Compositions and assays utilizing ADP or phosphate for detecting protein modulators

• US 7,202,051 B1
• Filed: 11/08/2005
• Issued: 04/10/2007
• Est. Priority Date: 05/18/1999
• Status: Expired due to Fees

• Current Assignee: Cytokinetics, Inc.
• Original Assignee: Cytokinetics, Inc.
• Inventors: Malik, Fady, Finer, Jeffrey T., Wood, Kenneth, Shumate, Christopher, Sakowicz, Roman
• Primary Examiner(s): Saidha; Tekchand
• Application Number:
  US11/270,193
• Time in Patent Office: 518 Days
• Field of Search: 435/21, 435/16, 435/17, 435/18, 435/196, 435/23, 435/24
• US Class Current: 435/21
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

Described herein are methods which identify candidate agents as binding to a protein or as a modulator of the binding characteristics or biological activity of a protein. Generally, the methods involve the use of ADP or phosphate. The assays can be used in a high throughput system to obviate the cumbersome steps of using gels or radioactive materials.

Claims

1. A high throughput assay method of identifying a candidate agent as a modulator of the function of a target protein wherein said method comprises:

a) adding a candidate agent to a mixture comprising a target protein which directly or indirectly produces ADP or phosphate under conditions which normally allow the production of ADP or phosphate;

b) subjecting the mixture to an enzymatic reaction which uses said ADP or phosphate as a substrate under conditions which normally allow the ADP or phosphate to be utilized; and

c) determining the level of activity of the enzymatic reaction wherein a change in said level between the presence and absence of said candidate agent indicates that said candidate agent is a modulator of said target protein function, wherein at least 1,000 assays and up to about 10,000 assays can be performed per hour and said assays are performed in microtiter plates having 96 wells or more.

2. The method of claim 1, wherein the mixture further comprises a surfactant or antifoam.

3. The method of claim 1, wherein the determining occurs by a fluorescent, luminescent, radioactive or absorbance readout.

4. The method of claim 1, wherein the enzymatic reaction is determined at multiple time points.

5. The method of claim 1, wherein a plurality of candidate agents are added.

6. The method of claim 1, wherein a plurality of candidate agents are added to a plurality of target proteins.

7. The method of claim 1, wherein the candidate agents are added using a robotic system.

8. The method of claim 1, wherein the adding step a and the subjecting step b are performed simultaneously.

9. A high throughput assay method of identifying a candidate agent as a modulator of the function of a target protein wherein said method comprises:

a) adding a candidate agent to a mixture comprising a target protein which directly or indirectly produces ADP or phosphate under conditions which normally allow the production of ADP or phosphate;

b) subjecting the mixture to an enzymatic reaction which uses said ADP or phosphate as a substrate under conditions which normally allow the ADP or phosphate to be utilized wherein said enzymatic reaction comprises a purine nucleoside phosphorylase and a purine analog; and

c) determining cleavage of the purine analog in the presence and absence of said candidate agent as a measure of phosphate production wherein a change in cleavage of the purine analog between the presence and absence of said candidate agent indicates that said candidate agent is a modulator of said target protein function, wherein at least 1,000 assays and up to about 10,000 assays can be performed per hour and the assays are performed in microtiter plates having 96 wells or more.

10. The method of claim 9, wherein the mixture further comprises a surfactant or antifoam.

11. The method of claim 9, wherein the determining occurs by a fluorescent, luminescent, radioactive or absorbance readout.

12. The method of claim 9, wherein the enzymatic reaction is determined at multiple time points.

13. The method of claim 9, wherein a plurality of candidate agents are added.

14. The method of claim 9, wherein a plurality of candidate agents are added to a plurality of target proteins.

15. The method of claim 9, wherein the candidate agents are added using a robotic system.

16. The method of claim 9, wherein the adding step a and the subjecting step b are performed simultaneously.