Tet(56-2) and Tet(56-3) Inhibitor Potency Assay

All experiments were prepared open to air in non-degassed buffer solutions at room temperature. Half-maximal inhibitory concentrations (IC₅₀) for the inhibition of Tet(56-2) and Tet(56-3) were determined from the velocities of substrate degradation in the presence of varying concentrations of inhibitor. Reaction samples were prepared in 100 mM TAPS buffer (pH 8.5) with 504 μM NADPH, 5.04 mM MgCl₂, 25.3 μM substrate, varying concentrations of inhibitor (0-227 μΜ), and 0.4 μM enzyme (final working concentrations). Reactions were initiated by the addition of enzyme and were monitored continuously via optical absorbance spectroscopy at 400 nm for 2 min (performed in triplicate as independent trials). Initial enzyme velocities were determined by linear regression using Agilent Cary WinUV Software over the linear range of the reaction (0 to 1 min). The velocities were plotted against the logarithm of inhibitor concentration, and apparent IC₅₀ values were determined using nonlinear regression analysis in GraphPad Prism 6. Each set of experiments included a no-TDase control reaction which was used as the full enzyme inhibition velocity and assigned to inhibitor concentration of 1 × 10¹⁵, and a no-inhibitor control which was assigned an inhibitor concentration of 1 × 10^–15. A no-tetracycline control was also performed to search for potentially competitive background signals generated from the enzymatic degradation of the inhibitor. For all inhibitor-enzyme combinations, the initial velocities of the no-tetracycline controls were negligible.