Kinase and phosphatase assays

• US 7,727,752 B2
• Filed: 07/14/2006
• Issued: 06/01/2010
• Est. Priority Date: 07/29/2003
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Somberg, Richard, Kleman-Leyer, Karen M., Lasky, David A., Klink, Tony A., Beebe, Jane A.
• Primary Examiner(s): Tsang; Cecilia
• Assistant Examiner(s): BRADLEY, CHRISTINA
• Application Number:
  US11/485,957
• Publication Number:
  US 20070059787A1
• Time in Patent Office: 1,418 Days
• Field of Search: None
• US Class Current: 435/194
• 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 method for identifying a modulator of activity of a kinase, comprising:

a) forming a kinase mixture comprising a protein kinase, a composition, and a test compound;

b) contacting the kinase mixture with a protease to form a protease mixture;

c) forming a control mixture comprising the protein kinase, the composition, and the protease; and

d) comparing a measurable property in the protease mixture to the measurable property in the 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, and

wherein the composition comprises;

(i) a peptide having a length from five to fifty amino acids, the peptide comprising a motif, wherein the motif is;

TX₁YVA, where X₁ can be G, A, or E (SEQ ID NO;

     10);

(ii) a first detectable moiety, wherein the first detectable moiety is associated with the peptide; and

(iii) a protease cleavage site, wherein the protease cleavage site is located in a position relative to the motif such that enzymatic modification of the motif alters the proteolytic cleavage of the peptide;

wherein the protein kinase is selected from the group consisting of CSF1R,FLT 3, and c-Kit.

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

3. The method of claim 1, 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.

4. The method of claim 1, wherein the peptide has a length selected from the group consisting of from 8 to 50 amino acids, from 8 to 25 amino acids and from 8 to 15 amino acids.

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

6. The method of claim 1, wherein the measurable property is an optical property, a magnetic property, or a radioactive property.

7. The method of claim 6, wherein the optical property is 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.

8. The method of claim 6, 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.

9. The method of claim 5, wherein the first or second detectable moiety is 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.

10. The method of claim 5, wherein the first detectable moiety, the second detectable moiety or the first and second detectable moiety is a member of a specific binding pair.

11. The method of claim 5, wherein the first detectable moiety and the second detectable moiety form a dark quenching RET pair or a FRET pair.

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

13. The method of claim 5, wherein the first or second detectable moiety is covalently linked to the peptide via a linker (L).

14. The method of claim 13, wherein the L is selected from the group consisting of GABA, diaminopentanyl, and aminohexanoyl.

16. The method of claim 1, wherein the motif comprises TX₁YVA, where X₁ can be G, A or E (SEQ ID NO:

10), and wherein X₁ is A.

15. A method for identifying a modulator of activity of a kinase, comprising:

a) forming a kinase mixture comprising a protein kinase, a composition, and a test compound;

b) contacting the kinase mixture with a protease to form a protease mixture;

c) forming a control mixture comprising the protein kinase, the composition, and the protease; and

d) comparing a measurable property in the protease mixture to the measurable property in the 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, and

wherein the composition comprises;

(i) a peptide having a length from five to fifty amino acids, the peptide comprising a motif, wherein the motif is TX₁YVA, where X₁ can be G, A, or E (SEQ ID NO;

     10);

(ii) a first detectable moiety, wherein the first detectable moiety is associated with the peptide; and

(iii) a protease cleavage site, wherein the protease cleavage site is associated with the peptide and 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,

wherein the protease cleavage site may overlap with or encompass the motif, and

wherein the protein kinase is selected from the group consisting of CSF1R, FLT3, and c-Kit.