| Assay Method Information | |
| | Biochemical Assay |
| Description: | Preferably, for the screening method above cited, both the PARP protein and the 5H-phenanthridin-6-one-derived probe of formula (IP) are pre-mixed, or the PARP protein and the test compound are pre-mixed. In a further preferred screening method, the PARP proteins are PARP-1, PARP-2 and PARP-3. The term PARP protein encompasses full-length native proteins as well as fragments thereof. More preferably, R11 is hydrogen or methyl, m is 0 or 1; when m is 1, n is 3 or 6, X′″ is trifluoroacetate. The 5H-phenanthridin-6-one-derived probe (IP) was selected for its capability of binding to the PARP proteins, both encompassing full-length native proteins and fragments thereof.The polarization signal can be measured, e.g., by a plate reader such as the Saphire2 (Tecan). Data analysis was performed, e.g., by using the Dynafit software. Displacement data were also fitted, e.g., by using Excel spreadsheet (Microsoft Inc. Seattle, USA) to a four parameter logistic model (4PL), or Hill-Slope model. The assay was used to test compounds of the present invention. The displacement ability of the test compounds of formula (I) is in correlation with the compounds affinity for the NAD pocket of the enzyme. Specific probes of formula (IP) used in the assay are: P1. 9-Dimethylamino-11,11-dimethyl-1-(3-{methyl-[(6-oxo-5,6-dihydro-phenanthridin-2-ylcarbamoyl)-methyl]-carbamoyl}-propyl-2,3,4,11-tetrahydro-naphtho[2,3-g]quinolinium trifluoroacetate; P2. 9-Dimethylamino-11,11-dimethyl-1-[3-(3-{[(6-oxo-5,6-dihydro-phenanthridin-2-ylcarbamoyl)-methyl]-amino}-propylcarbamoyl)-propyl])-2,3,4,11-tetrahydro-naphtho[2,3-g]quinolinium trifluoroacetate; P3. 9-Dimethylamino-11,11-dimethyl-1-[3-(6-{[(6-oxo-5,6-dihydro-phenanthridin-2-ylcarbamoyl)-methyl]-amino}-hexylcarbamoyl)-propyl]-2,3,4,11-tetrahydro-naphtho[2,3-g]quinolinium trifluoroacetate.A compound of formula (IP) as defined above can be prepared as described in WO 2010/133647.The assay is based on the use of a probe of formula (IP) that binds to the NAD binding pocket and takes advantage of the significant change in the polarization signal observed upon binding of the probe to PARP-1, -2 and -3. The ability of the probe of formula (IP) to bind full-length PARP-1, -2 and -3 has been previously reported (WO 2010/133647). The assay has been validated as described in WO 2010/133647. |
| Affinity data for this assay | |
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