Summary
The diarylquinoline R207910 (TMC207) is a promising candidate in clinical development for the treatment of tuberculosis. Though R207910-resistant mycobacteria bear mutations in ATP synthase, the compound's precise target is not known. Here we establish by genetic, biochemical and binding assays that the oligomeric subunit c (AtpE) of ATP synthase is the target of R207910. Thus targeting energy metabolism is a new, promising approach for antibacterial drug discovery.

Fig. 1. Binding studies using compound -linked affinity chromatography and BIAcore. (a) Affinity capture: membrane extracts from M. smegmatis were passed through an affinity resin and eluants were separated by two-dimensional gel electrophoresis. The indicated spots (circles) were identieid by mass spectrometry. (b) BIAcore: purified subunit c from wild-type (no symbols) or mutant (crosses) M. tuberculosis was injected onto BIAcore chip. Control data (circles) are from a chip without immobilized R207910 analog.

To identify drug-binding partners, we immobilized an amine analog of R207910 on Sepharose resin. Total membrane protein extract from M. smegmatis was passed through this affinity column, and two proteins of about 59 and 53 kDa, respectively, were eluted. We did not observe any non-specific binding to the control resin (without immobilized compound). The eluted fraction was resolved by two-dimensional gel electrophoresis and protein spots were excised and analyzed by mass spectrometry (Fig. 2a). We identified the alpha-subunit (59 kDa) and beta-subunit (52 kDa) of ATP synthase, the two largest subunits of the protein (Fig. 1a). Subunit c was not detected, probably because of its extreme hydrophobic character, which makes it prone to aggregation and therefore recalcitrant to gel electrophoresis.

Reference
A. Koul et al. Diarylquinolines target subunit c of mycobacterial ATP synthase. Nature Chemical Biology, 3 (6), (2007), 323-324