Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2003-06-20
2004-11-16
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S015000, C435S004000, C435S007500, C435S007700, C435S968000, C435S007920, C435S007940, C435S287200, C436S517000, C530S350000, C530S352000, C530S333000
Reexamination Certificate
active
06818413
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to novel fluorescence-based assays for kinases and phosphatases which can be used in high throughput screening.
BACKGROUND OF THE INVENTION
Eukaryotes employ phosphorylation and dephosphorylation of specific proteins to regulate many cellular processes (T. Hunter,
Cell
80:225-236 (1995); (Karin, M.,
Curr. Opin. Cell Biol
. 3:467-473 (1991)). These processes include signal transduction, cell division, and initiation of gene transcription. Thus, significant events in an organism's maintenance, adaptation, and susceptibility to disease are controlled by protein phosphorylation and dephosphorylation. These phenomena are so extensive that it has been estimated that humans have around 2,000 protein kinase genes and 1,000 protein phosphatase genes (T. Hunter,
Cell
80:225-236 (1995)), some of these likely coding for disease susceptibility. For these reasons, protein kinases and phosphatases are good targets for the development of drug therapies.
The most frequently used protein kinase and phosphatase screens employ either radioactive ATP or ELISAs. However, the use of radioactive ATP is undesirable due to the attendant costs of record-keeping, waste-disposal, and the fact that the assay format is not homogeneous. ELISAs are undesirable because they have a lower assay throughput due to the extra steps required for both washing and the enzyme reaction.
Fluorescence detection in the visible wavelengths offer an alternative to the use of radiotracers or ELISAs for kinase and phosphatase assays, as fluorescence offers detection limits comparable to those of radioactivity. Furthermore, this eliminates the cost of radioactive waste disposal. For example, the change in absorbance and fluorescence spectra of phosphotyrosine which occurs upon dephosphorylation has been used for the continuous monitoring of protein-tyrosine phosphatase (PTP) activity (Zhao, Z. et al.,
Anal. Biochem
. 202:361-366 (1993)). However, previously developed fluorometric assays for kinases and phosphatases have not been especially amenable to the requirements of high throughput screening.
Fluorescence detection frequently offers the advantage of using homogeneous assay formats (i.e.—“mix, incubate, and read”). Indeed, the high throughput screening (HTS) field is moving rapidly toward the use of fluorescence, luminescence, absorbance, and other optical methods. Two fluorescence techniques, fluorescence polarization (FP) and fluorescence resonance energy transfer (FRET) are finding widespread use for assays, both in the private sector for HTS, secondary assays including kinetics, SAR studies, etc., and in university laboratories. The use of FP is particularly desirable since its readout is independent of the emission intensity (Checovich, W. J., et al.,
Nature
375:254-256 (1995); Dandliker, W. B., et al.,
Methods in Enzymology
74:3-28 (1981)) and is thus insensitive to the presence of colored compounds that quench fluorescence emission. FRET, although susceptible to quenching, can also be used effectively, especially for continuous enzyme assays.
From the forgoing, it will be clear that there is a continuing need for the development of cost-effective, facile, and sensitive optical kinase and phosphatase assays for both high throughput screening (HTS) and secondary assays.
Information Disclosure
Checovich, W. J., et al.,
Nature
375:254-256 (1995).
Dandliker, W. B., et al.,
Methods in Enzymology
74:3-28 (1981).
E. Harlow and D. Lane, eds.,
Antibodies A Laboratory Manual
, Cold Spring Harbor Laboratory (1988).
T. Hunter,
Cell
80:225-236 (1995).
Leavine, L. M., et al.,
Anal. Biochem
. 247:83-88 (1997).
Owicki, J. C.,
Genetic Engineering News
17:27 (Nov. 1, 1997).
Rotman, B., et al.,
Proc. Nat. Acad. Sci
. 50:1-6 (1963).
Seethala, R. and R. Menzel,
A Fluorescence Polarization Tyrosine Kinase Assay for High Throughput Screening
, 3rd Annual Conference of The Society for Biomolecular Screening, San Diego, Calif., Sep. 22-25, 1997.
SUMMARY OF THE INVENTION
The invention relates to novel fluorescence-based assays for protein kinases and phosphatases which can be used in high throughput screening. The methods of the invention utilize a competitive immunoassay to determine the amount of substrate that is phosphorylated or dephosphorylated during the course of a kinase or phosphatase reaction to yield a product, as well as the phosphorylating or dephosphorylating activity of a kinase or phosphatase.
Thus, in one embodiment, the invention relates to a method of determining the phosphorylating activity of an enzyme comprising the steps of:
(a) combining the enzyme with
(i) a reporter molecule comprising a fluorescent label and a phosphorylated amino acid, wherein the amino acid is selected from the group consisting of serine, threonine and tyrosine;
(ii) a substrate molecule comprising the same amino acid that is phosphorylated in said reporter, wherein said substrate molecule is capable of being phosphorylated at said amino acid by said enzyme to yield a product;
(iii) an antibody which selectively binds to a molecule comprising the phosphorylated amino acid; and
(iv) a high-energy phosphate source;
(b) measuring the fluorescence polarization (FP), FQ, or fluorescence resonance spectroscopy (FCS) of the reporter following the combination of step (a); and
(c) using the FP, FQ, or FCS measurement of step (b) to determine the activity of the enzyme.
In another embodiment, the invention relates to a method for determining the dephosphorylating activity of an enzyme comprising the steps of:
(a) combining the enzyme with
(i) a reporter molecule comprising a fluorescent label and a phosphorylated amino acid, wherein the amino acid is selected from the group consisting of serine, threonine and tyrosine;
(ii) a substrate molecule comprising the same phosphorylated amino acid as said reporter, wherein said substrate molecule is capable of being dephosphorylated at said amino acid by said enzyme to yield a product; and
(iii) an antibody which selectively binds to a molecule comprising the phosphorylated amino acid;
(b) measuring the FP, FQ, or FCS of said reporter following the combination of step (a); and
(c) using the FP, FQ, or FCS measurement of step (b) to determine the activity of the enzyme.
The methods of the invention can also be used to determine the phosphorylation or dephosphorylation of a substrate molecule by an enzyme. Thus, in another embodiment, the invention relates to a method for determining the phosphorylation of a substrate molecule by an enzyme at an amino acid selected from the group consisting of serine, threonine and tyrosine, comprising the steps of:
(a) combining the substrate molecule with
(i) the enzyme
(ii) a reporter molecule comprising a fluorescent label and a phosphorylated amino acid, wherein the amino acid is the same amino acid which is phosphorylated in the reporter;
(iii) an antibody which selectively binds to a molecule comprising the phosphorylated amino acid; and
(iv) a high-energy phosphate source;
(b) measuring the FP, FQ, or FCS of the reporter following the combination of step (a); and
(c) using the FP, FQ, or FCS measurement of step (b) to determine whether the substrate molecule has been phosphorylated.
In another embodiment, the invention relates to a method for determining the dephosphorylation of a substrate molecule by an enzyme, wherein the substrate molecule comprises a phosphorylated amino acid, and wherein the amino acid is selected from the group consisting of serine, threonine and tyrosine, comprising the steps of:
(a) combining the substrate molecule with
(i) the enzyme;
(ii) a reporter molecule comprising a fluorescent label and a phosphorylated amino acid, wherein the reporter molecule comprises the same phosphorylated amino acid as the substrate molecule; and
(iii) an antibody which selectively binds to a molecule comprising the phosphorylated amino acid;
(b) measuring the FP, FQ, or FCS of the reporter following the combination of step (a); and
(c) using the FP, FQ, or FCS measurement of step (b) to determine w
Epps Dennis E.
Marschke Charles K.
Chin Christopher L.
Do Pensee T.
Kerber Lori K.
Pharmacia and Upjohn Company
Rehberg Edward F.
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