Kinase and phosphatase assays

• US 8,067,536 B2
• Filed: 03/22/2010
• Issued: 11/29/2011
• Est. Priority Date: 07/29/2003
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Lasky, David, Kleman-Leyer, Karen, Beebe, Jane, Somberg, Richard, Klink, Tony
• Primary Examiner(s): Bradley; Christina
• Application Number:
  US12/728,926
• Publication Number:
  US 20100240080A1
• Time in Patent Office: 617 Days
• Field of Search: None
• US Class Current: 530/328
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 2333/81 Protease inhibitors
• 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 33/573 for enzymes or isoenzymes

Abstract

Compositions, methods, and kits for detecting and monitoring kinase, phosphatase and protein post-translational modification activity are described. The compositions typically include a peptide, a detectable moiety, and a protease cleavage site. Modification of a peptide by a kinase, phosphatase or other protein post-translational modification alters the proteolytic sensitivity of the peptide, resulting in a change of a detectable property of the composition. Panel assays for determining substrates or modulators of kinase, phosphatase or other protein post-translational modification activity are also described.

Claims

1. A composition of matter capable of being a serine/threonine kinase substrate, comprising:

(a) a peptide having a length from eight to fifty amino acids, the peptide comprising FSVPPS*PD, where S* is a phosphorylated serine (SEQ ID NO;

85); and

(b) a first detectable moiety associated with the peptide.

2. The composition of claim 1, wherein the first detectable moiety is covalently linked to the peptide.

3. The composition of claim 1, wherein the peptide comprises a protease cleavage site.

4. The composition of claim 3, wherein the protease cleavage site is selected from the group consisting of a chymotrypsin protease cleavage site, a caspase 3 protease cleavage site, a cathepsin G protease cleavage site, a trypsin protease cleavage site, an elastase protease cleavage site, an endoproteinase Asp-N protease cleavage site, and an endoproteinase Glu-N protease cleavage site.

5. The composition of claim 1, wherein the peptide has a length selected from the group consisting of from 8 to 25 amino acids and from 8 to 15 amino acids.

6. The composition of claim 1, further comprising a second detectable moiety associated with the peptide.

7. The composition of claim 1, wherein the composition exhibits an optical property, a magnetic property, or a radioactive property.

8. The composition of claim 7, wherein the composition exhibits an optical property selected from the group consisting of a molar extinction coefficient at an excitation wavelength, a quantum efficiency, an excitation spectrum, an emission spectrum, an excitation wavelength maximum, an emission wavelength maximum, a ratio of excitation amplitudes at two wavelengths, a ratio of emission amplitudes at two wavelengths, an excited state lifetime, an anisotropy, a polarization of emitted light, a resonance energy transfer, and a quenching of emission at a wavelength.

9. The composition of claim 7, wherein the optical property is selected from the group consisting of a fluorescence excitation spectrum, a fluorescence emission spectrum, a fluorescence excitation wavelength maximum, a fluorescence emission wavelength maximum, a ratio of fluorescence excitation amplitudes at two wavelengths, a ratio of fluorescence emission amplitudes at two wavelengths, a fluorescence excited state lifetime, a fluorescence anisotropy, and a quenching of fluorescence emission at a wavelength.

10. The composition of claim 6, wherein the first or second detectable moieties are selected from the group consisting of 5-FAM, 6-FAM, 7-hydroxycoumarin-3-carboxamide, 6-chloro-7-hydroxycoumarin-3-carboxamide, fluorescein-5-isothiocyanate, dichlorotriazinylaminofluorescein, tetramethylrhodamine-5-isothiocyanate, tetramethylrhodamine-6-isothiocyanate, succinimidyl ester of 5-carboxyfluorescein, succinimidyl ester of 6-carboxyfluorescein, 5-carboxytetramethylrhodamine, 6-carboxymethylrhodamine, fluorescein maleimide, and 7-amino-4-methylcoumarin-3-acetic acid.

11. The composition of claim 6, wherein the first detectable moiety, the second detectable moiety or the first and second detectable moiety is a member of a specific binding pair.

12. The composition of claim 6, wherein the first detectable moiety and the second detectable probe moiety form a dark quenching RET pair or a FRET pair.

13. The composition of claim 6, wherein the first detectable moiety is 7-hydroxycoumarin-3-carboxamide and the second detectable moiety is 5-FAM.

14. The composition of claim 6, wherein the first or second detectable probe moiety is covalently linked to the peptide via a linker (L).

15. The composition of claim 14, wherein the L is selected from the group consisting of GABA, diaminopentanyl, and aminohexanoyl.

16. A method for identifying a modulator of activity of a serine/threonine kinase, comprising:

(a) forming a mixture of a serine/threonine protein kinase, a composition of claim 1, and a test compound;

(b) contacting the mixture with a protease to form a protease mixture; and

(c) comparing a measurable property in the protease mixture to the measurable property in a control mixture, wherein the test compound is identified as a modulator of activity of the kinase if the measurable property in the protease mixture is different from the measurable property in the control mixture.

17. A method for identifying a modulator of activity of a serine/threonine phosphatase, comprising:

(a) contacting a mixture of a serine/threonine protein phosphatase, a composition of claim 1, and a test compound to form a phosphatase mixture, wherein the composition of claim 1 comprises a phosphorylated threonine or serine amino acid;

(b) contacting the phosphatase mixture with a protease to form a protease mixture; and

(c) comparing a measurable property in the protease mixture to the measurable property in a control protease mixture, wherein the test compound is identified as a modulator of activity of the phosphatase if the measurable property in the protease mixture is different from the measurable property in the control mixture.

18. An article of manufacture comprising:

(a) packaging matter; and

(b) a composition of matter according to claim 1 associated with the packaging material.

19. The article of claim 18, further comprising at least one member selected form the group consisting of a serine/threonine protein kinase, a serine/threonine protein phosphatase, a protease and ATP.

20. The composition of claim 1, wherein the peptide comprises PRPFSVPPS*PDK (SEQ ID NO:

86).