( The DELFIA time-resolved fluo...)A DELFIA Assay for Inhibition of PARP

The DELFIA time-resolved fluorescence assay for inhibition of poly(ADP-ribose) polymerase (PARP) is a 96-well microplate assay that measures the inhibition of PARP-activated incorporation of biotinylated nicotinamide adenine diphsophate (NAD). The incorporation is detected using Europiumlabelled streptavidin. The method is a convenient way to screen test compounds for PARP activity in high throughput primary screening and secondary screening.

Poly(ADP-ribose) polymerase (PARP) is a nuclear protein involved in the response to DNA damage, where it catalyzes the polymerization of nicotinamide adenine diphsophate (NAD) into chains of poly(ADPribose) polymers¹. The polymerization occurs on a number of nuclear proteins, as well as on the PARP protein itself. Following activation by DNA strand breaks, PARP hydrolyzes NAD and catalyzes the formation of PARP onto itself and other nuclear proteins, with the release of nicotinamide. PARP binds to singleand double-stranded DNA breaks. When the enzyme undergoes auto-poly (ADPribosyl) ation, the automodified PARP dissociates from the DNA and becomes inactive².

Inhibitors of PARP have been shown to help prevent ischemic injury in the brain, increase apoptosis in cancer cells, prevent infiltration of neutrofils and subsequent inflammation, and to suppress the production of nitric oxide synthase in macrophages^3,4. In response to low to moderate levels of DNA damage, PARP is activated and NAD levels in the cells decrease. Experiments with PARP inhibitors suggest that PARP activity may be necessary to rescue cells after low to moderate levels of DNA damage. In contrast, when cells experience massive levels of DNA damage and DNA strand breaks, activation of PARP can lead to depletion of NAD and ATP, resulting in a marked decrease in energydependent processes and in DNA repair. In this situation, activation of PARP by massive DNA damage processes may actually be a suicide response, since it causes rapid NAD and ATP depletion and leads to cell death before the cell can repair the DNA damage.

METHODS

The DNA used in the polymerization reaction was fragmented by hydrodynamic shearing. The DNA was passed through the orifice of a 28-gauge hypodermic needle. This method of shearing generates 1.5-2.0 kb DNA fragments⁵. The polymerization reaction was performed in an uncoated 96-well microtiter plate. To the well was added: 2.5 μg histones (Trevigen/Upstate U.C. Biotechnology), 1 μg DNA (Sigma), 15 μM NAD + Bio-NAD (Trevigen) in a 17:1 or 1:1 ratio, 1 mM 3- aminobenzamide⁶ (inhibitor of PARP, Sigma) 0.5 U PARP enzyme (Trevigen) and PARP buffer (500 mM Tris-HCl, 250 mM MgCl₂). The reaction was performed at room temperature, for 30 minutes. The plate was then washed 2 times using the DELFIA Platewash. Eu-labelled streptavidin was diluted in DELFIA Assay Buffer to 200 ng/ well and incubated for 30 minutes at room temperature. The plate was washed 4 times using the DELFIA Platewash, prior to addition of 200 μL/well DELFIA Enhancment Solution. The fluorescence was measured using VICTOR² _TM plate reader.

The data shows that the DELFIA timeresolved fluorescence assay for inhibition of PARP allows for the measurement of the incorporation of biotinylated NAD. This technology is therefore suitable for the screening of inhibitors of PARP where the formation of poly(ADP-ribose) chains is inhibited.

REFERENCES:

1. Satoh, M. S. and Lindahl, T. 1994. Role of poly(ADP-ribose) formation in DNA repair. Nature 356, 356-358

2. Lautier, D., Langeux, J., Thibodeau, L., Menard, L. and Poirier, G. G. 1993. Molecular and biochemical features of poly(ADP-ribose) metabolism. Mol Cell Biochem 122, 171-193

3. Thiermann, C., Bowes, J., Myint, F. P. and Vane, J. R. 1997. Inhibition of the activity of poly(ADP ribose) synthease reduces ischemia-reperfusion injury in the heart and skeletal muscle. Proc. Natl. Acad. Sci. USA 94, 679-683

4. Heller, B., Wang, Z-Q., Wagner, E. F., Radons, J., Brkles, A., Fehsel, K., Burkart, V. and Kolb, H. 1995. Inactivation of the poly(ADP-ribose) polymerase gene affects oxygen radical and nitric oxide toxicity in islet cells. J. Biol. Chem. 270, 11176-11180

5. Davidson, P. F. 1959. The effect of hydrodynamic shear on the deoxyribonucleic acid from T2 and T4 bacteriophages. Proc. Natl. Acad. Sci. 45, 1560-1568

6. Purnell, M. R. and Whish, W. J. 1980. Novel inhibitors of poly(ADP-ribose) synthetase. Biochem. J . 185, 775-777