FLUORESCENCE POLARIZATION hERG ASSAY

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.

1 - 42 . (canceled) 
     
     
         43 . A hERG K +  channel-expressing cell population, wherein said cell population expresses at least about 100 μmol of hERG K +  channel per mg of total membrane protein. 
     
     
         44 . The hERG K +  channel-expressing cell population of  claim 43 , wherein cells thereof express a hERG K +  channel for which the hERG current as determined by patch clamping with a fully automated high throughput patch clamp system is in a range of about 1500 pA to about 2500 pA. 
     
     
         45 . The hERG K +  channel-expressing cell population of  claim 43 , wherein cells thereof are HEK 293 cells or CHO cells. 
     
     
         46 . The hERG K +  channel-expressing cell population of  claim 43 , wherein cells thereof have been transfected with an expression vector selected from the group consisting of:
 i) an isolated and purified nucleic acid comprising a nucleotide sequence which encodes a hERG K +  channel having an amino acid sequence that is at least 80% homologous to that of SEQ ID NO: 1 or a fragment thereof; and 
 ii) an isolated and purified nucleic acid comprising a nucleotide sequence which encodes a hERG K +  channel having the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. 
 
     
     
         47 . The hERG K +  channel-expressing cell population of  claim 46 , wherein the nucleic acid further comprises a nucleotide sequence which encodes an internal ribosomal entry site protein and a nucleotide sequence which encodes CD-8 plasma membrane protein. 
     
     
         48 . The hERG K +  channel-expressing cell population of  claim 47 , wherein the nucleotide sequences which encode the internal ribosomal entry site protein and the CD-8 plasma membrane protein are located successively downstream from the nucleotide sequence which encodes the hERG K +  channel. 
     
     
         49 . The hERG K +  channel-expressing cell population of  claim 48 , wherein expression of the hERG K +  channel is coupled to expression of the CD-8 plasma protein by means of the nucleotide sequence which encodes an internal ribosomal entry site protein. 
     
     
         50 . (canceled)