	US 7,588,899 B2
	Fluorescent probes for use in protein kinase inhibitor binding assay
Anthony S. Prokopowicz, III, Stormville, N.Y. (US); Martha Priscilla Brown, Southbury, Conn. (US); Jessi Marie Wildeson, Thomaston, Conn. (US); Scott Jakes, Southbury, Conn. (US); and Mark E. Labadia, Brookfield, Conn. (US)
Assigned to Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Conn. (US)
Filed on Jul. 25, 2006, as Appl. No. 11/492,654.
Application 11/492654 is a division of application No. 10/955129, filed on Sep. 30, 2004, granted, now 7,125,996, filed on Oct. 24, 2006.
Claims priority of provisional application 60/508539, filed on Oct. 03, 2003.
Prior Publication US 2006/0263841 A1, Nov. 23, 2006
Int. Cl. G01N 33/53 (2006.01); C12Q 1/48 (2006.01)

U.S. Cl. 435—7.2 [435/15]

12 Claims

1. A method for the identification of a potential kinase inhibitor agent comprising the steps of:

a) establishing the K_dof an active site probe for an ATP binding site, wherein the probe is a fluorescent probe;

b) contacting the active site probe with a protein comprising a kinase domain and measuring a signal from said probe to establish a base line level signal;

c) incubating the active site probe: kinase complex with a candidate inhibitor agent and measuring the signal from said probe;

d) comparing the signal from step b) with the signal from step c); and

e) deriving the K_dfor the candidate inhibitor agent based on the K_dof the probe for its ATP binding site and the dose response of the candidate inhibitor agent; under conditions where the probe can be displaced by the candidate inhibitor agent and wherein the active site probe binds to the ATP binding domain of said kinase, wherein the signals are measured as a shift in fluorescent polarization of the probe.