Proportion of mice with positive culture of lung 3 months after treatment completion (relapse).^*

<p>*Lung CFU count at start of treatment (D0): 7.1 log10 CFU.</p><p>Definition of abbreviations: J = TMC 207, R = Rifampin, M = Moxifloxacin, H = Isoniazid, Z = Pyrazinamide, A = amikacin, Et = ethionamide.</p>a<p>significantly more relapses than RHZ (p<0.05).</p>b<p>not significantly different from RHZ (p>0.05).</p

Experiment Design.

<p>Definition of abbreviations: J = TMC 207, R = Rifampin, M = Moxifloxacin, H = Isoniazid, Z = Pyrazinamide, A = amikacin, Et = ethionamide.</p><p>Mice were infected intravenously with 1.1×10⁶ of <i>M. tuberculosis</i> H37Rv.</p><p>Day -18: the day after infection, Day 0: start of treatment.</p><p>*Mice kept untreated for mortality assessment.</p><p>All drugs were given 5 times per week at the following doses: J, 25 mg/kg; R, 10 mg/kg; M, 100 mg/kg; H, 25 mg/kg; Z, 150 mg/kg; A, 150 mg/kg; Et, 50 mg/kg.</p

Multiplication of <i>M</i>. <i>leprae</i> organisms in mice treated by BDQ administered orally and the benefit of the combination of CFZ and BDQ (each mouse footpad is taken as a data point and the dotted line indicates the threshold of detection of <i>M</i>. <i>leprae</i>).

Multiplication of M. leprae organisms in mice treated by BDQ administered orally and the benefit of the combination of CFZ and BDQ (each mouse footpad is taken as a data point and the dotted line indicates the threshold of detection of M. leprae).</p

Bactericidal activity against <i>M</i>. <i>leprae</i> THAI53 of bedaquiline administered orally and bedaquiline long-acting measured in Swiss mice by the proportional bactericidal method.

Bactericidal activity against M. leprae THAI53 of bedaquiline administered orally and bedaquiline long-acting measured in Swiss mice by the proportional bactericidal method.</p

Multiplication of <i>M</i>. <i>leprae</i> organisms in nude mice to determine minimal effective dose of BDQ administered orally active against <i>M</i>. <i>leprae</i> and the benefit of the combination of CFZ and BDQ.

Multiplication of M. leprae organisms in nude mice to determine minimal effective dose of BDQ administered orally active against M. leprae and the benefit of the combination of CFZ and BDQ.</p

ATP synthesis inhibition by TMC207 at low proton motive force.

<p>(<b>A</b>) Inverted membrane vesicles from <i>Mycobacterium smegmatis</i> were diluted to 0.18 mg/ml in buffer containing 2 µM ACMA. To detect the proton motive force, quenching of ACMA fluorescence was investigated after addition of 5 mM succinate in the presence of increasing concentrations of the uncoupler SF6847. At the indicated time point, 1 µM of uncoupler SF6847 was added as control to collapse the proton gradient. (<b>B</b>) ATP synthesis by membrane vesicles of <i>M. smegmatis</i> (1 mg/ml) was measured in the presence of TMC207 and varying concentrations of uncoupler SF6847 to modulate the proton motive force. Samples were incubated at 37°C for 1 h in the presence of an ADP-regenerating system, and produced ATP was quantified spectrophotometrically by monitoring oxidation of glucose-6-phosphate with NADP⁺. As a control, 100 µM DCCD was added.</p

TMC207 and its target mycobacterial ATP synthase.

<p>(<b>A</b>) Structure formula of TMC207. (<b>B</b>) ATP synthase subunit composition with subunit c in grey. A homology model of a subunit c monomer from <i>Mycobacterium tuberculosis</i> is shown enlarged. The acidic residue Glu61, essential for proton transport, is depicted in red. Point mutations that influence mycobacterial sensitivity for TMC207 are indicated in colour.</p

TMC207 binds to a defined binding site in ATP synthase.

<p>(<b>A</b>) The dose-dependency of ATP synthesis inhibition by TMC207 in inverted membrane vesicles of <i>Mycobacterium smegmatis</i> was fitted with a one-site binding hyperbola (Y = 104.9X/6.3+X, R²>0.99) (<b>B</b>) Binding of purified ATP synthase subunit c from <i>Mycobacterium tuberculosis</i> to an amine analog of TMC207 linked onto a BIAcore chip was fitted using mono-exponential binding models (Association = Req*(1−exp(−1*53737X)) and Dissociation = 165.654*exp(−1*0.002295*(X−45)) R²>0.99) and (<b>C</b>) Binding of purified ATP synthase from <i>Bacillus</i> PS3 to an amine analog of TMC207 linked onto a BIAcore chip was fitted using mono-exponential binding models (Association = Req*(1−exp(−1*153.7X)) and Dissociation = 8575.97*exp(−1*0.0001030*(X−1187)) R²>0.99).</p

Electrostatic interactions are important for binding of TMC207.

<p>(<b>A</b>) ATP synthesis in the presence of TMC207 and increasing sodium chloride concentrations was measured for inverted membrane vesicles of <i>Mycobacterium smegmatis</i> (1 mg/ml). Samples were incubated at 37°C for 1 h in the presence of an ADP-regenerating system, and produced ATP was quantified spectrophotometrically by monitoring oxidation of glucose-6-phosphate with NADP⁺. As a control, 100 µM DCCD was added. (<b>B</b>) BIAcore binding studies. Purified subunit c from wild-type <i>Mycobacterium tuberculosis</i> was injected onto a chip with immobilized amine analog of TMC207 in the presence of 50, 150, and 300 mM NaCl at 37°C.</p

Acquired Resistance of <i>Mycobacterium tuberculosis</i> to Bedaquiline

<div><p>Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multi-drug resistant tuberculosis in decades. <i>In vitro</i> resistance to BDQ was previously shown to be due to target-based mutations. Here we report that non-target based resistance to BDQ, and cross-resistance to clofazimine (CFZ), is due to mutations in Rv0678, a transcriptional repressor of the genes encoding the MmpS5-MmpL5 efflux pump. Efflux-based resistance was identified in paired isolates from patients treated with BDQ, as well as in mice, in which it was confirmed to decrease bactericidal efficacy. The efflux inhibitors verapamil and reserpine decreased the minimum inhibitory concentrations of BDQ and CFZ <i>in vitro</i>, but verapamil failed to increase the bactericidal effect of BDQ in mice and was unable to reverse efflux-based resistance <i>in vivo</i>. Cross-resistance between BDQ and CFZ may have important clinical implications.</p></div