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High throughput assay systems and methods for identifying agents that alter surface expression of integral membrane proteins

A surface expression, membrane protein technology, applied in biochemical equipment and methods, microbial determination/inspection, measurement devices, etc., can solve problems such as proarrhythmia, short atrial APD, loss of efficacy, etc.

Inactive Publication Date: 2006-12-13
CHANXPRESS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because of its slow activation kinetics, I Ks Role in atrial repolarization may be limited due to relatively short atrial APD
Thus, although I Ks Blockers may provide unique benefit for ventricular arrhythmias, but their ability to affect supraventricular tachycardia (SVT) is thought to be minimal
[0027] Another major deficiency or limitation of most currently available class III antiarrhythmic drugs is that their effects are increased or become more pronounced during or during bradycardia or slow rhythms and this leads to their potential to cause proarrhythmias
On the other hand, they lose their primary efficacy during tachycardia or disease states when these agents or drugs are expected and most needed

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1 - Rescue of hERG-G601S surface expression with astemizole

[0102] HEK-293 cells stably expressing hERG-G601S-HA were plated in 96-well black plates (BD Discovery Labware) with clear bottom BIOCOAT poly-D-lysine cellware. Cells were plated in (8×10 4 cells / well) containing 10% FBS plus penicillin-streptomycin and geneticin (geneticin) (G418; 0.5mg / ml) in the complete culture solution of DMEM / F12, at 37°C / 5%CO before adding drugs 2 Incubate for 8 hours. Stock solutions (200 μM, 1 mM and 5 mM) of the drugs (astemizole, norastemizole, fexofenadine) were prepared. Working dilutions (200 nM, 1 μM and 5 μM) were prepared in DMEM / F 12 containing 10% FBS just before addition. Vehicle consisted of 0.1% DMSO in DMEM / F12 containing 10% FBS. The medium soaking the cells was removed and replaced with control medium (100 μl / well) containing drug or vehicle. There are 3 test wells and 3 control wells for each concentration of each drug. Prior to the start of the surfac...

Embodiment 2

[0106] Example 2 - Rescue of drug removal with astemizole that removes the binding site in hERG

[0107] HEK-293 cells in 35 mm dishes were transiently transfected with cDNA encoding hERG-G601S-HA or hERG-G601S / F656C-HA. Astemizole (1 [mu]M) was added to some plates and vehicle control (DMSO) to others 24 hours after transfection and these plates were incubated overnight.

[0108] Following incubation, cells were fixed with paraformaldehyde, and HA markers for surface expression were processed as described above (except for a proportional increase in reagent volume). Chemiluminescence signals were measured with a TD20 / 20 luminometer (Turner Biosystems).

[0109] The obtained data showed that astemizole could not rescue the expression of the double mutant hERG-G601S / F656C, but could indeed rescue the expression of hERG-G601S, indicating that it is a potent hERG blocker.

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PUM

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Abstract

Disclosed are high throughput assay systems and methods for identifying agents that alter the level of surface expression of integral membrane proteins, such as cardiac ion channels, in mammalian cells. Also disclosed are therapeutic methods of using agents identified using such methods.

Description

[0001] This application is a continuation-in-part of Application Serial No. 10 / 619,184, filed July 15,2003. technical field [0002] The present invention relates to high throughput assay systems and methods for identifying agents that alter the surface expression levels of integral membrane proteins, such as cardiac ion channels, in mammalian cells. The invention also relates to methods of treatment using agents identified by this method. Background technique [0003] A. Determination [0004] Human-Ether-a-go-go Related Gene (hERG) is the gene that produces cardiac repolarization current I Kr The pore-like potassium channel subunit consists of six transmembrane segments (S1-S6) and cytoplasmic amino- and carboxy-termini. hERG has been linked to congenital and drug-induced long QT syndrome, a serious and potentially fatal heart disease. [0005] Mutations in hERG produce functionally defective channels and / or trafficking-deficient channels, which both reduce I Kr curren...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/495C12Q1/02G01N33/53G01N33/50G01N33/566G01N33/567G01N33/68
CPCG01N33/502G01N33/5044G01N33/5008G01N33/6872G01N33/566
Inventor 阿瑟·M·布朗埃克哈德·菲克尔芭芭拉·A·威布尔
Owner CHANXPRESS
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