Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of the 3′-ester bond of cyclic AMP (cAMP) and cyclic GMP (cGMP), important second messengers in the transduction of a variety of extracellular signals. There is growing interest in the study of PDEs as drug targets for novel therapeutics. We describe the development of a homogeneous fluorescence polarization assay for PDEs based on the strong binding of PDE reaction products (i.e., AMP or GMP) onto modified nanoparticles through interactions with immobilized trivalent metal cations. This assay technology (IMAP) is applicable to both cAMP- and cGMP-specific PDEs. Results of the assay in 384- and 1536-well microplates are presented.
References
1.
Porath J: IMAC-immobilized metal ion affinity based chromatography. Trends Anal Chem1988;7:254-259.
2.
Arnold FH: Metal-affinity separations: a new dimension in protein processing. Bio/Technology1991;9:151-156.
3.
Muszynska G, Andersson L, Porath J: Selective adsorption of phosphoproteins on gel-immobilized ferric chelate. Biochemistry1986;25:6850-6853.
Andersson L: Recognition of phosphate groups by immobilized aluminium (III) ions. J Chromatogr1991;539:327-334.
6.
Huang W, Zhang Y, Mark D, et al: A homogeneous fluorescence polarization assay adaptable for a range of serine/threonine and tyrosine kinases. (Manuscript submitted, 2001.)
7.
Perry MJ, Higgis GA: Chemotherapeutic potential of phosphodiesterase inhibitors. Curr Opin Chem Biol1998;2:472-481.
8.
Conti M: Phosphodiesterase and cyclic nucleotide signaling in endocrine cells. Mol Endocrinol2000;14:1317-1327.
9.
Conti M, Jin S-LC: The molecular biology of cyclic nucleotide phosphodiesterases. Prog Nucleic Acid Res Mol Biol1999;63:1-38.
10.
Johnson JD, Walters JD, Mills JS: A continuous fluorescence assay for cyclic nucleotide phosphodiesterase hydrolysis of cyclic GMP. Anal Biochem1987;162:291-295.
11.
Hiratsuka T: New fluorescent analogs of cAMP and cGMP available as substrates for cyclic nucleotide phosphodiesterase. J Biol Chem1982;257:13554-13558.
12.
Kawski A: Excitation energy transfer and its manifestation in isotropic media. Photochem Photobiol1983;38:487-504.
13.
Takase Y, SaeKi T, Fujimoto M, et al: Cyclic GMP phosphodiesterase inhibitors. 1. The discovery of a novel potent inhibitor, 4-((3,4-(methylenedioxy)benzyl)amino)-6,7.8-trimethoxyquinazoline. J Med Chem1993;36:3765-3770.
14.
Jin S-LC.Bushnik T, Lan L, et al: Subcellular localization of rolipram-sensitive, cAMP-specific phosphodiesterase. J Biol Chem1998;273:19672-19678.
15.
Yan C, Zhao AZ, Bently JK, et al: The calmodulin-dependent phosphodiesterase gene PDE1C encodes several functionally different splice variants in a tissue-specific manner. J Biol Chem1996;271:25699-25706.
16.
Zhang J, Chung TDY, Oldenburg KR: A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Bioniol Screen1999;4:67-73.
17.
Owicki J: Fluorescence polarization and anisotropy in high throughput screening: perspectives and primer. J Biomol Screen2000;5:297-306.
