Bedaquiline

CB37, CWHM-728 (CB81), CWHM-935, CWHM-936, CWHM-941, CWHM-950, CWHM-937, CWHM-946, CWHM-951, and CWHM-942 were purchased from ChemBridge Corporation. Synthesis of the compounds CWHM-1069, CWHM-1020, CWHM-1022, CWHM-1021, CWHM-1304, CWHM-1303, CWHM-1306, and CWHM-1023 is described in Text S1 in the supplemental material, and liquid chromatography-mass spectrometry (LC-MS), ¹H nuclear magnetic resonance (NMR), and ¹³C NMR analyses were done on CWHM-1023 to confirm the purity and identity of the synthesized compound (Fig. S3 and S4). Q203 was acquired from Enamine (catalog number EN-300-218150), and both bedaquiline (catalog number 465749185) and thioridazine (catalog number 1662504) were purchased from Sigma-Aldrich; all three compounds were tested in the MABA for comparative purposes.

SQ109 was provided by Sequella, Inc. (Rockville, MD). SQ109-d4 was synthesized by the group of Clifton E. Barry, NIH-NIAID (Bethesda, MD). Linezolid, moxifloxacin, and bedaquiline were purchased from Sigma-Aldrich (St. Louis, MO), Chem-Impex (Woodale, IL), and Chem Shuttle (Hayward, CA), respectively. Linezolid-d8 and moxifloxacin-d4 were purchased from Toronto Research Chemicals (North York, ON), and bedaquiline-d8 was purchased from Clear Synth (Mumbai, India). All antibiotics tested in pairwise combinations were purchased from Sigma-Aldrich.

Potassium dihydrogen phosphate and dipotassium hydrogen phosphate were obtained from Merck (Kenilworth, NJ, USA). Liquid chromatography-mass spectrometry (LC-MS)-grade acetonitrile (acetonitrile) was purchased from Anatech (Johannesburg, South Africa). Bedaquiline, high-performance liquid chromatography (HPLC)-grade dimethyl sulfoxide (DMSO), and formic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). The compounds PhX1, PhX2, PhX6, PhX8, PhX10, PhX14, PhX15, and DPINH were prepared as reported previously (30 (link), 106 (link)– (link)110 (link)). Compounds were synthesized according to the methodologies reported by Crossley et al. unless otherwise stated (106 (link), 111 (link)). RMB041 and WHN296 were synthesized according to previously reported methodologies (29 (link), 112 (link)). Water was purified using a Milli-Q purification system (Millipore, Bedford, MA, USA).

In-vitro evolution experiments were done with the H37Rv reference strain (ATCC 27294). Bedaquiline was purchased from Janssen-Cilag (Neuss, Germany) and clofazimine from Sigma (C8895-1G; Darmstadt, Germany) and both were reconstituted from powder in dimethyl sulphoxide and stored at –20°C.
Bacteria were exposed in liquid culture to Bedaquiline or clofazimine concentrations that were lower than the corresponding MICs, over five bacterial passages (appendix 1 p 12). The bacteria were plated on selective agar plates supplemented with the MIC of Bedaquiline or clofazimine. Mutant colonies were selected from these plates for population sequencing and characterisation. Further details are given in appendix 1 (p 2).

The selected compounds, bedaquiline (Sigma), clofazimine (Sigma), and moxifloxacin (Sigma), were first solubilized in 100% dimethyl sulfoxide (DMSO; Sigma) at a concentration of 20 mM and stored at −20°C until further use. To determine the activity of the compounds against the mycobacterial strains, both Mab WT and Mab∆mtr mutants in their logarithmic phase were diluted to an OD₆₀₀ of 0.05 with 7H9 supplemented with 10% ADS, 0.2% glycerol, and 0.5% tyloxapol and inoculated in a 96-well plate. Then, each compound was added in a one-over-three dilution to the 96-well plates containing the bacteria to reach a final concentration starting from 100 µM and a maximal final DMSO concentration of 1%. The 96-well plates were incubated at 37°C for 3 days to allow for exposure to the compounds. After incubation, 0.001% (wt/vol) of resazurin (Sigma) was added to each well after which the plates were incubated again overnight at 37°C. Finally, the viability of the mycobacterial strains was assessed by measuring the fluorescence signal emitted by each well at an excitation and emission of 550 and 590 nm, respectively, with the use of a Tecan plate reader (Infinite F plex).

Antimicrobial sensitivity assays for M. tuberculosis have been performed as previously described (Goodsmith et al., 2015 (link)). Mycobacteria strains were grown to early log phase and were diluted to an optical density of 0.02 in 7H9 media containing 0.05% Tween 80 and 10% OADC. Then the bacterial strains were exposed to two-fold dilutions of the following antimicrobials; rifampicin, vancomycin, bedaquiline, streptomycin, ofloxacin, isoniazid, Capreomycin, amikacin, and ethambutol (Sigma-Aldrich, United States). The minimum inhibitory concentrations (MICs) were recorded as the minimum concentrations at which the growth was inhibited by at least 90%, as compared to a control containing no antibiotic.
For M. smegmatis, the MICs at which no bacterial growth was observed were recorded after 3–5 days of incubation. For M. tuberculosis, MICs at which no bacterial growth was observed were recorded after approximately 2 weeks of incubation.

Ceftibuten, cefixime, ceftazidime (hydrate), ceftriaxone (sodium), ethionamide, gentamycin, kanamycin (sulfate), levofloxacin, meropenem (trihydrate), penicillin (G sodium), rifampicin (all purchased from Sigma-Aldrich), bedaquiline, delamanid, linezolid, macozinone, pretonamid, streptomycin (sulfate salt), sutezolid, SQ109, 9-chloro-3,4-dihydro-chromeno[2′,3′:4,5]pyrimido[2,1-b][1,3]thiazine-6,7-dione (TBA161) and its derivatives 3,4-dihydro-chromeno[2′,3′:4,5]pyrimido[2,1-b][1,3]thiazine-6,7-dione (TBA161-A), 9-bromo-3,4-dihydro-chromeno[2′,3′:4,5]pyrimido[2,1-b][1,3]thiazine-6,7-dione (TBA161-B), 9,11-dichloro-3,4-dihydro-chromeno[2′,3′:4,5]pyrimido[2,1-b][1,3]thiazine-6,7-dione (TBA161-C) and 7-chloro-3-isopropyl-2-(methylsulfanyl)-chromeno[2,3-d]pyrimidine-4,5-dione (TBA161-D) were all purchased from MedChemExpress, and solubilized and stored according to the manufacturers' recommendations.
The TB Alliance compound library was a gift from TBAlliance (New York, USA). The library consists of 1392 compounds that were previously shown to inhibit Mtb H37Rv viability in vitro. Compounds of this library were stored as stock solutions (10 mM) in DMSO at −80°C.