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