Kinase and phosphatase assays
View Patent ↗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.
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:
TX 1 YVA, where X 1 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 CSF 1 R,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.
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 TX 1 YVA, where X 1 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.
16. The method of claim 1 , wherein the motif comprises TX 1 YVA, where X 1 can be G, A or E (SEQ ID NO: 10), and wherein X 1 is A.