Fig. 2

Biochemical target validation of TriSLas and impact on the Mtb electron transport chain. (a) Compound 12 biochemical inhibition of NADH oxidation by recombinant Mtb MBP-Ndh (filled circles, solid line), MBP-Ndh(Y403C) (filled triangles, dashed line), and MBP-Ndh(Q334P) (filled squares, dotted line) overexpressed and purified using E. coli. Data are presented as a mean ± SD of at least three independent biological replicates. (b) Compound 12 biochemical inhibition of NADH oxidation by recombinant Mtb MBP-Ndh (filled circles, solid line) and MBP-NdhA (filled triangles, dashed line) overexpressed and purified using M. smegmatis. (c) Lineweaver–Burk plot showing noncompetitive inhibition of Ndh by 12 relative to menadione (n ≥ 3), (d) Lineweaver–Burk plot showing noncompetitive inhibition of Ndh by 12 relative to NADH (n ≥ 3), (e) Mtb NADH/NAD⁺ ratios measured following 2 and 24 h exposure to 11 (green-blue, 600 nM, 4× MIC), 12 (dark blue, 600 nM, 4× MIC), and bedaquiline (green, 1 μM, 4× MIC), compared to unexposed DMSO controls (black) (n ≥ 3), and (f) Mtb ATP concentrations measured following 24 h exposure to either 11 (green-blue), 12 (dark blue), and bedaquiline (green) (n ≥ 3).