Article Title: Combining amoxicillin and relebactam provides a new therapeutic option for Mycobacterium abscessus infection
Figure Lengend Snippet: Biochemical analysis of relebactam inhibition of M. abscessus β -lactamase, Bla Mab . Our novel Thin Layer Chromatography (TLC) assay (2a) exhibiting the activity of Bla Mab in the turnover of penicillin V (high R f value) to penicilloic acid (lower R f value). In the absence, or termination of activity of Bla Mab (by boiling (100 °C) for 1 h) or addition of known inhibitor avibactam ( Lefebvre et al., 2017 ) (200 μg/mL) no lower R f value spot corresponding to penicilloic acid is seen on the TLC plate. The addition of relebactam to the reaction between Bla Mab and penicillin V also results in the absence of the lower Rf value spot, suggesting inhibition of Bla Mab (2a). This observed inhibition is validated by a spectrophotometric analysis, where the turnover of nitrocefin (100 μM) by Bla Mab (0.25 nM) was monitored at 486 nm with a varying concentration of relebactam (0, 0.1, 1, 10 and 100 μM). The increase in concentration of relebactam resulted in partial inhibition of nitrocefin turnover at 1 μM and total abrogation at 10 μM (2b). The initial velocity (v i ) of the reaction between Bla Mab and nitrocefin was monitored for a range of substrate concentrations (1-500 μM) and relebactam concentrations (0, 0.5, 0.75, 1 and 2.5 μM). This data was plotted v i vs [S] in order to determine K m values (2c/f). The values for K obs were obtained as previously described ( Dubée et al., 2014 ) and plotted against relebactam concentrations ([I]) to deduce a carbamylation rate ( K 2 / K i ) for Bla Mab (2d). The kinetics of Bla Mab decarbamylation were assessed to show the recovery of nitrocefin hydrolysis by Bla Mab after inhibition by relebactam in order to derive a K off value (2e). Kinetic parameters were derived as described previously (2f) ( Dubée et al., 2014 ).
Article Snippet: The hydrolysis of nitrocefin was monitored at 486 nm using a Multiskan Go plate reader (Thermo Scientific).
Techniques: Inhibition, Thin Layer Chromatography, Activity Assay, Concentration Assay, Derivative Assay