Selectivity of TMC207 towards mycobacterial ATP synthase compared with that towards the eukaryotic homologue

Abstract

The diarylquinoline TMC207 kills Mycobacterium tuberculosis by specifically inhibiting ATP synthase. We show here that human mitochondrial ATP synthase (50% inhibitory concentration [IC(50)] of >200 microM) displayed more than 20,000-fold lower sensitivity for TMC207 compared to that of mycobacterial ATP synthase (IC(50) of 10 nM). Also, oxygen consumption in mouse liver and bovine heart mitochondria showed very low sensitivity for TMC207. These results suggest that TMC207 may not elicit ATP synthesis-related toxicity in mammalian cells. ATP synthase, although highly conserved between prokaryotes and eukaryotes, may still qualify as an attractive antibiotic target.

FIG. 1.

Effect of TMC207 on ATP synthesis by mitochondria from a human cell line. ATP synthesis in the presence of TMC207 was measured for mitochondria (250 μg/ml) and SMPs (150 μg/ml) from a human cancer cell line (A) and compared to that for inverted membrane vesicles of Mycobacterium smegmatis (B). Samples were incubated at 25°C for 1 h in the presence of an ADP-regenerating system, and produced ATP was quantified spectrophotometrically by monitoring the oxidation of glucose-6-phosphate with NADP⁺. As controls, DCCD (100 μM for M. smegmatis and 5 μM for human mitochondria and SMPs) and oligomycin (1 μM) were used.

FIG. 2.

Effect of TMC207 on respiratory function in mitochondria isolated from mouse and bovine tissue. Oxygen consumption coupled to ATP synthesis in the presence of TMC207 was measured for mitochondria from mouse liver, mitoplasts from mouse liver, and mitochondria from bovine heart (each at a final protein concentration of 1 mg/ml). The oxygen concentration was measured using a Clark electrode at 37°C in a medium with 20 mM Tris-HCl, pH 7.3, 85 mM KCl, 5 mM KH₂PO₄, 2.3 mM MgCl₂, 25 mM creatine, and 25 mM phosphocreatine in the presence of an ADP-regenerating system and the indicated concentrations of TMC207. The membrane was energized by an addition of succinate, and complex I was inhibited by rotenone. Inhibition of ATP synthase and respiratory chain enzymes was determined as a decrease in the state III oxygen consumption rate. As a control, oligomycin (0.6 μM) was used. Each graph shows mean values of three independent experiments with standard deviations.

FIG. 3.

Multiple sequence alignment for subunit c of ATP synthase. Amino acid sequences of ATP synthase subunit c from mycobacteria and eukaryotic species are compared. Species abbreviations: M.tub, M. tuberculosis (Swiss-Prot accession no. Q10598); M.sme, M. smegmatis (EMBL accession no. AJ862722); M.xen, M. xenopi (GenBank accession no. DQ306893); H.sap, Homo sapiens (residue 60 to 136; RefSeq accession no. NM_001002027); B.tau, Bos taurus (bovine, residue 60 to 136; RefSeq accession no. NP_788822); and M.mus, Mus musculus (mouse, residue 60 to 136; RefSeq accession no. NM_007506). For comparison, the two transmembrane helices found in the structure of the Escherichia coli enzyme are indicated below. The N-terminal part of the mitochondrial precursor sequence for the bovine and mouse subunit c is not shown. Amino acid residues found to be important for drug sensitivity in M. tuberculosis (positions 63 and 66, numbering for M. tuberculosis) are indicated by boxes. The glutamic acid residue E61 is an essential residue for proton translocation.