PARP1 enzyme inhibition assays

The inhibition of the tested compounds on PARP1 enzymatic activity in a cell-free system was measured with enzyme-linked immunosorbent assays (ELISA)^45,46,47. Briefly, NAD⁺ (6 μmol/L) and the activator deoxyoligonucleotide (100 μg/mL) were diluted in 70 μL of reaction buffer and added to each well of a histone-pre-coated 96-well plate, and this was followed by the addition of 10 μL of the compound or a solvent control. The reaction was started by addition of 20 μL of recombinant human PARP1 (10 ng/well) for 1.5 h. After cells were washed with PBST, 100 μL of anti-PARP polyclonal antibody (Trevigen) was added, and the mixture was incubated for another 1.5 h. After washing the cells, the second antibody, goat anti-rabbit IgG horseradish peroxidase, was added, and the mixture incubated for an additional 30 min. Finally, a solution of 0.03% H₂O₂ and 2 mg/mL OPD in 0.1 mol/L citrate buffer (pH 5.4) was added, and the mixture was incubated for 10 min. The reaction was stopped by the addition of 2 mol/L H₂SO₄. A490 was measured with a multi-well spectrophotometer (SPECTRA MAX190). The inhibition rate of PARP1 enzymatic activity was calculated as (A490_control-A490_treated/A490_control)×100%. The concentration required to inhibit 50% of PARP1 enzymatic activity (IC₅₀) was determined with the Logit method.