Tyrosine kinase biosensors and methods of use
The claims describe a process for designing and validating peptide-based substrates for a specified kinase. The process uses a rational approach to design peptides with a specific amino acid that is phosphorylated by a target tyrosine kinase, and upon phosphorylation shows an increased affinity for a lanthanide ion that results in an enhanced fluorescent signal. The process uses kinase specific input data sets, encodes the input into positional scoring matrices, analyses the positional scoring matrices in order to generate a candidate list of peptide sequences, compares the peptide sequences in the candidate list, substitutes residues at non-selective positions in selections from the candidate list with terbium binding residues, and then screens the substituted selections with different kinases in vitro.
1. A method for designing peptide substrates phosphorylated by a single target kinase family which, upon phosphorylation, binds and enhances lanthanide ion sensitization, binding affinity and luminescent lifetime without the need for sensitizing agents, the method comprising:
(a) curating kinase specific input data sets consisting of validated endogenous sequences for peptide substrates and the target kinase family interacting partner sequences;
(b) encoding the input data sets from step (a) into positional scoring matrices;
(c) manipulating the positional scoring matrices generated in step (b) to identify positions required for kinase selectivity, scoring amino acids overrepresented 4 residues upstream and downstream of a phosphorylated residue;
(d) generating a candidate list of peptide sequences based on combinatorial peptide sequences of a result in step (c);
(e) comparing the combinatorial peptide sequences of step (d) to the combinatorial peptide sequences for at least one other kinase;
(f) using the results of steps (c)-(e), substitute residues at non-selective positions in selections from the candidate list of peptide sequences from step (e) with terbium binding residues by comparing sequence similarity scores to a known terbium binding motif; and
(g) screening the selections from the candidate list of peptide sequences with substituted residues from step (f) in vitro for the ability to bind terbium in a phosphorylation dependent manner.
2. The method of claim 1 , wherein the peptide substrate is a substrate for a tyrosine kinase.
3. The method of claim 1 , wherein the peptide substrate is a substrate for a serine/threonine kinase.
4. The method of claim 2 , wherein the tyrosine kinase is selected from the group consisting of Abl, Arg, Btk, Csk, Fyn, Hck, Jak2, Lck, Lyn, Pyk2, Src, Syk, and Yes.
5. The method of claim 4 , wherein the tyrosine kinase is Abl.
6. The method of claim 4 , wherein the tyrosine kinase is Arg.
7. The method of claim 4 , wherein the tyrosine kinase is Btk.
8. The method of claim 4 , wherein the tyrosine kinase is Csk.
9. The method of claim 4 , wherein the tyrosine kinase is Fyn.
10. The method of claim 4 , wherein the tyrosine kinase is Jak2.
11. The method of claim 4 , wherein the tyrosine kinase is Lck.
12. The method of claim 4 , wherein the tyrosine kinase is Lyn.
13. The method of claim 4 , wherein the tyrosine kinase is Pyk2.
14. The method of claim 4 , wherein the tyrosine kinase is Src.
15. The method of claim 4 , wherein the tyrosine kinase is Syk.
16. The method of claim 4 , wherein the tyrosine kinase is Hck.
17. The method of claim 4 , wherein the tyrosine kinase is Yes.