Fluorescent probes for use in protein kinase inhibitor binding assay
AssigneeBoehringer Ingelheim Pharmaceuticals, Inc.
InventorsAnthony S. Prokopowicz, III, Martha Priscilla Brown, Jessi Marie Wildeson, Scott Jakes, Mark E. Labadia
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Abstract
The invention provides methods relating to a novel screening assay format that can be applied to broad members of the protein kinase gene family. The assay uses a series of labeled, active site probes described herein that can be displaced by an inhibitor agent. The Kd for the inhibitor compound is derived based on the Kd of the probe for the kinase and the dose response of the inhibitor agent. The invention also provides active site probes suitable for use with the screening method.
Claims3
A fluorescent probe having the following general formula (I):
- R1-L-X—Y (I)
- wherein
- R1 is a nitropyrimidine of formula (III):
- wherein R4 is selected from phenyl and naphthyl and is optionally substituted with one to three groups selected from Cl, F, Br, I, —CH3, —CN, —CO2CH3, —C(O)NR5R6, —NO2, —OH, —NH2, and —CF3;
- L is
- a direct bond or a C1–C16 alkylene group, a C2–C16 alkenylene group or a C2–C16 alkynylene group, wherein one or more of the available —CH2— groups present in the C1–C16 alkylene group, C2–C16 alkenlene group or C2–C16 alkynylene group is optionally and independently replaced with —O—, —C(O)—, —S(O)p— wherein p is 0 to 2, or —N(R2)—;
- X is selected from the group consisting of O, S, —N(R2)C(O)—, —C(O)N(R2)—, —N(R2)C(S)—, —C(S)N(R2)—, —N(R2)C(S)NH—, —NHC(S)N(R2)—, —N(R2)C(O)NH—, —NHC(O)N(R2), —SO2NR2—, —NR2SO2—, —CH2N(R2)—, —N(R2)CH2—, —CH2S—, —SCH2—, —C(O)CH2S—, SC(O)CH2—, —NHCH2CH2S—, —SCH2CH2NH—, —NC(O)O—, —ONC(O)—, —C(O)O—, —OC(O)—, —NH—N═C(R2)—, —C(R2)═N—NH—, —NHCH(R2)—, and —CH(R2)NH—;
- R2 is H or C1–3alkyl;
- and
- Y is a fluorescent label which fluoresces at a wavelength above about 450 nm.
A fluorescent probe having the following general formula (I):
- R1-L-X—Y (I)
- wherein
- R1 is a nitropyrimidine of formula (III):
- wherein R4 is selected from phenyl and naphthyl and is optionally substituted with one to three groups selected from Cl, F, Br, I, —CH3, —CN, —CO2CH3, —C(O)NR5R6, —NO2, —OH, —NH2, and —CF3;
- L is
- a direct bond or a C1–C16 alkylene group, a C2–C16 alkenylene group or a C2–C16 alkynylene group, wherein one or more of the available —CH2— groups present in the C1–C16 alkylene group, C2–C16 alkenylene group or C2–C16 alkynylene group is optionally and independently replaced with —O—, —C(O)—, —S(O)p— wherein p is 0 to 2, or —N(R2)—;
- X is selected from the group consisting of O, S, —N(R2)C(O)—, —C(O)N(R2)—, —N(R2)C(S)—, —C(S)N(R2)—, —N(R2)C(S)NH—,—NHC(S)N(R2)—, —N(R2)C(O)NHh—, —NHC(O)N(R2), —SO2NR2—, —NR2SO2—, —CH2N(R2)—, —N(R2)CH2—, —CH2S—, —SCH2—, —C(O)CH2S—, —SC(O)CH2—, —NHCH2CH2S—, —SCH2CH2NH—, —NC(O)O—, —ONC(O)—, —C(O)O—, —OC(O)—, —NH—N═C(R2)—, —C(R2)═N—NH—, —NHCH(R2)—, and —CH(R2)NH—;
- R2 is H or C1-3alkyl;
- and
- Y is a fluorescent label chosen from rhodamine and rhodamine derivatives, fluorescein and fluorescein derivatives, 4,4-difluor-4-bora-3a,4a-diaza-s-indacene and its derivatives, cyanine dyes, and Alexa Fluor® dyes.
Dependencies are inferred from the claim text.