FLUORESCENCE-BASED ASSAY FOR DETECTING COMPOUNDS FOR MODULATING THE SODIUM-CALCIUM EXCHANGER (NCX) IN "FORWARD MODE"
Transporters are an emerging target family with enormous potential, offering scientific and economic opportunities. The Sodium/Calcium exchanger is an important mechanism for removing Ca 2+ from diverse cells. In heart, it extrudes Ca 2+ that has entered through Ca 2+ channels to initiate contraction, while Na + enters the heart cell. It is of considerable interest to identify compounds that modulate the activity of Sodium/Calcium exchangers. The present invention is directed to a fluorescence-based assay for detecting NCX “forward mode” modulating compounds. It further refers to a kit of parts comprising cells expriming NCX and the use of the kit of parts to test a compound for activity as an agonist or antagonist of NCX.
1 . An assay for determining the activity of NCX protein, said assay comprising:
a) providing cells expressing NCX;
b) providing a luminescent colored substance for determining intracellular calcium;
c) contacting cells with a NCX activity activator; and
d) comparing the calcium mediated change in the luminescent signal from said colored substance to a luminescent signal produced in a control experiment.
2 . The assay according to claim 1 , wherein the NCX protein is a NCX protein selected from the group consisting of NCX1, NCX2 and NCX3.
3 . The assay according to claim 1 , wherein the NCX protein is of mammalian origin.
4 . The assay according to claim 1 , wherein the cells are selected from the group consisting of: CHO, HEK, COS7 and JURKAT cells.
5 . The assay according to claim 1 , wherein said colored substance is added to the cells as a dye precursor capable of entering the cells and being hydrolyzed to a dye, whereby the dye complexes with calcium in said cells and provides a luminescent signal.
6 . The assay according to claim 1 , wherein said luminescent signal is fluorescence and said assay employs a FLIPR device.
7 . The assay according to claim 5 , wherein said dye precursor is an acetoxymethylester derivate.
8 . The assay according to claim 5 , wherein said dye is the calcium sensitive fluorescence dye fluo-4.
9 . The assay according to claim 1 , wherein said NCX activity activator is ionomycin.
10 . The assay according to claim 1 further comprising providing a compound to be tested for activity as an agonist or antagonist of NCX.
11 . The assay according to claim 1 wherein said comparing facilitates diagnosis of a disease associated with a NCX altered expression.
12 . An assay for determining activity of NCX protein in response to the addition of a compound, said assay comprising:
a) providing cells expriming NCX;
b) providing a luminescent colored substance for determining intracellular calcium;
c) contacting cells with a compound, wherein said cells have been treated, prior to treating with said compound, with a NCX activity activator; and
d) comparing the calcium mediated change in the luminescent signal from said colored substance to a luminescent signal produced in a control experiment.
13 . An assay according to claim 12 , wherein the NCX protein is a NCX protein selected from the group consisting of NCX1, NCX2 and NCX3.
14 . The assay according to claim 12 , wherein the NCX protein is of mammalian origin.
15 . The assay according to claim 12 , wherein the cells are selected from the group consisting of: CHO, HEK, COS7 and JURKAT cells.
16 . The assay according to claim 12 , wherein said colored substance is added to the cells as a dye precursor capable of entering the cells and being hydrolyzed to a dye, whereby the dye complexes with calcium in said cells and provides a luminescent signal.
17 . The assay according to claim 12 , wherein said luminescent signal is fluorescence and said assay employs a FLIPR device.
18 . The assay according to claim 16 , wherein said dye precursor is an acetoxymethylester derivate.
19 . The assay according to claim 16 , wherein said dye is the calcium sensitive fluorescence dye fluo-4.
20 . The assay according to claims 12 , wherein said compound is a NCX antagonist.
21 . The assay according to claim 12 , wherein said NCX activator is ionomycin.
22 . A kit comprising:
a) lyophilized cells expriming NCX protein;
b) a colored substance;
c) a compound buffer; and
d) a colored substance buffer.
23 . The kit according to claim 22 , wherein said colored substance is the calcium sensitive fluorescence dye fluo-4.
24 . The kit according to claim 22 , wherein the NCX protein is a NCX protein selected from the group consisting of NCX1, NCX2 and NCX3.
25 . The kit according to claims 22 , wherein the NCX protein is of mammalian origin.
26 - 27 . (canceled)
28 . The assay according to claim 3 , wherein the NCX protein is selected from the group consisting of rat, mouse, dog, bovine, pig, ape and human NCX protein.
29 . The assay according to claim 14 , wherein the NCX protein is selected from the group consisting of rat, mouse, dog, bovine, pig, ape and human NCX protein.
30 . The kit according to claim 25 , wherein the NCX protein is selected from the group consisting of rat, mouse, dog, bovine, pig, ape and human NCX protein.