| Assay Method Information | |
| | hDHODH inhibition assay |
| Description: | The enzymatic inhibition assay was optimized for being performed on a 96 well plate and to achieve higher throughput. For each well of the plate a total volume of 200 μL was used: 5 μL of purified GST-hDHODH; 60 μL of 2,6-dichloroindophenol (DCIP) 500 μM; μL of coenzyme Q10 enzyme 100 μM; 20 μL of dihydroorotate (DHO) 500 μM; Tris-HCl pH8 up to a final volume of 200 μL. Inhibitory activity was assessed by monitoring the reduction of DCIP, which is associated with the oxidation of dihydroorotate as catalysed by the DHODH enzyme. The enzyme was pre-incubated for 5 min at 37° C. in Tris-HCl pH8 with coenzyme Q10, with DCIP (50 μM) and with the compounds to be tested used at different concentrations (final DMSO concentration 0.1% v/v). The reaction was initiated by the addition of DHO (500 μM), and the absorbance kinetic reduction was monitored at λ=650 nm using a multi-plate reader (Tecan, M1000Pro). In order to assess the minimum and maximum absorbance values of the enzymatic reaction, a Min control value was obtained by measuring the absorbance without DHO. Similarly, a Max value was obtained by measuring the absorbance with DHO, but none inhibitor. A blank reduction calculation was also performed by measuring the absorbance values using 180 μL of Tris-HCl and 20 μL of coenzyme Q10. The Instrument was set to read the absorbance values every 10 s for a total read time of 10 minutes at 37° C. The initial rate was measured in the first 5 min (s=10 400 M-1 cm-1) and an IC50 value was calculated, n using GraphPad Prism 7 software |
| Affinity data for this assay | |
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