Cell-signaling assays

• US 6,806,053 B1
• Filed: 01/23/2001
• Issued: 10/19/2004
• Est. Priority Date: 06/09/1999
• Status: Expired due to Fees

• Current Assignee: Molecular Devices
• Original Assignee: LJL Biosystems, Inc. (Molecular Devices)
• Inventors: Sportsman, J. Richard, Kauvar, Lawrence M.
• Primary Examiner(s): Brumback, Brenda
• Assistant Examiner(s): NICHOLS, CHRISTOPHER J
• Application Number:
  US09/768,661
• Publication Number:
  US 20040203000A1
• Time in Patent Office: 1,365 Days
• Field of Search: 435/7.1, 435/70.21, 436/518, 530/334, 530/300, 530/350
• US Class Current: 435/7.1
• CPC Class Codes: B01L 3/50853 with covers or lids
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 2035/00237 Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 2035/0405 manipulating closing or opening means, e.g. stoppers, screw caps, lids or covers
• CPC Class Codes: G01N 2035/0425 Stacks, magazines or elevators for plates
• CPC Class Codes: G01N 2333/9121 with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 35/028 having reaction cells in the form of microtitration plates
• CPC Class Codes: G01N 35/1011 Control of the position or alignment of the transfer device
• CPC Class Codes: G01N 35/1074 arranged in a two-dimensional array

Abstract

Assays for detecting the presence and activity of cell-signaling components. These assays include luminescence polarization assays for detecting cell-signaling nucleotides and modulators of receptors and enzymes related to the generation and activity of such nucleotides.

Claims

1. A method of identifying a compound as a modulator of G-protein-linked receptor activity that generates or consumes a cyclic nucleotide through the action of a cyclase or phosphodiesterase, comprising:

conducting a G-protein-linked-receptor mediated reaction that generates or consumes a cyclic nucleotide in the presence of a candidate compound;

contacting, in vitro, a product of the reaction with a luminescent tracer and with the opposite member of a specific binding pair to the cyclic nucleotide, wherein the tracer and the cyclic nucleotide compete for binding to the opposite member of the specific binding pair;

illuminating the tracer with polarized light, wherein the light is capable of inducing emission of polarized light from the tracer;

detecting the extent of polarization of light emitted from the tracer; and

identifying the candidate compound as a modulator of the reaction based on the extent of polarization of the emitted light.

2. The method of claim 1, wherein the cyclic nucleotide is selected from the group consisting of cAMP and cGMP.

3. The method of claim 1, wherein the opposite member of a specific binding pair is an immunological binding partner.

4. The method of claim 1, wherein the extent of polarization is determined using a function selected from the group consisting of polarization and anisotropy.

5. The method of claim 1, wherein the extent of polarization of the emitted light is inversely correlated with the concentration of the cyclic nucleotide.

6. The method of claim 1, further comprising determining the concentration of the cyclic nucleotide.

7. The method of claim 1, wherein the reaction is conducted using whole cells.

8. The method of claim 1, wherein the reaction is conducted using lysed cells.

9. The method of claim 1, wherein the reaction generates a cyclic nucleotide.

10. The method of claim 9, wherein the reaction generates a cyclic nucleotide through the action of a cyclase.

11. The method of claim 1, wherein the reaction consumes a cyclic nucleotide.

12. The method of claim 11, wherein the reaction consumes a cyclic nucleotide through the action of a phosphodiesterase.

13. The method of claim 1, further comprising repeating the steps of conducting, contacting, illuminating, and detecting in the absence of a candidate compound, wherein the step of identifying the candidate compound as a modulator includes comparing the extent of polarization of the emitted light based on the reaction conducted in the presence of the candidate compound to the extent of polarization of the emitted light based on the reaction conducted in the absence of the candidate compound.

14. The method of claim 13, the reaction generating a cyclic nucleotide, wherein an increase in the extent of polarization when the reaction is conducted in the presence of the candidate compound in comparison with the extent of polarization when the reaction is conducted in the absence of the candidate compound identifies the candidate compound as an inhibitor of the reaction, and wherein a decrease in the extent of polarization when the reaction is conducted in the presence of the candidate compound in comparison with the extent of polarization when the reaction is conducted in the absence of the candidate compound identifies the candidate compound as an agonist of the reaction.

15. The method of claim 13, the reaction consuming a cyclic nucleotide, wherein an increase in the extent of polarization when the reaction is conducted in the presence of the candidate compound in comparison with the extent of polarization when the reaction is conducted in the absence of the candidate compound identifies the candidate compound as an agonist of the reaction, and wherein a decrease in the extent of polarization when the reaction is conducted in the presence of the candidate compound in comparison with the extent of polarization when the reaction is conducted in the absence of the candidate compound identifies the candidate compound as an inhibitor of the reaction.

16. The method of claim 1, the reaction generating a cyclic nucleotide, wherein the step of conducting the reaction includes providing a nucleotide triphosphate.

17. The method of claim 1, the reaction consuming a cyclic nucleotide, wherein the step of conducting the reaction includes providing the cyclic nucleotide.

18. The method of claim 1, further comprising repeating the steps of conducting, contacting, illuminating, detecting, and identifying for a different candidate compound.

19. The method of claim 18, at least one of the steps being performed using a microplate, wherein a different well of the microplate is used for each different candidate compound.

20. The method of claim 1, wherein at least one of the steps of conducting, contacting, illuminating, detecting, and identifying is performed using a microplate.

21. The method of claim 1, the step of conducting the reaction being performed in a reaction volume, wherein the step of contacting includes adding the luminescent tracer and the opposite member of a specific binding pair to the reaction volume.

22. The method of claim 1, wherein the luminescent tracer comprises a cyclic nucleotide coupled to a luminophore.

23. The method of claim 1, the step of conducting a reaction being performed inside a cell, further comprising the step of lysing the cell to obtain the product of the reaction prior to the steps of contacting, illuminating, detecting, and identifying.

24. The method of claim 1 wherein the G-protein-linked receptor generates cAMP through G-protein activation and adenylate cyclase activity.

25. The method of claim 1, wherein the candidate compound is a modulator of the G-protein-linked receptor.