| Assay Method Information | |
| | In Vitro Scintillation Proximity Assay (SPA) |
| Description: | Compounds of the present invention were tested in an in vitro assay based on SPA technology with Ni Flash plates (96 or 384 well).In principle, the assay relies upon SPA technology for the detection of auto-poly(ADP-ribosyl)ation of TANK-2 protein using [3H]-nicotinamide adenine dinucleotide ([3H]-NAD+) as ADP-ribosyl donor.A stock solution of 100 nM [3H]-NAD+/NAD (0.1 mCi/ml, supplier: Perkin Elmer) and 25 μM NAD (Sigma) was made in assay buffer (60 mM Tris/HCl, pH 7.4; 0.9 mM DTT; 6 mM MgCl2). The TANK-2 enzyme was produced as described in EP1238063. 60 μl of assay buffer, together with 1 μl of compound in DMSO, 20 μl of [3H]-NAD+/NAD and 20 μl of TANK-2 enzyme (final concentration 8 μg/ml) was added per well into a 96-well Ni-coated flash plate (Perkin Elmer). After incubation of the mixture for 120 minutes at room temperature, the reaction was terminated by adding 60 μl of stopsolution (42.6 mg NAD in 6 ml H2O). The plates were covered with a plate sealer and placed in a TopCountNXT (Packard) for scintillation counting. Values were expressed as counts per minute (cpm). For each experiment, controls (containing TANK-2 enzyme and DMSO without compound), a blank incubation (containing DMSO but no TANK-2 enzyme or compound) and samples (containing TANK-2 enzyme and compound dissolved in DMSO) were run in parallel. All compounds tested were dissolved and eventually further diluted in DMSO. In first instance, compounds were tested at a concentration of 10−5M. When the compounds showed activity at 10−5 M, a dose-response curve was made wherein the compounds were tested at concentrations between 10−5M and 3×10−8M. In each test, the blank value was subtracted from both the control and the sample values. The control sample represented maximal TANK-2 enzyme activity. For each sample, the amount of cpm was expressed as a percentage of the mean cpm value of the controls. When appropriate, IC50-values (concentration of the drug needed to reduce the TANK-2 enzyme activity to 50% of the control) were computed using linear interpolation between the experimental points just above and below the 50% level. Herein the effects of test compounds are expressed as pIC50 (the negative log value of the IC50-value). As reference compounds, 3-aminobenzamide and 4-amino-1,8-naphtalimide were included to validate the SPA assay. Herein the assay was described using 96-well plates. In the assay using 384-well plates the same final concentrations were used and volumes were adapted. If 96-well plate results were available these results were incorporated in Table-2, otherwise the results from the 384-well plate assay were shown. |
| Affinity data for this assay | |
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