Fluorogenic peptide substrate arrays for highly multiplexed, real-time monitoring of kinase activities
OwnerGunjan Tiwari, Jiangtao Yu +1inventors
InventorsGunjan Tiwari, Jiangtao Yu, Steven Sundberg
Life of this patent
Estimating the term…
Abstract
The embodiments of the invention relate to a biomolecule microarray comprising a plurality of different polypeptides, where the polypeptides contain a kinase recognition sequence connected via a linker sequence to a metal binding amino acid that undergoes chelation enhanced fluorescence upon binding to a divalent cation. The embodiments further relate to kits comprising the microarray, and methods of using the microarray to screen for kinase activity, and to screen for inhibitors of kinase activity.
Claims31
A positionally addressable array comprising 10 or more different polypeptides immobilized on a substrate, wherein the polypeptides comprise a kinase recognition sequence joined by a linker sequence to a metal binding amino acid comprising a compound having the formula (I)
- wherein at least one R group is —SO2X, where X is OR″ or —NR″R′″;
- R′ is hydroxy, amino, or thiol;
- R″ is C1-6 alkyl;
- R′″ is hydrogen or alkyl;
- and
- N is 1, 2, or 3;
- and
- wherein compound (I) undergoes chelation-enhanced fluorescence (CHEF) upon binding to Mg2+.
A method for identifying a target of a kinase, comprising
- contacting the kinase with a positionally addressable array comprising at least 10 different polypeptides immobilized on a substrate, wherein the polypeptide comprises a kinase recognition site joined by a beta-turn sequence to a metal binding amino acid, and
- identifying a polypeptide on the positionally addressable array that shows greater than an approximately 2 fold increase in fluorescence after being contacted with the kinase, wherein the increase in fluorescence indicates that the polypeptide is a target for the kinase.
A method for identifying a compound that modulates kinase activity, comprising
- contacting a kinase with a positionally addressable array comprising at least 10 different polypeptides immobilized on a substrate, wherein the polypeptide comprises a kinase recognition site joined by a linker polypeptide to a metal binding amino acid, wherein said contact is in the presence of a test compound, a divalent cation, and ATP, and
- determining whether fluorescence of the polypeptide is modulated in the presence of the test compound, wherein a change in fluorescence is an indication that the test compound modulates kinase activity.
Dependencies are inferred from the claim text.