Development of a miniaturized assay for the high-throughput screening program for poly(ADP-ribose) polymerase-1.
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. PARP inhibitors may have potential therapeutic benefit in the treatment of myocardial ischemia, stroke, and neurodegenerative disease. With these emerging medicinal interests, various screening programs have identified small molecules that inhibit PARP with reasonable potencies. However, the increasing numbers of diverse small molecules generated through combinatorial chemistry necessitate the use of robust assays with good sensitivity and specificity for use as a high-throughput screening (HTS) program. Here, we report the development and the validation of a nonisotopic PARP-1 assay suitable for HTS by converting a biotinylated NAD-based colorimetric assay to a miniaturized 384-well plate format. Comparing with the conventional methods, this miniaturized PARP-1 inhibition assay was equally sensitive with excellent reproducibility and cost-effectiveness. Because nonisotopic PARP-1 inhibition assays are widely used, the methodology described in this article can expand the feasibility of this assay as a high-throughput assay for screening of PARP-1 inhibitors from a random chemical library.
Lee
S
S
Department of Biotechnology and Informatics, College of Engineering, Sangmyung University, Cheonan, Republic of Korea.
Koo
H-N
HN
Lee
B H
BH
D019921:Biotinylation; D003124:Colorimetry; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry; D020128:Inhibitory Concentration 50; D008904:Miniaturization; D009243:NAD / Q000737:chemistry; D000071137:Poly (ADP-Ribose) Polymerase-1; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry
D004791:Enzyme Inhibitors; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D009243:NAD; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases