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US 7,070,938 B2Granted
Method to detect modulators of VEGF kinase domain
AssigneeOrtho-McNeil Pharmaceutical, Inc.
InventorsDana L. Johnson, Stuart L. Emanuel
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Abstract
The present invention relates to assays for the detection of compounds with pharmacological activity, particularly for the detection of modulators of rat vascular endothelial growth factor receptor (rat VEGF-R2) kinase domain.
Claims2
A method to measure the effect of a putative VEGF-R2 kinase activity modulating compound comprising the steps, in order:
- A) providing a test compound, a rat VEGF-R2 kinase fusion protein (SEQ ID NO:4) comprising N-terminal hexahistidine linked to amino acids 786–1343 of the rat VEGF-R2 protein in a solution suitable to provide rat VEGF-R2 catalytic activity and containing 33P-γ-ATP as the source of phosphate;
- B) contacting the compound and the kinase fusion protein for sufficient time to provide a 33P phosphorylated substrate;
- C) isolating the phosphorylated kinase fusion protein by affinity capture using a multiwell assay plate;
- D) removing remaining 33P-γ-ATP by first aspirating the aqueous solution and then washing the plate with a phosphate buffered saline solution containing a divalent cation chelator in a concentration of about 1 mM to 100 mM;
- and
- E) detecting a change in kinase activity by monitoring the rate or absolute amount of 33P transfer to the kinase fusion protein by autophosphorylation in the presence of the compound.
3 more elements
A method to measure the effect of a putative VEGF-R2 kinase activity modulating compound comprising the steps, in order:
- A) providing a test compound, a rat VEGF-R2 kinase fusion protein (SEQ ID NO:4) comprising amino acids 786–1343 of the rat VEGF-R2 protein, and a biotinylated rat VEGF-R2 substrate in a solution suitable to provide rat VEGF-R2 catalytic activity and containing 33P-γ-ATP as the source of phosphate;
- B) contacting the compound, kinase fusion protein, and substrate for sufficient time to provide a 33P phosphorylated substrate;
- C) isolating the phosphorylated substrate by affinity capture using an avidin or streptavidin coated multiwell assay plate;
- D) removing remaining 33P-γ-ATP by first aspirating the aqueous solution and then washing the plate with a phosphate buffered saline solution containing a divalent cation chelator in a concentration of about 1 mM to 100 mM;
- and
- E) detecting a change in kinase activity by monitoring the rate or absolute amount of 33P transfer to the substrate in the presence of the compound.
3 more elements
Dependencies are inferred from the claim text.