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. 2020 Dec 7;13(1):e13207. doi: 10.15252/emmm.202013207

Figure 4. The combination ND‐011992/Q203 is bactericidal against replicating and non‐replicating M. tuberculosis H37Rv and shows potency in vivo .

Figure 4

  1. Bactericidal activity of ND‐011992 and ND‐011992‐Q203 combination in replicating M. tuberculosis H37Rv. Bedaquiline (BDQ) was used at 500 nM, and Q203 at 100 nM. Inoc.: starting inoculum; DMSO: vehicle control. Bacteria viability was determined by enumerating colony‐forming units (CFU) on nutrient‐agar plates after 15 days of incubation. Dotted line represents 90% reduction in CFU/ml compared with the initial inoculum at time 0. *P < 0.05; **P < 0.01 unpaired Student’s t‐test, one‐tailed; n = 3; comparing Q203 alone vs Q203 + ND‐011992. Exact P‐values (from left to right): 0.0346, 0.0143, 0.00016, 0.000052.
  2. Bactericidal activity of ND‐011992 and ND‐011992‐Q203 combination in nutrient‐starved M. tuberculosis H37Rv. Bedaquiline (BDQ) was used at 500 nM, isoniazid was used at 1 μM, Q203 was used at 100 nM, and ND‐011992 was used at 3, 6, 12, and 30 μM. Bacteria viability was determined by enumerating colony‐forming units (CFU) after 15 days of incubation. Dotted line represents 90% reduction in CFU/mL compared with the initial inoculum at time 0. **P < 0.01 unpaired Student’s t‐test, one‐tailed; n = 3; comparing Q203 alone vs Q203 + ND‐011992. Exact P‐values (from left to right): 0.0098, 0.000027, 0.000067.
  3. Bactericidal activity of ND‐011992 and ND‐011992‐Q203 combination in M. tuberculosis H37Rv under hypoxia. BDQ was used at 500 nM, isoniazid at 1 μM, metronidazole (MTZ) at 200 μM, Q203 at 100 nM, and ND‐011992 at 3, 6, 12, and 30 μM. Bacteria viability was determined by enumerating colony‐forming units (CFU) after 15 days of incubation. Dotted line represents 90% reduction in CFU/mL compared with the initial inoculum at time 0.**P < 0.01 unpaired Student’s t‐test, one‐tailed; n = 3; comparing Q203 alone vs. Q203 + ND‐011992. Exact P‐values (from left to right): 0.0092, 0.00010, 0.00079.
  4. Efficacy of ND‐011992‐Q203 combination treatment in a mouse model of acute tuberculosis. Bacterial load (CFU) was enumerated in the lungs of mice before treatment, and after 5 days of treatment with either the vehicle control (brown squares), Q203 at 5 mg/kg (red squares), ND‐011992 at 25 mg/kg (green triangles), or ND‐011992 at 25 mg/kg with Q203 at 5 mg/kg (orange diamonds). **P = 0.0006 unpaired Student’s t‐test, two‐tailed; n = 4; comparing Q203 alone vs. Q203 + ND‐011992.

Data information: Data are expressed as the mean ± SD of triplicates for each data point. Experiments in (A), (B), and (C) were performed with 3 technical replicates and independently repeated at least once. In vivo experiment in (D) was performed with 4 replicates for all treatment groups, and 3 replicates to elucidate bacterial load before initiation of treatment. The experiment was repeated once.