01 (50 mg/kg) or isoniazid (25 mg/kg) once daily, by gavage, 5 days/week for 4 weeks. At the end of the experiment, CFU counts in the lungs were determined. While isoniazid and BTZ043 were effective, leading to ∼3.2-log-unit decreases in the numbers of CFU over 4 weeks, compared to the untreated control group, PyrBTZ01 did not provide any protection to the treated mice, and their microbiological and pathological responses were the same as those of the untreated animals.
DISCUSSION
The pyrrole-benzothiazinone family of compounds described here (in particular, PyrBTZ01 and PyrBTZ02) provides new insights into the chemical and pharmacological requirements for DprE1 inhibition in mycobacteria. Of note, these compounds are the first non-nitro-benzothiazinones that demonstrate significant mycobacterial activity in vitro. Several noncovalent DprE1 inhibitors have been described recently (12, 18, 20, 21, 25), and these display MIC values close to those of PyrBTZ01 and PyrBTZ02 presented in this work. In particular, TCA1 (18) and the azaindoles (26) were reported to be efficacious in a mouse model of TB, although those compounds are less potent than BTZ043 or PBTZ169 in vitro (1, 3).
The less favorable MIC values of PyrBTZ01 and PyrBTZ02, compared to their nitro-containing counterparts BTZ043 and PBTZ169, are reflected by considerably higher IC50s against purified M. tuberculosis DprE1 in both its wild-type and BTZ-resistant forms (Table 1). Genetic studies showed that PyrBTZ resistance in M. tuberculosis stemmed from mutations of the active site Cys387 residue, as was reported earlier for the nitro-BTZ counterparts. This was unexpected, since resistant mutants of M. tuberculosis generated against several other noncovalent DprE1 inhibitors, including TCA1 and carboxyquinoxalines, displayed mutations elsewhere, namely, at amino acid residues Gly15, Tyr314, and Leu386 (12, 18, 20). However, molecular docking studies show that PyrBTZ inhibitors occupy roughly the same positions as BTZ043 and PBTZ169 in DprE1, with the pyrrole group being close to and likely clashing with Cys387 (Fig. 4). One possible consequence of this would be the trifluoromethyl group failing to enter the pocket below Cys387 as deeply as the corresponding group on BTZ043 or PBTZ169. This pocket is lined by His132, Gly133, Lys134, Lys367, and Phe369, and the trifluoromethyl moiety forms van der Waals interactions with these residues, as well as forming a hydrogen bond with the amide of Asn385 (21, 27). It is conceivable that the trifluoromethyl group on the pyrrole derivatives fails to make these interactions and this, especially the missing covalent semimercaptal bond, leads to PyrBTZ01 and PyrBTZ02 binding much more weakly to their target than do the nitro-benzothiazinones. This explanation is fully consistent with the respective MICs and IC50s and indicates that the trifluoromethyl moiety is important for stabilizing the interactions between BTZ compounds and DprE1.
PyrBTZ01 and PyrBTZ02 are analogs of BTZ043 and PBTZ169, respectively, that differ solely by replacement of the DprE1-reactive nitro group by a pyrrole. Their in vitro ADME profiles display only minor differences (Table 3), and the in vivo PK profiles of PyrBTZ01, BTZ043, and PBTZ169 in mice are essentially identical (1, 3). Thus, the lack of in vivo efficacy of PyrBTZ01 was not due to insufficient exposure, as satisfactory plasma Cmax and area under the curve values were reached (Fig. 5). The use of similar doses of BTZ043 and PBTZ169 resulted in survival of all treated animals and 1- to 3-log-unit reductions in the bacterial burdens in the lungs in acute and chronic models of TB (1, 3), whereas PyrBTZ01 showed no antituberculosis activity under the same conditions. The combined results suggest that successful inhibition of DprE1 during infection in mammals requires prolonged occupancy of the active site, which may be best achieved by formation of a covalent adduct.
PK profiles in plasma following oral administration of PyrBTZ01 (50 mg/kg) or BTZ043 (25 mg/kg) to BALB/c mice. Data for BTZ043 in the plot were reported previously (3).
ACKNOWLEDGMENTS
We thank Stefanie Boy-Röttger for technical assistance, Laure Menin for performing mass spectrometry, and Kai Johnsson for fruitful discussions.
The research leading to these results received funding from the European Community Seventh Framework Programme (grant 260872). João Neres was the recipient of a Marie Curie Fellowship from the European Commission (DPRETB project, grant 252802).
FOOTNOTES
- Received 2 April 2015.
- Returned for modification 28 April 2015.
- Accepted 7 May 2015.
- Accepted manuscript posted online 18 May 2015.
‡ For this virtual institution, see http://www.mm4tb.org.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.00778-15.
- Copyright © 2015, American Society for Microbiology. All Rights Reserved.
