| Assay Method Information | |
| | Biochemical Evaluation of MBP-Ndh Inhibition |
| Description: | To define the activity of compounds on purified MBP-Ndh, a biochemical assay was setup to monitor MBP-Ndh dependent oxidation of NADH in the presence of an electron acceptor menadione, similar to methods described previously (Murugesan, D. et al. 2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and Mycobacterium tuberculosis: Structure-Activity Relationships, Mechanism of Action and Absorption, Distribution, Metabolism, and Excretion Characterization. ACS Infect. Dis. 4, (2018)). Briefly, to evaluate the IC50 of the compounds, the compounds were transferred to a black 384-well plate with transparent bottom using Echo liquid handling acoustic technology (Labcyte) and backfilled to 500 nL with DMSO. Using a Viafill liquid dispenser (INTEGRA Biosciences) a 45 μL mixture was added to these wells containing a Mtb recombinant MBP tagged Ndh-2 enzyme (either 70 nM recombinant MBP-Ndh produced in E. coli-; 0.66 nM recombinant MBP-Ndh produced in M. smegmatis, or 30 nM recombinant MBP-NdhA produced in M. smegmatis, concentration adjusted to have similar rate of NADH oxidation) and NADH (500 μM) in 50 mM HEPES buffer (pH 7.1 with K2HPO4). Following a pre-incubated (25° C., 15 min), enzyme activity was initiated by the addition of menadione (5 μL, 100 μM final concentration (dispensed into wells using ENVISION, Perkin Elmer), and the kinetics of NADH oxidation monitoring at 340 nm (measured every 60 sec, ENVISION plate reader, Perkin Elmer). The rate of NADH oxidation was calculated as the slope of the linear decrease in 340 nm signal using Microsoft Excel, and enzyme inhibition parameters determined using Graphpad Prism v. 9. IC50 are reported as mean and standard deviation of at least 3 independent experiments. |
| Affinity data for this assay | |
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