Compositions and assays utilizing ADP or phosphate for detecting protein modulators

• US 7,378,254 B2
• Filed: 01/18/2007
• Issued: 05/27/2008
• 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:
  US11/655,733
• Publication Number:
  US 20070254310A1
• Time in Patent Office: 495 Days
• Field of Search: 435/21, 435/15, 435/16, 435/17, 435/18, 435/19, 435/196
• 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 method of modulating polymerization or depolymerization of a cytoskeletal filament protein, wherein the filament protein comprises at least one of tubulin and actin, the method comprising providing a compound that modulates polymerization or depolymerization of the cytoskeletal filament protein, wherein the compound is characterized by

a) adding a test compound to a mixture comprising the filament protein under conditions which normally promote either polymerization or depolymerization of the filament protein;

b) adding a motor protein to the mixture, wherein the motor protein has ATPase activity, and the ATPase activity is activated by polymerized filament protein, and wherein the motor protein is added to the mixture under conditions which normally allow the production of ADP or phosphate by said motor protein; and

c) determining the ATPase activity of the motor protein by detecting the formation of ADP or phosphate, wherein a change in ATPase activity in the presence of the compound compared to the absence of said compound is an indication that the test compound is suitable for use as the modulator of filament protein polymerization or depolymerization.

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

3. The method of claim 1, wherein said activity of said motor protein is determined at multiple time points.

4. The method of claim 1, wherein a plurality of test compounds are added.

5. The method of claim 4, wherein a plurality of test compounds and a plurality of motor proteins are added.

6. The method of claim 1, wherein said compounds are added using a robotic system.

7. The method of claim 1, wherein the motor protein is a kinesin.

8. The method of claim 7, wherein the motor protein is Kin2, chromokinesin, Kif1A, KSP, CENP-E, MCAK, HSET, or Kif15.

9. The method of claim 1, wherein the motor protein is myosin.

10. The method of claim 1, wherein the step of determining the activity of said motor protein comprises coupling an enzymatic reaction that utilizes ADP or phosphate to the oxidation of NADH;

and measuring NADH oxidation as a measure of ADP production.