Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2000-03-07
2001-12-11
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S007100, C435S007200, C435S325000, C435S007210, C435S007400, C435S029000, C435S069100, C435S455000, C435S456000, C435S348000, C435S254110, C435S007720, C435S254200, C435S320100, C435S183000
Reexamination Certificate
active
06329137
ABSTRACT:
BACKGROUND OF THE INVENTION
Protein tyrosine phosphatases (PTPs) (EC 3.1.3.48) dephosphorylate tyrosyl residues, acting in concert with protein tyrosine kinases (PTKs) to control tyrosine phosphorylation of proteins in the cell. This reversible phosphorylation at tyrosyl residues has been found in many signal transduction pathways, such as those involved in the action of growth factors, control of cellular proliferation, differentiation and metabolism (1, 2). Although PTPs are very different in size and structural organization, they contain a unique and highly conserved active site sequence (1,2,6). Representative examples of PTPs are CD45 and PTP1B. CD45, is a transmembrane receptor-type PTP that activates protein tyrosine kinase p561ck and p59fyn, and is involved in T cell (3,4) and B cell(5) signaling. PTP1B is a nonreceptor PTP containing a single catalytic domain and has been implicated in insulin-resistant states and type 2 diabetes (6,7-9).
Specific and selective PTP inhibitors have a potential value as biological tools to reveal the function of individual phosphatases in cellular signal transduction pathways and as disease-modulating drugs. To date, sodium orthovanadate (vanadate), pervanadate and phenylarsine oxide are known PTP inhibitors. None of these exhibit tyrosine phosphatase selectivity. Small peptides containing the hydrolytically stable difluorophosphonomethyl phenylalanine were found to be inhibitors of PTP1B (10). Available cell-based systems that can be used for the identification of a broad range of selective PTP inhibitors employ downstream readouts for the quantitation of PTP activity.
A limitation in this type of assay is that test inhibitors can interact at sites in the signal transduction pathway other than at the enzyme of interest, thus giving rise to an erroneous outcome, such as a false positive result for a test inhibitor. To date there are no known assays without intermediate steps, having an immediate end-point and that allow for a direct quantitation of PTP activity.
Previously, an in vitro intact cell assay for screening nonsteroidal anti-inflammatory drug inhibition of human cyclooxygenase was developed. The assay utilizes the baculovirus expression system in sf9 insect cells (11).
Consequently, one object of the present invention was to develop an assay for PTP activity that overcomes the defects in the prior art.
Another object was to provide an assay for PTP activity that is reliable and reproducible.
More particularly, an object of the present invention was to select cell-permeable PTP substrates such that the hydrolysis of these substrates provides a direct readout of the phosphatase activity.
These and other objects will become apparent to those of ordinary skill from the teachings provided herein.
The application refers to a number of publications, the content of which is hereby incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
A method for assaying protein tyrosine phosphatase (PTP) activity in a eukaryotic host cell is disclosed comprising the steps of:
(a) transforming a host cell with a vector containing a nucleotide sequence encoding a PTP to provide a transformed host cell line;
(b) incubating said transformed host cell with a substrate to said PTP activity; and
(c) comparing the amount of PTP activity in the transformed host cell to a standard.
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Cromlish Wanda
Kennedy Brian
Guzo David
McGinnis James L.
Merck Frosst Canada & Co.
Rose David L.
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