Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-09-29
2002-12-24
Celsa, Bennett (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S005000, C435S006120, C435S007500, C435S007720, C435S007910, C435S091100, C435S091200, C435S091500, C435S091500, C436S171000, C436S172000
Reexamination Certificate
active
06498005
ABSTRACT:
BACKGROUND OF THE INVENTION
Protein tyrosine and serine/threonine kinases are an important class of enzymes involved in the regulation of a number of biological processes. These enzymes are an increasingly significant target for new drug design. Methods for the rapid, high-throughput screening of chemical libraries for the identification of new inhibitory structures against these enzymes are actively being pursued.
Traditionally, the enzyme activity of protein tyrosine and serine/threonine kinases has been assayed by following the transfer of a radioactive phosphate group from &ggr;
32
P ATP to the tyrosine, serine or threonine residue of a suitable protein or peptide substrate (See, e.g., Witt, J. J. and Roskoski, R., Jr. (1975)
Anal. Biochem
. 66, 253-258 and Casnellie, J. E. (1991)
Methods Enzymol
. 200, 115-120). Following the phosphorylation reaction, the labeled product has to be separated from excess labeled ATP. This approach requires the use of radioactivity, involves multiple steps and is poorly suited for high-throughput screening applications. The scintillation proximity based approach represents an improvement, but it still has all the disadvantages of radioactive assays.
Several non-radioactive kinase assay approaches have been described that rely on the use of anti-phosphotyrosine antibodies. In the method of Braunwalder et al. (See, e.g., Braunwalder, A. F., Yarwood, D. R., Sills, M. A., and Lipson, K. E.
Anal. Biochem
. (1996) 238, 159-164), these antibodies are labeled with an Eu chelate. The substrate is immobilized to the surface of an ELISA plate, and the product of phosphorylation is detected using time-resolved fluorescence following incubation with the labeled antibodies. The main disadvantage of this method is its heterogeneous nature, which does not easily permit the detailed enzymological characterization of the kinase. As an alternative, a completely homogeneous approach has been described, where the binding of the anti-phosphotyrosine antibodies to the reaction product is detected by fluorescence polarization (See, e.g., Seethala, R. and Menzel, R.
Anal. Biochem
. (1997) 253, 210-218, and Seethala, R. and Menzel, R.
Anal. Biochem
. (1998) 255, 257-262). These methods may not work as well for serine/threonine kinase due to the lack of similar, high-affinity anti-phosphoserine/threonine antibodies.
Kinases can also assayed by separating the substrate from the phosphorylated product. This is generally done by a number of different methods, such as HPLC; capillary electrophoresis, TLC, ion-exchange chromatography etc. However, the need for an additional separation step represents a complication for high throughput screening applications.
It would generally be desirable to provide alternative approaches to assaying kinases as well as other enzymes having similar complications, which methods are adaptable to high-throughput screening methods. The present invention meets these and other needs.
SUMMARY OF THE INVENTION
In a first aspect, the present invention provides a method of assaying an enzyme-mediated coupling reaction between a first and a second reactant. The method comprises contacting the first reactant with the second reactant in the presence of the enzyme. The second reactant comprises a thiol derivative to yield a first product comprising a thiol derivative. The thiol derivative is then detected in the first product.
Another aspect of the present invention is a method of identifying a phosphorylatable substrate for a kinase enzyme. The method provides a phage display peptide library wherein each peptide in the library comprises a conserved phosphorylatable amino acid residue. The phage display library reacts with the kinase and ATP&ggr;S and contacted with a biotinylated haloacetate. Any biotinylated phage is immobilized on a solid support. DNA from any phage immobilized on the solid support is isolated and sequenced. A phosphorylatable peptide sequence is determined from a sequence of the DNA isolated from the phage.
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Jeong Sang
Nikiforov Theo T.
Caliper Technologies Corp.
Celsa Bennett
Filler Andrew L.
Murphy Matthew B.
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