Mycobacterium tuberculosis H37Rv( MIC50=2.7 nM )

Telacebec (Q203; IAP6) is an imidazopyridine amide (IAP) compound that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. It has the potential for the treatment of tuberculosis. Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of Q203. Q203 is active against the reference strain Mycobacterium tuberculosis H37Rv with MIC50s of 2.7 nM in culture broth medium and 0.28 nM inside macrophages. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, these data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis.

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Telacebec (Q203; IAP6) was active against the reference strain M. tuberculosis H37Rv at a minimum concentration required to inhibit the growth of 50% of organisms (MIC50) of 2.7 nM in culture broth medium and at a MIC50 of 0.28 nM inside macrophages (Fig. 1b,c). The MIC50 in culture broth medium was determined by four different techniques (see Online Methods) in three centers, resulting in comparable values. Furthermore, we confirmed the activity of Q203 in liquid broth medium by CFU determination on agar plates [1].

To gain insight into the mode of action of Telacebec (Q203; IAP6) and identify its molecular target, we selected spontaneous-resistant mutants to two different IPA derivatives, IPA04 and IPA05 (Supplementary Fig. 5). After confirmation of stable genotypic resistance to Q203 (Fig. 3a), we subjected six spontaneous-resistant mutants from independent biological replicates to whole-genome sequencing. The mutants showed a consistent increase in MIC50 for Q203 of several orders of magnitude but remained susceptible to standard antituberculars. We identified a single amino acid substitution in the cytochrome b subunit (qcrB, also known as Rv2196 of the cytochrome bc1 complex in all six mutants (Fig. 3b). Sequence analysis of qcrB in an additional 18 (of 18 tested) independent, spontaneous-resistant mutants confirmed that mutation of Thr313 to either alanine or isoleucine (Fig. 3b) was associated with resistance to Telacebec (Q203; IAP6). Furthermore, the re-introduction of mutation Ala313 by homologous recombination in parental M. tuberculosis H37Rv conferred resistance to Q203 (Fig. 3a), demonstrating that this substitution is directly and specifically involved in the mechanism of resistance to the compound. Combined analysis of the six independent mutant-selection experiments at a concentration of 1 μM determined the spontaneous rate of mutation to IPA04 and IPA05 to be 2.4 × 10−8, which indicates a low probability of emergence of resistant mutants. Spontaneous-resistant mutants selected directly on Q203 were also highly resistant to Q203 (Supplementary Fig. 6) and harbored a polymorphism T313A in qcrB, whereas we identified no mutation in qcrB in two pan-susceptible and three XDR clinical isolates (Supplementary Fig. 7). A similar polymorphism in qcrB was recently identified in Mycobacterium bovis bacillus Calmette-Guérin (BCG) mutants selected on a related IPA derivative18 that was less active than Q203 in inhibiting the growth of M. tuberculosis in vitro and not optimized for clinical use [1].

Notably, several residues that have key roles in the binding of QP site inhibitors (for example, stigmatellin) or that are implicated in resistance to such inhibitors, or both, are located at the ef region (Supplementary Fig. 8), suggesting a mode of action for Telacebec (Q203; IAP6) similar to nonselective inhibitors acting at the QP site. Given the key role of cytochrome bc1 in the respiratory electron transport chain, we tested whether Telacebec (Q203; IAP6) may interfere with ATP synthesis in M. tuberculosis. We found that Q203 triggered a rapid reduction in intracellular ATP at an IC50 of 1.1 nM (Fig. 3c). Under similar experimental conditions (see Online Methods), moxifloxacin or streptomycin did not reduce the ATP pool size, whereas bedaquiline did (IC50 of 27.7 nM). Finally, Q203 was able to interfere with ATP homeostasis in hypoxic nonreplicating tuberculosis at an IC50 less than 10 nM (Fig. 3d). The rapid inhibition of ATP synthesis at low concentration strongly suggests that the inhibition of cytochrome bc1 activity is the primary mode of action of Q203 [1].

Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Q203 has a bioavailability of 90% and a terminal half-life of 23.4 h. The volume of distribution is moderate (5.27 l per kg body weight), and the systemic clearance is low (4.03 mL/min per kg). After 4 weeks of treatment, reductions of 90%, 99% and 99.9% in M. tuberculosis H37Rv bacterial load is observed in the groups treated with Q203 at 0.4, 2 and 10 mg per kg body weight, respectively

Q203 is active against the reference strain Mycobacterium tuberculosis H37Rv with MIC50s of 2.7 nM in culture broth medium and 0.28 nM inside macrophages.

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Microsomal stability assay. [1]
Compounds (2 μM final in 0.2% DMSO) were incubated with 0.5 mg mL−1 human (pool of 200, mixed gender), male dog, male rat or male mouse liver microsomes in potassium phosphate buffer. The reaction was initiated by the addition of NADPH and stopped either immediately or at 10, 20, 30 or 60 min for a precise estimate of clearance. A triple quadrupole Quattro Premier mass spectrometer with electrospray ionization (ESI) was employed for sample analysis. Samples were passed through trapping cartridges (Acquity BEH RP18 50 mm × 2.1 mm, 1.7 μm, Waters, Milford, MA) followed by an analytical column. The percentage of remaining compound was calculated by comparing with the initial quantity at 0 min. Half-life was then calculated using first-order reaction kinetics.

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CYP450 inhibition assays. [1]
The assay used individual fluorescent probe substrates with individual recombinant human cytochrome P450 (rhCYP) isozymes and a fluorescent detection according to previously published methods36. The probe substrates (in 0.5% DMSO) used for each isozyme were as follows: 7-benzyloxy-4-(trifluoromethyl)-coumarin for CYP3A4, 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) for CYP2D6, 3-cyano-7-ethoxycoum (CEC) for 1A2 and 2C19 and 7-methoxy-4-(trifluoromethyl)-coumarin (MFC) for 2C9. Fluorescence was measured using Victor3 V multilabel plate reader. The IC50 was determined using an eight-point concentration curve with threefold serial dilution.

High-content screening assay in infected macrophages.[1]
The assay was performed as previously described9,30. Briefly, Raw 264.7 cells were infected with M. tuberculosis H37Rv-GFP at a multiplicity of infection of 2:1 and dispensed into 384-well plates. After 5 d of infection, macrophages were stained with Syto 60. Image acquisition was performed on an EVOscreen Mark III platform integrated with Opera. Bacterial load and macrophage number were quantified using proprietary image analysis software.

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Minimum inhibitory concentration determination.[1]
H37Rv-GFP was dispensed into 384-well plates in 7H9 medium without glycerol. Mycobacterial growth was determined by measuring fluorescence intensity at 488 nm after 5 d of incubation. Alternatively, the MICs were determined using the resazurin susceptibility assay or a turbidity-based assay in 384-well plates. The MIC50 were determined using an eight- or ten-point concentration curve with threefold serial dilution.

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Cytotoxicity.[1]
Cytotoxicity was tested against the human cell lines SH-SY5Y (brain), HEK293 (kidney) and HepG2 (liver) using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assay as previously described.

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ATP depletion assay on M. tuberculosis H37Rv.[1]
M. tuberculosis H37Rv was exposed to the test compounds for 24 h (aerobiosis) or 5 d (anaerobiosis), mixed with an equal volume of BacTiter-Glo reagent and incubated in the dark for 10 min. Luminescence was recorded on a Victor3 V multilabel plate reader.

Pharmacokinetics.[1]
BALB/c mice and Sprague Dawley rats were used for pharmacokinetic studies. Compounds were given at a dose of 2 mg per kg body weight intravenously or 10 mg per kg body weight orally. Otherwise stated, the compounds [Telacebec (Q203; IAP6)] were formulated in 20% TPGS (D-α tocopheryl polyethylene glycol 1000 succinate) for repeated-dose studies and in 40% PEG400, pH4 for single-dose studies. Blood samples were taken through the caudal vena cava using 1-mL syringes before perfusion. Samples were collected from three mice or rats at 0.5, 1, 2, 6, 12, 24 and 48 h post-dose. Blood samples were centrifuged at 3,200g for 10 min at 4 °C. Following centrifugation, plasma was collected and frozen until further analysis. Compound concentrations were determined by LC-MS.

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In vivo efficacy in the mouse model of tuberculosis.[1]
The acute model was performed as previously described. Briefly, mice were infected with a high dose of M. tuberculosis H37Rv. Dosing was initiated 6 d after infection. Drugs [Telacebec (Q203; IAP6)] were administered orally for 3 d. Bacterial load in the lungs of infected mice was determined by colony-forming unit (CFU) enumeration. For the established mouse model, BALB/c mice were infected with 2 × 102 to 2 × 103 CFU of M. tuberculosis H37Rv by the intranasal route. Treatment was initiated 3 weeks after infection. Drugs were formulated in 20% TPGS and administered by oral gavage for 28 d, five times per week. Bacterial load in the lungs of infected mice was determined by CFU enumeration. For histopathology analysis, segments of the lungs were fixed with 10% neutral formalin, embedded in paraffin and processed for histology. Sections (5 μm) were stained with H&E. Histologic sections were used for morphologic analysis of the size and number of granulomas using an image analyzer.

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Rats: Sprague Dawley rats are used for pharmacokinetic studies. Compounds (Q203) are given at a dose of 2 mg per kg body weight intravenously or 10 mg per kg body weight orally. The compounds (Q203) are formulated in 20% TPGS (d-α tocopheryl polyethylene glycol 1000 succinate) for repeated-dose studies and in 40% PEG400, pH4 for single-dose studies. Blood samples are taken through the caudal vena cava using 1-mL syringes before perfusion. Samples are collected from three mice or rats at 0.5, 1, 2, 6, 12, 24 and 48 h post-dose. Blood samples are centrifuged at 3,200g for 10 min at 4 °C. Following centrifugation, plasma is collected and frozen until further analysis. Compound concentrations are determined by LC-MS; Mice: Efficacy of Q203 in a mouse model of established tuberculosis is studied. Bacterial loads are enumerated in the lung of infected mice after 14 d and 28 d of treatment. Q203 is used at 0.4, 2 and 10 mg per kg body weight. Bedaquiline and isoniazid (INH) are used as positive controls at 6.5 and 15 mg per kg body weight, respectively. Five mice per group and per time point are used.

Sprague Dawley rats

The metabolic stability of Telacebec (Q203; IAP6) in microsomes and cryopreserved hepatocytes from human, monkey, rat and dog origin was high (Supplementary Table 5), suggesting that Telacebec (Q203; IAP6) may achieve good blood exposure in humans. Because any new antitubercular drug will be given clinically in combination with other medications, the absence of drug-drug interactions is crucial. Telacebec (Q203; IAP6) did not inhibit any of the cytochrome P450 (CYP450) isoenzymes tested, nor did it induce human pregnane X receptor (hPXR) activation (Supplementary Table 5). In addition, it was not a substrate or an inhibitor for the efflux transporter P-glycoprotein (Supplementary Table 5), indicating that it has low potential for drug-drug interaction.[1]

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Next, we determined the pharmacokinetic profile of Telacebec (Q203; IAP6) in mice (Supplementary Table 6). Telacebec (Q203; IAP6) had a bioavailability of 90% and a terminal half-life of 23.4 h. The volume of distribution was moderate (5.27 l per kg body weight), and the systemic clearance was low (4.03 ml min−1 kg−1). The drug concentration in lungs was two- to threefold higher than in the serum (Supplementary Table 7), which is a desirable property for an antitubercular drug15. Given its desirable pharmacokinetic and safety profile, we assessed Telacebec (Q203; IAP6) for in vivo efficacy. We initially evaluated Telacebec (Q203; IAP6) in an acute mouse model of tuberculosis16. It promoted a reduction in bacterial load of more than 90% at a dose of 10 mg per kg body weight, an effect comparable to that of bedaquiline or isoniazid (Fig. 2a). We further evaluated Telacebec (Q203; IAP6) in a mouse model of established tuberculosis. After 4 weeks of treatment, we observed reductions of 90%, 99% and 99.9% in M. tuberculosis H37Rv bacterial load in the groups treated with v at 0.4, 2 and 10 mg per kg body weight, respectively (Fig. 2b). Telacebec (Q203; IAP6) was slow acting compared to isoniazid; the reduction in bacterial number was less than one order of magnitude in the first 2 weeks of treatment, but it was more than two orders of magnitude in the following 2 weeks. This profile might be explained by its pharmacokinetic properties or by its mode of action. Of note, bedaquiline displayed a similar time-dependent efficacy (Fig. 2b). We also observed that Telacebec (Q203; IAP6) reduced the formation of lung granulomatous lesions (Fig. 2 c–i). In untreated mice, the lung sections contained multiple tuberculosis granulomatous foci (Fig. 2c), consisting predominantly of lymphocytes surrounding intra-alveolar macrophages (Fig. 2f). In isoniazid-treated groups, we observed a reduction in the size of the granulomatous foci; however, the number of the inflammatory lesions was comparable to that in the untreated control group (Fig. 2d,g,i). In contrast, we observed only a limited number of small granulomatous foci in the lungs of the mice treated with Telacebec (Q203; IAP6) (Fig. 2e–i). Notably, other very effective drugs, such as bedaquiline, also have a strong beneficial effect on lung pathology [1].

Given that successful treatment of tuberculosis lasts at least six months, the safety profile of a clinical candidate is of utmost importance. To evaluate the cytotoxicity of Telacebec (Q203; IAP6), we measured the minimum concentration that induces cell death in three eukaryotic cell lines. We did not observe cytotoxicity in any of the cell lines up to a concentration of 10 μM (Fig. 1d), which gives Telacebec (Q203; IAP6) a selectivity index of >3,700. We used a hERG potassium channel patch-clamp assay to test whether Q203 would cause QT interval prolongation as a result of hERG potassium channel inhibition. Q203 did not inhibit hERG, suggesting a low risk for cardiotoxicity (Supplementary Table 5). In addition, Q203 had no genetic toxicity in a mini-Ames mutagenicity test and in micronucleus formation assays (Supplementary Table 5). To test for acute toxicity in mice, we administered a high dose of Q203 and observed the mice for 2 weeks. The mice tolerated, without clinical signs of toxicity, a single oral administration of 1,000 mg per kg body weight of Q203, a dose that resulted in a maximum serum concentration of 14.8 μg ml−1 at 24 h and serum concentrations of >3 μg ml−1 for at least 10 d (Supplementary Fig. 3). Furthermore, in a rat long-term administration study, Q203 was well tolerated without body weight loss (Supplementary Fig. 4) or clinical signs of toxicity when administered daily at a dose of 10 mg per kg body weight for 20 d. These data showed that Q203 was well tolerated at a prolonged exposure level.[1]

[1]. Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis. Nat Med. 2013 Sep;19(9):1157-60.

New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious public health challenge worldwide. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tuberculosis therapy with a success rate comparable to that of current therapies for drug-susceptible tuberculosis. The last decade has seen the discovery of new agent classes for the management of tuberculosis, several of which are currently in clinical trials. However, given the high attrition rate of drug candidates during clinical development and the emergence of drug resistance, the discovery of additional clinical candidates is clearly needed. Here, we report on a promising class of imidazopyridine amide (IPA) compounds that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. The optimized IPA compound Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of this compound. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, our data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis.[1]

C29H28CLF3N4O2

557.01

556.185

C, 62.53; H, 5.07; Cl, 6.36; F, 10.23; N, 10.06; O, 5.74

1334719-95-7

1334719-95-7;1566517-83-6 (Ditosylate);

68234908

White to off-white solid powder

1.3±0.1 g/cm3

1.615

7.32

1

7

7

39

796

0

ClC1C([H])=C([H])C2=NC(C([H])([H])C([H])([H])[H])=C(C(N([H])C([H])([H])C3C([H])=C([H])C(=C([H])C=3[H])N3C([H])([H])C([H])([H])C([H])(C4C([H])=C([H])C(=C([H])C=4[H])OC(F)(F)F)C([H])([H])C3([H])[H])=O)N2C=1[H]

OJICYBSWSZGRFB-UHFFFAOYSA-N

InChI=1S/C29H28ClF3N4O2/c1-2-25-27(37-18-22(30)7-12-26(37)35-25)28(38)34-17-19-3-8-23(9-4-19)36-15-13-21(14-16-36)20-5-10-24(11-6-20)39-29(31,32)33/h3-12,18,21H,2,13-17H2,1H3,(H,34,38)

6-chloro-2-ethyl-N-[[4-[4-[4-(trifluoromethoxy)phenyl]piperidin-1-yl]phenyl]methyl]imidazo[1,2-a]pyridine-3-carboxamide

Q-203 free base; Q 203; Telacebec; 1334719-95-7; Q203; Q-203; 6-chloro-2-ethyl-n-(4-(4-(4-(trifluoromethoxy)phenyl)piperidin-1-yl)benzyl)imidazo[1,2-a]pyridine-3-carboxamide; MMV687696; 55G92WGH3X; CHEMBL3298910;

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: ~20 mg/mL ( 35.9 mM)
Water: ~20 mg/mL

Solubility in Formulation 1: 2 mg/mL (3.59 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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