Development of a high-throughput screening-amenable assay for human poly(ADP-ribose) polymerase inhibitors.
BACKGROUND
Poly(ADP-ribose) polymerase (PARP) plays a pivotal role in the repair of DNA strand breaks. However, excessive activation of PARP causes a rapid depletion of intracellular energy, leading to cell death. Inhibitors of PARP have been shown to reduce infarct size in animal models of myocardial ischemia. PARP inhibitors may have potential therapeutic benefit in the treatment of myocardial ischemia, stroke, head trauma, and neurodegenerative disease, and as an adjunct therapy with chemotherapeutic agents/radiation in cancer therapy.


METHODS
Assays reported in the literature and commercially available PARP assay kits are labor-intensive, use radioactive reagents, use antibodies, and are not readily amenable to high throughput screening (HTS) [corrected]. Here we report the development and the validation of a nonradioactive PARP assay suitable for HTS. This is a biotinylated NAD-based colorimetric assay in a 96-well plate format.


RESULTS
The assay is sensitive, reproducible, and easy to use. The IC(50) values generated for the known PARP inhibitors are in agreement with those generated using the commercial radioactive kit and those reported in the literature.


CONCLUSIONS
The present study demonstrates a sensitive and reproducible methodology capable of screening human PARP inhibitors in high-throughput format.
Brown
Janice A
JA
Department of Cardiovascular and Metabolic Diseases, MS: 8220-3118, Pfizer Global Research and Development, Pfizer Inc., Eastern Point Road,Groton, CT 06340, USA.
Marala
Ravi B
RB
D020727:ADP-Ribosylation Factors / Q000378:metabolism; D001549:Benzamides / Q000494:pharmacology; D019921:Biotinylation; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004987:Ethers / Q000494:pharmacology; D006801:Humans; D006845:Hydrocarbons, Fluorinated / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D008403:Mass Screening / Q000379:methods*; D009243:NAD / Q000378:metabolism; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis*; D011799:Quinazolines / Q000494:pharmacology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001549:Benzamides; D004791:Enzyme Inhibitors; D004987:Ethers; D006845:Hydrocarbons, Fluorinated; C115774:NU 1025; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011799:Quinazolines; D009243:NAD; C078742:fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether; C025160:3-aminobenzamide; D011065:Poly(ADP-ribose) Polymerases; D020727:ADP-Ribosylation Factors