Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2002-05-21
2003-04-15
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S021000, C435S023000, C435S024000, C435S004000, C435S174000, C435S183000, C435S007100
Reexamination Certificate
active
06548266
ABSTRACT:
Multicellular organisms must coordinate the functions of various cells and tissues. Cell-to-cell communication usually occurs through secretion of molecules by one cell, which molecules then bind to receptors on another cell. Phosphorylation of proteins is a post-translational mechanism used by cells to selectively transmit regulatory signals from the receptors outside of the target cell into the nucleus. The molecules mediating these biochemical reactions are predominantly phosphorylation enzymes, a group that includes protein kinases, which catalyze the addition of phosphate; and protein phosphatases, which catalyze the removal of phosphate. These phosphorylation enzymes are defined by the substrate upon which they act and by their apparent function. Substrate specificity is determined by the enzyme interaction with key amino acid sequences at certain portions of the substrate protein.
Secondary signal transduction molecules generated by protein kinases or protein phosphatases lead to a ‘cascade’ of signaling which, in turn, regulates numerous cell functions including cell division, differentiation, transformation and death. Inhibition of specific protein kinases and phosphatases provides a means of intervening in these signaling pathways. Defects in signaling mediated by phosphorylation enzymes are associated with a variety of pathological or clinical conditions, including cancer, diabetes, autoimmune and immunodeficiency diseases, and neural disorders. Assays for specific protein kinase or phosphatase inhibitors enable the discovery of therapeutic agents for the treatment of conditions characterized by defects in protein phosphorylation.
Assays currently used for screening drugs and ligands that act on phosphorylation enzymes, in general, involve exposing whole cells or their constituents to a test substance, and measuring either phenotypic changes in the cell culture, or biochemically analyzing cell extracts to assess the levels and identities of phosphorylated proteins. Large numbers of natural and synthetic compounds are typically screened before a potential therapeutic agent is found. The screening steps are preferably performed by a robot to incorporate high speed with relative consistency. “High throughput screening” is a term given to semi-automated procedures that assay large numbers of samples at a time. Cell-free assays offer enhanced specificity and higher throughput over whole cell assays.
Assays are typically conducted in multiwell polystyrene plates. Standard microtiter plates offer small volumes as well as a large number of wells which can be assayed simultaneously. One drawback to the assays for kinase and phosphatase inhibitors is the difficulty in obtaining a definitive signal from the wells. The target enzymes are in many cases very hard to isolate and only small amounts are available. Subsequently, a very important target may be limited as a screening tool due to its scarcity. A two-plate screening procedure, in which the inhibition of a target phosphorylation enzyme is measured over a range of concentrations, overcomes some of these limitations, however, disadvantages associated with this procedure include the cost of using two plates per assay, transfer inefficiency and limited throughput due to the requirement for manual washing. Substrate immobilization on the surface of a microtiter plate removes the latter limitation and increases assay throughput, however, limited binding capacity on the plate surface, relatively low substrate activity and steric hindrance can make it difficult to achieve a high signal output.
Further development of techniques for rapidly screening large numbers of variously-reactive phosphorylation substrates and inhibitors would be of great benefit in drug discovery.
RELEVANT LITERATURE
U.S. Pat. No. 5,759,787 describes the use of a chemiluminescent protein kinase assay using biotinylated substrate peptides captured on a streptavidin coated microtiter plate, and monoclonal antibodies to detect their phosphorylation. U.S. Pat. No. 5,763,198 describes a high-throughput screening assay using antiphosphate antibodies bound to plates to bind phosphorylated proteins. Forrer et al. (1998)
Biol Chem
379:1101-1111 disclose an enzyme-linked immunosorbent assay (ELISA) for measurement of the enzyme activity of the MAP kinases that involves immobilization of the respective kinase substrates on microtiter plates. An ELISA-based assay to measure trkA tyrosine kinase activity using a GST-fusion substrate immobilized on a microtiter plate is described by Angeles, et al. (1996)
Anal Biochem
236:49-55. In-gel detection of protein kinase activities toward oligopeptides is disclosed in Kameshita et al. (1996)
Anal Biochem
237:198-203.
Kameshita et al. (1997)
J Biochem
122:168-172 describe a peptide conjugate as a highly specific substrate for MAP kinase.
SUMMARY OF THE INVENTION
Improved methods are provided for assaying compounds that modulate phosphorylation. The methods of the invention find particular use in high-throughput screening systems, gel assays and affinity chromatography. The substrate for an enzyme is immobilized by covalently linking it to a polymer and attaching the modified substrate to the surface of a solid support, which may be a plate, column resin, etc. The resulting increased enzyme-substrate contact leads to enhanced phosphorylation and increased sensitivity in detection. In one embodiment of the invention, a simplified and fully automated one-plate assay procedure is provided. The assay involves addition of the phosphorylation reaction mixture containing the enzyme and radiolabelled phosphate to the immobilized substrate and measurement of phosphorylation by scintillation counting.
In another embodiment of the invention, the modified substrate is phosphorylated and retained in a gel matrix, and phosphorylation is measured by autoradiography. In yet another embodiment of the invention, the modifed substrate is immobilized on beads to form an enzyme affinity matrix. The subject methods are useful for the rapid screening of inhibitors of phosphorylation, which may lead to the discovery of potential therapeutic agents.
REFERENCES:
patent: 5759787 (1998-06-01), Strulovici
patent: 5763198 (1998-06-01), Hirth et al.
patent: 6054280 (2000-04-01), Lemmon et al.
Angeles, Thelma S. et al.,Enzyme-Linked Immunosorbent Assay for TRKA Tyrosine Kinase Activity, Analytical Biochemistry, (Sep. 12, 2995), V. 236, pp. 49-55 Article No. 0130, Academic Press, Inc.
Forrer, Patrik et al.,Enzyme-Linked Immunosorbent Assay for Measurement of JNK, ERK, and P38 Kinase Activities, Biol. Chem., (1998), V. 379, pp. 1101-1111, Walter de Gruyter-Berlin-New York.
Kameshita, Isamu et al.,Detection of Protein Kinase Activities Toward Oligopeptides in Sodium Dodecyl Sulfate-Polyacrylamide Gel1, Analytical Biochemistry, (Nov. 13, 1995), V. 237, pp. 198-203, Academic Press, Inc.
Kameshita, Isamu et al.,A New Peptide Conjugate as a Highly Specific Substrate for Map Kinase1, J. Biochem., (Feb. 19, 1997), V. 122, pp. 168-172.
Williams, Richard T. et. al.,Identification Assay and Purification of a CDC2-Activating Threonine-161 Protein Kinase from Human Cells, Archives of Biochemistry and Biophysics, (Jun. 21, 1994), V. 314, pp. 99-106, No. 1, Academic Press, Inc.
Woodgett, James Robert et al.,Use of Peptide Substrates for Affinity Purification of Protein-Serine Kinases, Analytical Biochemistry, (Feb. 9, 1989), V. 180, pp. 237-241, Academic Press, Inc.
Sanghera Jasbinder
Wang Shisen
Zhang Zaihui
Bozicevic Field & Francis LLP
Kinetek Pharmaceuticals, Inc.
Leary Louise N.
Sherwood Pamela J.
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