Enzymatic assays (InhA)

InhA activity was assessed by a spectrophotometric assay that followed the oxidation of NADH at λ = 340 nm in the presence of 2-trans-octanoyl-CoA. Compounds were dissolved in 100% DMSO. The enzyme reaction contains 30 mM PIPES pH 7.5, 50 mM NaCl, 1% (v/v) DMSO and 0.1 mM EDTA. InhA (100 nM) was pre-incubated for 10 min at room temperature with 0.25 mM NADH and compounds at a final concentration of 100 μM in 150 μl reaction volume. The reaction is started by the addition of 2-trans-octanoyl-CoA at a final concentration of 1.5 mM, prepared as described previously (He et al., 2006). The reactions were followed for 20 min using a spectrophotometer plate reader (CLARIOstar—BMG LABTECH). Inhibition percentage was calculated based on the initial rates of reaction. All experiments were performed in triplicate.