| Assay Method Information | |
| | DHODH Kinetic Analysis |
| Description: | The 50% inhibitory concentration (IC50) for the described compounds was determined using the 2,6-dichloroindophenol (DCIP) assay to monitor the DHODH reaction rate at 25° C. in assay buffer (100 mM HEPES, pH 8.0, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100 reduced, 20 μM CoQD, 200 μM L-DHO and 5-20 nM enzyme) as described in Malmquist et al., 2008, supra. Inhibitor stocks (100 mM) were prepared in DMSO in amber bottles. A 3-fold dilution series was generated in DMSO from these stocks and then dispensed into assay buffer via a 1/100 dilution to generate a final concentration range of 0.001-100 μM (1% DMSO final). Data were collected using triplicate technical replicates and where indicated additional biological replicates were collected (the number of biological replicates is provided in parenthesis). IC50's were determined by fitting the data to log (inhibitor) vs. response equation Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((X−Log IC50))) in GraphPad Prism. Compounds in the invention show potent activity versus P. falciparum DHODH but importantly, unlike DSM265, they show equivalent activity on P. vivax and P. falciparum DHODH (see above methods for protein purification and enzyme assays)(Table 2). Furthermore, the compounds of the invention do not inhibit any of the tested mammalian DHODHs including human DHODH and they also retain their selectivity versus DHODHs from important toxicologic models (including mouse, rat and dog). |
| Affinity data for this assay | |
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