Decaprenyl-phosphoribose-epimerase (DprE1)

PBTZ169 (also known as Macozinone, an 8-Nitro-benzothiazinones (BTZs) analog) is a novel inhibitor of decaprenyl-phosphoribose-epimerase (DprE1) that displays nanomolar bactericidal activity against Mycobacterium tuberculosis in vitro. DprE1 is an essential enzyme involved in the cell wall biosynthesis of Corynebacterineae. 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. When tested against thirty Nocardia brasiliensis isolates, the MIC50 and MIC90 values for PBTZ169 were 0.0075 and 0.03 μg/mL, respectively. Because Nocardia is a potential intracellular bacterium, a THP-1 macrophage monolayer was infected with N. brasiliensis HUJEG-1 and then treated with PBTZ169, resulting in a decrease in the number of colony-forming units (CFUs) at a concentration of 0.25X the in vitro value. The in vivo activity was evaluated after infecting female BALB/c mice in the right hind food-pad. After 6 weeks, treatment was initiated with PBTZ169 and its activity was compared with the first generation compound, BTZ043. Both BTZ compounds were administered at 100 mg/kg twice daily by gavage, and sulfamethoxazole/trimethoprim (SXT), at 100 mg/kg sulfamethoxazole, was used as a positive control. After 22 weeks of therapy, only PBTZ169 and SXT displayed statistically significant activity.

The in vivo activity was evaluated after infecting female BALB/c mice in the right hind food-pad. After 6 weeks, treatment was initiated with PBTZ169 and its activity was compared with the first generation compound, BTZ043. Both BTZ compounds were administered at 100 mg/kg twice daily by gavage, and sulfamethoxazole/trimethoprim (SXT), at 100 mg/kg sulfamethoxazole, was used as a positive control. After 22 weeks of therapy, only PBTZ169 and SXT displayed statistically significant activity. PBTZ169 can be suspend in 0.25% hydroxy-propylmethyl-cellulose. The administertration for PBTZ169 is 100 mg/kg by gavage. The MIC50 and MIC90 values were 0.0075 and 0.030 μg/mL, respectively. The MIC for PBTZ169 for N. brasiliensis HUJEG-1 was 0.0037 μg/mL.

---

---

Effect of BTZs in the mycetoma animal model [1]
When PBTZ169 and BTZ043 were administered at 100 mg/kg twice daily by gavage (Fig 4), only the former showed a statistically significant effect compared to the saline control (P = 0.017). No significant difference was detected in the BTZ043-treated group (P = 0.667). The mouse group treated with SXT showed a statistically significant difference compared to the control group (P = 0.007).

---

Benzothiazinones are highly potent drug candidates for the treatment of tuberculosis and other actinobacterial infections. Because of the nanomolar activity of benzothiazinones, we expected excellent in vivo activity. At 100 mg/kg twice daily, we observed a therapeutic effect, but only with PBTZ169. In M. tuberculosis, a microorganism with a thicker and more hydrophobic cell-wall than N. brasiliensis, BTZ043 at 50 mg/kg once daily resulted in a significant decrease in the lung and spleen bacterial burden. PBTZ169 is a more effective drug, and in the mouse model of infection, it significantly decreases the amount bacilli at 25 mg/kg once daily compared with BTZ043 [1].

---

Efficacy of PBTZ169 against M. marinum in zebrafish [3]
To establish whether BTZ derivatives have the potential to cure other mycobacterial infections we tested their efficacy against M. marinum using the zebrafish embryo model as this has proved to be a powerful tool for assessing the effect of TB drugs (Davis et al, 2002; Adams et al, 2011). This model assesses simultaneously the effect of compounds on host survival, host pathology and bacterial burden. Embryos were infected with M. marinum strains E11 or M, producing the fluorescent protein mCherry, and observed by fluorescence microscopy. Treatment of infected zebrafish embryos with increasing concentrations of PBTZ169 or BTZ043 led to a decrease in the bacterial burden after 5 days, as measured by the amount of fluorescent pixels present in the embryos (Fig 4A). Whereas infection with M. marinum M (or E11 data not shown) resulted in substantial bacterial clustering (Fig 4B, C), almost no bacteria were present when infected zebrafish embryos were treated with 25 or 50 nM PBTZ169 or BTZ043 (Fig 4D, E, F). Treatment of embryos with either compound at 5 nM had no significant effect on the infection (Fig 4A, C).

To confirm the bactericidal effect, zebrafish embryos were infected with M. marinum strains M and E11, and then exposed to 25 nM PBTZ169 for 5, 4 or 3 days with the drug added at 0, 1 or 2 days post-infection, respectively. The number of colony forming units (CFU) per embryo was determined and compared to the level of fluorescence. A decrease of about 3 and 2 log units was observed in the number of CFU for the M. marinum strains M and E11, respectively (Fig 5B, D) independently of the duration of treatment. Decreasing bacterial viability was mirrored by a sharp decrease in fluorescence although more scatter was seen at later time-points (Fig 5A, C).

On examination of infected zebrafish embryos treated with BTZ043 it was observed that the compound affected embryo development (Fig 6A–G) especially at concentrations above 25 nM. Administration of BTZ043 to the embryos 1 day after fertilization resulted in defects in notochord development and a slightly shortened Anterior-Posterior axis (Fig 6C, D) with 60.4% (n = 29 out of 48) of embryos affected after treatment with 25 nM BTZ043 and 76.7% (n = 23 out of 30) after treatment with 50 nM BTZ043 (Fig 6G). These defects were also observed when uninfected embryos were exposed to BTZ043. However, no developmental defects were seen after treatment with PBTZ169 at the same concentrations (compare Fig 4E, F) or even at 10 μM.

---

Comparative efficacy of PBTZ derivatives in vivo [3]
The in vivo efficacy of PBTZ169 and four other candidates was assessed in the murine model of chronic TB after low-dose aerosol infection of BALB/c mice and treatment at 50 mg/kg, the recommended dose for BTZ043. Compared to the untreated control group, the bacterial burden in the lungs and spleens of BTZ043-treated mice was 0.6 and 1.7 logs lower, respectively (Fig 7A). All five PBTZs were active in both organs and not inferior to BTZ043. Strikingly, PBTZ169 and PBTZ134 reduced the bacterial burden in the spleens 10-fold more than BTZ043 did. Furthermore, PBTZ169 had significantly greater bactericidal activity in the lungs reducing the number of CFU by >0.5 log in comparison to BTZ043 at the same dose (Fig 7A). This activity was also equivalent to that of INH, suggesting that PBTZ169 was the most potent of all the BTZs in vivo.

---

Combination studies in the mouse model of chronic TB [3]
If PBTZ169 is to be used in a new regimen for TB treatment in humans it is important to demonstrate the efficacy of appropriate drug combinations in animal models. Consequently, we assessed the combination found to be synergistic in vitro in the murine model of chronic TB after low-dose aerosol infection. PBTZ169 was tested alone (at 25 mg/kg), in combination with BDQ (25 mg/kg) and pyrazinamide (PZA; 150 mg/kg), and with both drugs together, against M. tuberculosis H37Rv. The reduction in the bacterial burden in the lungs and spleens was measured after 4 and 8 weeks of treatment and compared to that obtained with the standard three drug therapy comprising INH, RIF and PZA at concentrations of 25, 10 and 150 mg/kg, respectively. As can be seen in Fig 9, the combination of PBTZ169 and BDQ was more effective than the standard treatment in reducing the number of CFU in both organs after 1 month of treatment (P values = 0.004 for the lung, 0.002 for the spleen) whereas the addition of PZA did not further improve the potency of the combination at this stage (P = 0.003 for the spleen). The number of bacteria remaining in the lungs of mice treated with the experimental combination was below the limit of detection used at this time-point (<200 CFU). After 2 months of treatment (Fig 9), only the triple combination PBTZ, BDQ and PZA was significantly better than RHZ both in the lungs (P = 0.046) and in the spleen (P = 0.015; Supplementary Table 6). The efficacy of the combination of PBTZ, BDQ and PZA was thus superior to the standard triple therapy INH, RIF and PZA in the chronic model of TB.

MIC determination for PBTZ169 [1] We used the broth microdilution method based on the CLSI M24-A document that we previously described. As external controls, we used Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213. Because of the high susceptibility of Nocardiae, the concentrations ranged from 0.125 μg/mL to 0.0002 μg/mL.

---

PBTZ169, inhibit decaprenylphosphoryl-β-d-ribose 2′-oxidase (DprE1) and display nanomolar bactericidal activity against Mycobacterium tuberculosis in vitro.

---

MIC determination for BTZ169 [1]
We used the broth microdilution method based on the CLSI M24-A document that we previously described. As external controls, we used Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213. Because of the high susceptibility of Nocardiae, the concentrations ranged from 0.125 μg/mL to 0.0002 μg/mL.

---

Drug susceptibility testing. [2]
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. [2]
The C387G and C387S mutant DprE1 proteins were generated using the pET28a-M. tuberculosis DprE1 plasmid 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. 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 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.

---

Metabolic stability in vitro. [2]
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 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.

---

Biochemistry and structural biology [3]
NfnB assays were performed at 25°C, as outlined previously (Manina et al, 2010). Briefly, compounds were added to a reaction mixture containing NfnB (6 μM), NADH (150 μM), 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 5% glycerol. Full details of the purification and crystallization of M. tuberculosis DprE1 are given in the supporting information. DprE1 inhibition was assessed following incubation with BTZ043 or PBTZ169 (0–20 μM) for 5 min, using a peroxidase-coupled assay with Amplex Red as a substrate. The enzyme (5 μM) was incubated at 30°C with inhibitor and 200 μM FPR, in 50 mM glycylglycine pH 8.4, 100 mM NaCl. An aliquot (5 μL) was taken after 5 min incubation and diluted assay mixture (final volume 50 μL) to give final concentrations of 400 μM FPR, 0.2 μM horseradish peroxidase and 50 μM Amplex Red and 0.5 μM DprE1. The peroxidase activity was then assessed by continuous measurement of the fluorescence with excitation/emission wavelengths of 560/590 nm, respectively. Analysis of DprE1-PBTZ169 complexes by mass spectrometry was performed as reported previously, now using M. tuberculosis DprE1 (Neres et al, 2012).

When tested against thirty Nocardia brasiliensis isolates, the MIC50 and MIC90 values for PBTZ169 were 0.0075 and 0.03 μg/mL, respectively. Because Nocardia is a potential intracellular bacterium, a THP-1 macrophage monolayer was infected with N. brasiliensis HUJEG-1 and then treated with PBTZ169, resulting in a decrease in the number of colony-forming units (CFUs) at a concentration of 0.25X the in vitro value.

---

Preparation of a unicellular Nocardia suspension [1]
Because N. brasiliensis grows as filaments, a unicellular suspension was prepared as published previously. N. brasiliensis HUJEG-1 was cultured on Sabouraud agar for 1 week and then sub-cultured in brain heart infusion at 37°C in a shaker at 110 rpm for 72 hrs. The bacterial mass was then separated by centrifugation and washed four times with saline. After grinding in an Evelham-Potter device, the suspension was centrifuged twice at 100 ×g; the supernatant was the unicellular suspension. The bacterial concentration was determined by plating on BHI agar with 5% sheep blood, and the suspension was stored in 20% glycerol at -70°C until use.

---

THP-1 macrophage assays [1]
The human monocyte cell line THP-1 was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS; Gibco-BRL) and 1 mM sodium pyruvate. To transform the cells into macrophages, the cells were sub-cultured four times without sodium pyruvate. The cell density was then determined in a hemocytometer, and the cell suspension was diluted as required in complete RPMI 1640 supplemented with 10% FCS and 6.25 ng/mL phorbol-12-myristate 13-acetate to obtain a density of 4 x 105 cells/mL. A 1 mL aliquot of the cell suspension was seeded into each well of a 24-well microplate, and the cell cultures were washed twice with RPMI 1640 every 48 h for no longer than 4 days.

---

Determination of the intracellular activity of BTZ [1]
The technique used has been published previously. Briefly, a 3:1 multiplicity of infection (MOI) was used to determine the effect of antimicrobials on Nocardia intracellular growth. Two hours after infecting the monolayer, the medium was discarded and the monolayer was washed twice with warm PBS, pH 7.4. PBTZ was added at 0.25X, 1X, 4X and 16X the MIC in RPMI 1640 with 10% FCS and incubated for 6 h at 37°C in 5% CO2. We cannot use rifampin as an intracellular active control because N. brasiliensis is a naturally resistant bacteria. Instead, we used DA-7218, an oxazolidinone drug that previously demonstrated good intracellular and in vivo activity against N. brasiliensis. The culture medium was discarded, and 1 mL of cold distilled water was added and incubated for 15 min. To release the intracellular bacteria, the monolayer was disrupted by pipetting up and down several times, and the suspension was collected in 1.5 mL Eppendorf tubes. Nocardia growth was quantified on BHI agar.

---

Decaprenylphosphoryl-β-d-ribose epimerization by M. tuberculosis H37Ra cells. [2]
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), as described elsewhere. 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. 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 in CHCl3/CH3OH/NH4OH/1 M ammonium acetate/H2O (180:140:9:9:23 [vol/vol]) and visualized by autoradiography (Biomax MR-1 film). The intensity of the bands was quantified using ImageJ software (NIH).

---

Cytotoxicity studies. [2]
The cytotoxicity of the compounds was measured as described previously, 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).

Drugs [1]
BTZ043 and PBTZ169 were suspended in 0.25% hydroxy-propylmethyl-cellulose.

---

Plasma quantitation of BTZs [1]
To quantitate the plasma levels in mice, we administered the compounds to 8–12-week-old female BALB/c mice by gavage using BTZ043, PBTZ169 or SXT, all at 100 mg/kg. Blood samples from the periorbitary plexus were collected at 0, 20, 40, 60, 120, 240, 360, 480, and 600 min. The concentrations of BTZ043, PBTZ169 and SXT were analyzed using a high-pressure liquid chromatography method developed in our laboratory.

---

Efficacy in mice [1]
Eight- to twelve-week-old female BALB/c mice were infected with N. brasiliensis HUJEG-1. Experimental mycetoma was produced by injecting 20 mg (wet weight) of a N. brasiliensis suspension into the left hind footpad, as previously described. Four weeks later, therapy was initiated. Groups of 15 animals were tested. One group of animals received saline solution by gavage as a negative control. The remainder were treated with PBTZ169, BTZ043, or SXT at 100 mg/kg administered twice daily by gavage for 10 weeks. The latter was used a positive control of experimental therapy. After 2 weeks of rest, the compounds were administered for a final period of 6 weeks. The effect of the drugs on the development of mycetomatous lesions was assessed by a blind reader using a previously published scale. Potential differences among the groups against a control inoculated with saline solution were established using a variance test analysis.

---

PBTZ derivatives were suspended in 0.5% carboxymethyl cellulose for the comparative efficacy studies. PBTZ169 and BDQ were suspended in 20% hydroxypropyl-β-cyclodextrin (pH = 3.0) for in vivo combination studies. Solutions of compounds for administration to mice were prepared weekly and stored at 4°C for all compounds but BDQ which was prepared monthly and stored at 4°C. PZA, INH, and RIF were suspended in water.[3]

Plasma levels of drugs [1]
BTZ043 plasma levels in mouse were previously published. At a dose of 100 mg/kg, it reaches a concentration of 4.06 μg/mL (Tmax of 40 min); which is quite similar to the levels in plasma observed in our case (Fig 2). In the case of PBTZ169, it presented a Cmax of 1.74 micrograms/mL, with a Tmax of 40 min (Fig 3). In Fig 3, we also present the plasma concentrations of SXT at 100 mg/kg, presenting a maximum concentration of 553.88 μg /mL 40 min after drug administration. The t ½ was 1.66 h, and the AUC was 1507.69 mg/L*h.

---

PyrBTZs and BTZ043 present similar absorption-distribution-metabolism-excretion/toxicity (ADME/T) profiles. [2]
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, neither PyrBTZ01 nor PyrBTZ02 was mutagenic when tested in the SOS chromotest.

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.

---

Dose escalation study and comparative pharmacokinetics [3]
PBTZ169 was selected for further investigation and its efficacy in vivo compared to that of BTZ043 in a dose escalation study using the chronic model of TB. PBTZ169 was administered at 5, 10, 25, 50 and 100 mg/kg, whereas BTZ043 was given at 50 mg/kg. Four weeks of treatment with BTZ043 reduced the bacterial burden in the lungs and spleens by 1 log (Fig 7B). PBTZ169 was active at all the concentrations tested and had significantly greater bactericidal activity than BTZ043 at the same dose. Lowering the dose of PBTZ169 decreased the activity, but there was no significant difference in the bacterial burden in the lungs of mice treated with PBTZ169 at 5 mg/kg and BTZ043 at 50 mg/kg. Above 25 mg/kg, PBTZ169 was significantly better than BTZ043 at lowering the number of CFU in the spleen. PBTZ169 at 25 mg/kg displayed comparable bactericidal activity in both the lungs and spleens of mice (Fig 7B) to the frontline drug isoniazid (INH). [1]
To investigate whether differential exposure was responsible for the differences observed between BTZ043 and PBTZ169 efficacy in vivo, compound pharmacokinetics were measured in mice orally dosed with 25 mg/kg of the respective compounds. The better efficacy of PBTZ169 cannot be accounted for by differences in the pharmacokinetics of the two compounds since, except for the faster uptake of PBTZ169 (Supplementary Fig 4), these behaved in a similar manner (Supplementary Table 5).

In vitro ADME/T characterization [3]
The potential cytotoxicity of BTZ043 and PBTZ169 was assessed using the HepG2 human cell line (Supplementary Table 4). PBTZ169 was found to be 10-times less cytotoxic (TD50 of 58 μg/ml) compared to BTZ043 (TD50 of 5 μg/ml). Both compounds thus have excellent selectivity indices of >10 000. On incubation with human or mouse microsomes, both BTZ043 and PBTZ169 showed medium clearance values (Supplementary Table 4).

[1]. PLoS Negl Trop Dis.2015 Oct 16;9(10):e0004022.

[2]. Antimicrob Agents Chemother.2015 Aug;59(8):4446-52.

[3]. EMBO Mol Med. 2014 Mar;6(3):372-83..

Macozinone is under investigation in clinical trial NCT03036163 (Phase 1 Study of PBTZ169).

---

Background: Mycetoma is a neglected, chronic, and deforming infectious disease caused by fungi and actinomycetes. In Mexico, N. brasiliensis is the predominant etiologic agent. Therapeutic alternatives are necessary because the current drug regimens have several disadvantages. Benzothiazinones (BTZ) are a new class of candidate drugs that inhibit decaprenyl-phosphoribose-epimerase (DprE1), an essential enzyme involved in the cell wall biosynthesis of Corynebacterineae.

Methodology/principal findings: In this study, the in vitro activity of the next generation BTZ, PBTZ169, was tested against thirty Nocardia brasiliensis isolates. The MIC50 and MIC90 values for PBTZ169 were 0.0075 and 0.03 μg/mL, respectively. Because Nocardia is a potential intracellular bacterium, a THP-1 macrophage monolayer was infected with N. brasiliensis HUJEG-1 and then treated with PBTZ169, resulting in a decrease in the number of colony-forming units (CFUs) at a concentration of 0.25X the in vitro value. The in vivo activity was evaluated after infecting female BALB/c mice in the right hind food-pad. After 6 weeks, treatment was initiated with PBTZ169 and its activity was compared with the first generation compound, BTZ043. Both BTZ compounds were administered at 100 mg/kg twice daily by gavage, and sulfamethoxazole/trimethoprim (SXT), at 100 mg/kg sulfamethoxazole, was used as a positive control. After 22 weeks of therapy, only PBTZ169 and SXT displayed statistically significant activity.

Conclusion: These results indicate that DprE1 inhibitors may be useful for treating infections of Nocardia and may therefore be active against other actinomycetoma agents. We must test combinations of these compounds with other antimicrobial agents, such as linezolid, tedizolid or SXT, that have good to excellent in vivo activity, as well as new DprE1 inhibitors that can achieve higher plasma levels. [1]

---

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.[2]

---

The benzothiazinone lead compound, BTZ043, kills Mycobacterium tuberculosis by inhibiting the essential flavo-enzyme DprE1, decaprenylphosphoryl-beta-D-ribose 2-epimerase. Here, we synthesized a new series of piperazine-containing benzothiazinones (PBTZ) and show that, like BTZ043, the preclinical candidate PBTZ169 binds covalently to DprE1. The crystal structure of the DprE1-PBTZ169 complex reveals formation of a semimercaptal adduct with Cys387 in the active site and explains the irreversible inactivation of the enzyme. Compared to BTZ043, PBTZ169 has improved potency, safety and efficacy in zebrafish and mouse models of tuberculosis (TB). When combined with other TB drugs, PBTZ169 showed additive activity against M. tuberculosis in vitro except with bedaquiline (BDQ) where synergy was observed. A new regimen comprising PBTZ169, BDQ and pyrazinamide was found to be more efficacious than the standard three drug treatment in a murine model of chronic disease. PBTZ169 is thus an attractive drug candidate to treat TB in humans.[3]

C20H23F3N4O3S

456.48

456.144

C, 52.62; H, 5.08; F, 12.49; N, 12.27; O, 10.51; S, 7.02

1377239-83-2

57331386

Solid powder

1.5±0.1 g/cm3

555.6±60.0 °C at 760 mmHg

289.8±32.9 °C

0.0±1.5 mmHg at 25°C

1.660

3.83

0

8

3

31

715

0

O=C1C2=CC(C(F)(F)F)=CC([N+]([O-])=O)=C2SC(N3CCN(CC4CCCCC4)CC3)=N1

BJDZBXGJNBMCAV-UHFFFAOYSA-N

InChI=1S/C20H23F3N4O3S/c21-20(22,23)14-10-15-17(16(11-14)27(29)30)31-19(24-18(15)28)26-8-6-25(7-9-26)12-13-4-2-1-3-5-13/h10-11,13H,1-9,12H2

2-(4-(cyclohexylmethyl)piperazin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 5~6.4 mg/mL ( 10.95~14.02 mM)
Water: <4 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)