Probing the interaction of the diarylquinoline TMC207 with its target mycobacterial ATP synthase

Abstract

Infections with Mycobacterium tuberculosis are substantially increasing on a worldwide scale and new antibiotics are urgently needed to combat concomitantly emerging drug-resistant mycobacterial strains. The diarylquinoline TMC207 is a highly promising drug candidate for treatment of tuberculosis. This compound kills M. tuberculosis by binding to a new target, mycobacterial ATP synthase. In this study we used biochemical assays and binding studies to characterize the interaction between TMC207 and ATP synthase. We show that TMC207 acts independent of the proton motive force and does not compete with protons for a common binding site. The drug is active on mycobacterial ATP synthesis at neutral and acidic pH with no significant change in affinity between pH 5.25 and pH 7.5, indicating that the protonated form of TMC207 is the active drug entity. The interaction of TMC207 with ATP synthase can be explained by a one-site binding mechanism, the drug molecule thus binds to a defined binding site on ATP synthase. TMC207 affinity for its target decreases with increasing ionic strength, suggesting that electrostatic forces play a significant role in drug binding. Our results are consistent with previous docking studies and provide experimental support for a predicted function of TMC207 in mimicking key residues in the proton transfer chain and blocking rotary movement of subunit c during catalysis. Furthermore, the high affinity of TMC207 at low proton motive force and low pH values may in part explain the exceptional ability of this compound to efficiently kill mycobacteria in different microenvironments.

Figure 1. TMC207 and its target mycobacterial ATP synthase.

(A) Structure formula of TMC207. (B) ATP synthase subunit composition with subunit c in grey. A homology model of a subunit c monomer from Mycobacterium tuberculosis 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.

Figure 2. ATP synthesis inhibition by TMC207 at low proton motive force.

(A) Inverted membrane vesicles from Mycobacterium smegmatis 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) ATP synthesis by membrane vesicles of M. smegmatis (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.

Figure 3. Effect of TMC207 on mycobacterial ATP synthesis at low pH.

ATP synthesis in the presence of TMC207 and varying external pH values was measured for Mycobacterium smegmatis inverted membrane vesicles (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.

Figure 4. Electrostatic interactions are important for binding of TMC207.

(A) ATP synthesis in the presence of TMC207 and increasing sodium chloride concentrations was measured for inverted membrane vesicles of Mycobacterium smegmatis (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) BIAcore binding studies. Purified subunit c from wild-type Mycobacterium tuberculosis was injected onto a chip with immobilized amine analog of TMC207 in the presence of 50, 150, and 300 mM NaCl at 37°C.

Figure 5. TMC207 binds to a defined binding site in ATP synthase.

(A) The dose-dependency of ATP synthesis inhibition by TMC207 in inverted membrane vesicles of Mycobacterium smegmatis was fitted with a one-site binding hyperbola (Y = 104.9X/6.3+X, R²>0.99) (B) Binding of purified ATP synthase subunit c from Mycobacterium tuberculosis 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 (C) Binding of purified ATP synthase from Bacillus 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).