Fluorescence polarization hERG assay
AssigneeLife Technologies Corporation
InventorsDavid Piper, Kurt Vogel, Mohammed Saleh Shekhani, Stephen Hess, Steve Duff, Thomas Livelli, Zhong Zhong
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Abstract
Disclosed are assays, methods, and kits for the screening of test compounds for their capability to induce cardiotoxicity in a subject. In particular, whether a test compound has the effect to prolong the Q-T interval as measured by an electrocardiogram in a human. The assays, methods, and kits disclosed herein make use of the binding interaction between novel fluorescent tracers and the hERG K+ channel, and the propensity of a test compound to influence that binding interaction.
Claims34
An assay for screening a test compound, wherein said assay is a binding assay using a fluorescent tracer and a source of a hERG K+ channel or fragment thereof, comprising:
- a) incubating an assay sample comprising the fluorescent tracer, the source of the hERG K+ channel or fragment thereof, an assay buffer, and a test compound;
- and
- b) measuring the amount of the fluorescent tracer bound to the hERG K+ channel or fragment thereof in the sample,
- wherein the fluorescent tracer is a compound of structural formula:
- or a pharmaceutically acceptable salt thereof, wherein:
- Ar is a benzo ring;
- R1 and R2 are independently hydrogen or —NHSO2CH3;
- the ring system comprising W, X, and Y 6-membered ring system wherein Y is —O—, W is C═O, —CH2—, C═NOH, —NH—, CHNH2, ═CH—, or ═N—, and X is —CH2— or ═CH—;
- B is a 6-membered N-containing ring;
- L is —(CR4R5)m-Q—(CR4R5)q—NH—[CZ—(CR4R5)u-(D)w]z—, wherein
- R4 and R5 are independently hydrogen or C1-6 alkyl,
- Q is a bond, —O—, C═O, CHOH, —NR5— or —S(O)n—,
- Z is ═O or ═S,
- D is —O—, —S(O)n—, —NR5—, or —NR5SO2—,
- m and q are independently 1 to about 5,
- n is 0, 1, or 2,
- u is 0 to about 7,
- w is 0 or 1,
- z is 1 or 2;
- and
- R3 is a fluorescent dye.
A kit for screening test compounds, said kit comprising:
- a) a fluorescent tracer or a pharmaceutically acceptable salt thereof;
- b) a source of the hERG K+ channel or fragment thereof;
- and
- c) an assay buffer
- wherein the fluorescent tracer is a compound of structural formula:
- wherein:
- Ar is a benzo ring;
- R1 and R2 are independently hydrogen or —NHSO2CH3;
- the ring system comprising W, X, and Y is a 6-membered ring system wherein Y is —O—,
- W is C═O, —CH2—, C═NOH, —NH—, CHNH2, ═CH—, or ═N—, and X is —CH2— or ═CH—;
- B is a 6-membered N-containing ring;
- L is —(CR4R5)m-Q-(CR4R5)q—NH—[CZ—(CR4R5)u-(D)w]z-, wherein
- R4 and R5 are independently hydrogen or C1-6 alkyl,
- Q is a bond, —O—, C═O, CHOH, —NR5— or —S(O)n—,
- Z is ═O or ═S,
- D is —O—, —S(O)n—, —NR5—, or —NR5SO2—,
- m and q are independently 1 to about 5,
- n is 0, 1, or 2,
- u is 0 to about 7,
- w is 0 or 1,
- z is 1 or 2;
- and
- R3 is a fluorescent dye.
Dependencies are inferred from the claim text.