We have developed a novel assay for measuring the activity of an enzyme that transfers multiple adenine-containing groups to an acceptor protein. The assay is based on fluorescence polarization (FP) technology in a 1536-well plate format. In the assay, a long wavelength fluorescence tracer, Texas Red (Rhodamine), was covalently conjugated to adenine of the donor substrate through a C6 spacer arm. As a result of the transfer of the adenine-containing moieties to the acceptor protein substrate, the rotational correlation time of the Texas Red conjugate increased, hence increasing the degree of fluorescence polarization. The pharmacological profile and kinetics of the enzyme measured according to the FP method were consistent with those determined previously by conventional analysis. We have successfully executed a 250,000-compound high throughput screening program based on the FP assay method. The quality and validity of the assay were verified by a variety of statistical analyses.
References
1.
Boyer, P.D. (ed.). (1973). The Enzyme, Vols. VIII and IX: Group Transfer. Academic Press, New York.
Yablonski, D., Kuhne, M.R., Kadlecek, T., Weiss, A. (1998). Uncoupling of nonreceptor tyrosine kinases from PLC-yl in an SLP-76-deficient T cells. Science281:413-416.
4.
Burbaum, J.J. (1998). Miniaturization technologies in HTS: How fast, how small, how soon?Drug Discovery Today3:313-322.
5.
Abriola, L., Chin, M., Fuerst, P., Schweitzer, R., Sills, M.A. (1999). Digital imaging as a detection method for a fluorescent protease assay in 96-well and miniaturized assay plate formats. J. Biomol. Screen.4:121-127.
6.
Maffia, A.M., III, Kariv, K., Oldenburg, K.R. (1999). Miniaturization of a mammalian cell-based assay: Luciferase reporter gene readout in a 3 microliter 1536-well plate. J. Biomol. Screen.4:137-142.
7.
Hill, D.C. (1998). Trends in development of high throughput screening technologies for rapid discovery of novel drug. Curr. Opin. Drug Discovery Dev.1:92-97.
8.
Stahl, W., Major, J. (1999). What is the future of high throughput screening?J. Biomol. Screen.4:117-119.
9.
Auer, M., Moor, K.J., Meyer-Almes, F.J., Guenther, R., Pope, A.J., Stoeckli, K.A. (1998). Fluorescence correlation spectroscopy: Lead discovery by miniaturized HTS. Drug Discovery Today3:457-465.
10.
Oldenburg, K.K., Zhang, J., Chen, T., Maffia, A., III., Blom, K.F., Combs, A.P., Chung, T.D.Y. (1998). Assay miniaturization for ultra-high throughput screening of combinational and discrete compound libraries: A 9600-well (0.2 microliter) assay system. J. Biomol. Screen. 3:55-62.
11.
Vollert, H. (1999). HMR's pilot screens in 1536: How robust is screening in high density plates? In Proceedings of the IBC's 2nd Annual International Conference on Miniaturization Technologies, Berkeley, CA.
12.
Binnie, A. (1998). Assay miniaturization for combinatorial chemistry. In Proceedings of the IBC's Drug Discovery Technology '98 meeting, Boston, MA.
13.
Schullek, J.R., Butler, J.H., Ni, Z.J., Chen, D., Yuan, Z. (1997). A high-density screening format for encoded combinational libraries: Assay miniaturization and its application to enzymatic reactions. Anal. Biochem.246:20-29.
14.
Pope, A.J., Moore, K.J. (1998). Homogeneous fluorescence readouts for miniaturized high throughput screening: Theory and practice. Proceeding, The Society for Biomolecular Screening. Baltimore, MD.
15.
Bosworth, N., Towers, P. (1989). Scintillation proximity assay. Nature341:167-168.
16.
Cantor, C., Schimmel, P. (eds.). (1980). Biophysical Chemistry, Part II: Techniques for the Study of Biological Structure and Function. W.H. Freeman, San Francisco.
17.
Seethala, R., Menzel, R. (1997). A homogeneous, fluorescence polarization assay for Scr-family tyrosine kinases. Anal. Biochem.253:210-218.
18.
Wild, D. (ed.). (1994). The Immunoassay Handbook. Stockton Press, New York.
Rogers, M.V. (1997). Light on high-throughput screening: Fluorescence-based assay technologies. Drug Discovery Today2:156-160.
21.
Zhang, J., Chung, T.D.Y., Oldenburg, K.R. (1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen.4:67-73.
22.
Dunnington, D.J., Fogel, P.Designing assays for high throughput screening. In Principles and Practice of High Throughput Screening, K. Murray (ed.). Blackwell Publications, Oxford, UK (in press).
23.
Neter, J., Wasserman, W., and Kuther, M.H. (1985). Applied linear statistical models: Regression, analysis of variance, and experimental designs. Irwin Publications, Inc., Homewood, IL.
