Biosensor and uses thereof

Abstract

Compositions include biosensors and enzyme substrates for use in the biosensors. Methods of identifying modulators of enzymatic activity include the biosensors.

Description

DOCKET: 348358.18802 BIOSENSOR AND USES THEREOF The present application claims the benefit of U.S. provisional application no. 63 / 685,235 filed August 20, 2024, which is incorporated by reference herein in its entirety. BACKGROUND

[0001] Ca2+ / Calmodulin-dependent protein kinase II (CaMKII) is a highly validated cause or contributor to major cardiac illnesses, including heart failure, myocardial infarction, and arrhythmias. Excessive CaMKII activity causes intracellular Ca2+dysregulation, inflammation, maladaptive transcription, and cell death. Yet, there are currently no approved CaMKII inhibiting therapies. Thus, development of safe and effective CaMKII inhibitors is a translational priority. SUMMARY

[0002] We now provide a novel biosensor comprising: an enzyme substrate, a detectably labelled protein, and a phospho-amino acid binding protein.

[0003] In certain embodiments, the enzyme substrate comprises one or more peptides having at least 70% sequence identity to any of the following SEQ ID NOS: 1 to 11: MGKIYRLRTQDDGEGGTGG (SEQ ID. NO.1); MLRRHTVEDAVGGTGG (SEQ ID. NO.2); MPALVRRHTLEDRSGGTGG (SEQ ID. NO.3); MHRQETVDDLKGGTGG (SEQ ID. NO.4); MHRQETVEDLKGGTGG (SEQ ID. NO.5); MSSLARQRTLEDEEGGTGG (SEQ ID. NO.6); MSSLARQRTLEDEGGTGG (SEQ ID. NO.7); MSSLARQRTLEGGTGG (SEQ ID. NO.8); MSSLARQRTLEDEEGTGG (SEQ ID. NO.9); 161750686 175727263.1MSSLARQRTLEDEETGG (SEQ ID. NO.10); MSSLARQRTLEDEEG (SEQ ID. NO.11).

[0004] In certain embodiments, the enzyme substrate comprises one or more peptides having at least 75, 80, 85, 90, 95, 97, 98 or 99% sequence identity to any of the following SEQ ID NO: 1 to 11: MGKIYRLRTQDDGEGGTGG (SEQ ID. NO.1); MLRRHTVEDAVGGTGG (SEQ ID. NO.2); MPALVRRHTLEDRSGGTGG (SEQ ID. NO.3); MHRQETVDDLKGGTGG (SEQ ID. NO.4); MHRQETVEDLKGGTGG (SEQ ID. NO.5); MSSLARQRTLEDEEGGTGG (SEQ ID. NO.6); MSSLARQRTLEDEGGTGG (SEQ ID. NO.7); MSSLARQRTLEGGTGG (SEQ ID. NO.8); MSSLARQRTLEDEEGTGG (SEQ ID. NO.9); MSSLARQRTLEDEETGG (SEQ ID. NO.10); MSSLARQRTLEDEEG (SEQ ID. NO.11).

[0005] In certain embodiments, the enzyme substrate comprises one or more peptides of any following SEQ ID NO: 1 to 11: MGKIYRLRTQDDGEGGTGG (SEQ ID. NO.1); MLRRHTVEDAVGGTGG (SEQ ID. NO.2); MPALVRRHTLEDRSGGTGG (SEQ ID. NO.3); MHRQETVDDLKGGTGG (SEQ ID. NO.4); MHRQETVEDLKGGTGG (SEQ ID. NO.5); 2 175727263.1MSSLARQRTLEDEEGGTGG (SEQ ID. NO.6); MSSLARQRTLEDEGGTGG (SEQ ID. NO.7); MSSLARQRTLEGGTGG (SEQ ID. NO.8); MSSLARQRTLEDEEGTGG (SEQ ID. NO.9); MSSLARQRTLEDEETGG (SEQ ID. NO.10); MSSLARQRTLEDEEG (SEQ ID. NO.11).

[0006] In certain embodiments, a method of identifying modulators of kinases comprises contacting a biosensor or a cell expressing a biosensor, with one or more candidate agents, wherein the biosensor comprises an enzyme substrate that comprises one or more peptides having at least 70% sequence identity to any of SEQ ID NOS: 1-11, a detectably labelled protein, and a phospho-amino acid binding protein; assaying for the presence or absence of a signal from the detectable label; and, identifying modulators of kinases. In certain embodiments, a signal from the detectable label is identified as an activator of the kinase. In certain embodiments, the absence of a signal from the detectable label is identified as an inhibitor of the kinase. In certain embodiments, the enzyme substrate that comprises one or more peptides having at least 80%, 90%, 95%, 97%, 95% pr 100% sequence identity to any of SEQ ID NOS: 1-11. In certain embodiments, the detectably labelled protein comprises a radio labeled molecule, a fluorophore, a radiochemical, a luminescent compound, an electron-dense reagent, an enzyme, biotin, a radioactive compound, a non-radioactive compound, digoxigenin or a hapten. In certain embodiments, the detectably labelled protein comprises a fluorophore.

[0007] In certain embodiments, a host cell comprises an expression vector encoding any one or more of SEQ ID NOS: 1 to 11, or a sequence having 70, 80, 9095, or 98 % identify to any of SEQ ID NOS: 1 to 11.

[0008] In certain embodiments, a kit comprises a synthetic biosensor, an expression vector encoding SEQ ID NOS: 1 to 11, or a sequence having 70, 80, 9095, or 98 % identify to any of SEQ ID NOS: 1 to 11.

[0009] Suitable biosensor systems, including where the present enzyme substrate peptides (such as those of SEQ ID NOS:1-11) may be incorporated are disclosed in in 3 175727263.1PCT / US2023 / 016315 (WO2023 / 183625), which is incorporated by reference herein in its entirety.

[0010] In a further aspect, a biosensor is provided (which may suitably comprise: i) an enzyme substrate, ii) a detectably labelled protein, and iii)a phospho-amino acid binding protein) as disclosed herein and be encoded by a nucleic acid sequence of any of the following SEQ ID NOS: 12-22. The sensors of SEQ ID NOS.12-22 suitably comprise an enzyme substrate peptide of SEQ ID NOS.1-11 respectively, i.e. a sensor of SEQ ID NO: 12 suitably comprises an enzyme substrate peptide of SEQ ID NO.1; a sensor of SEQ ID NO: 13 suitably comprises an enzyme substrate peptide of SEQ ID NO.2; a sensor of SEQ ID NO: 14 suitably comprises an enzyme substrate peptide of SEQ ID NO.3; a sensor of SEQ ID NO: 15 suitably comprises an enzyme substrate peptide of SEQ ID NO.4; a sensor of SEQ ID NO: 16 suitably comprises an enzyme substrate peptide of SEQ ID NO.5; a sensor of SEQ ID NO: 17 suitably comprises an enzyme substrate peptide of SEQ ID NO.6; a sensor of SEQ ID NO: 18 suitably comprises an enzyme substrate peptide of SEQ ID NO.7; a sensor of SEQ ID NO: 19 suitably comprises an enzyme substrate peptide of SEQ ID NO.8; a sensor of SEQ ID NO: 20 suitably comprises an enzyme substrate peptide of SEQ ID NO.9; a sensor of SEQ ID NO: 21 suitably comprises an enzyme substrate peptide of SEQ ID NO.10; and a sensor of SEQ ID NO: 22 suitably comprises an enzyme substrate peptide of SEQ ID NO.11. SEQ ID NO:12: ATGGGAAAGATTTATAGACTTAGAACGCAGGACGATGGGGAAGGCGGCACCGGC GGCAGCGAGCTCAGCTTCTGCAACGTCTATATCAAGGCCGACAAGCAGAAGAACG GCATCAAGGCGAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCT CGCCTACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCC GACAACCACTACCTGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGC GCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG GACGAGCTGTACAAGGGCGGTACCGGAGGGAGCATGGTGAGCAAGGGCGAGGAG CTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCC ACAAGTTCAGCGTGTCCGGCGAGGGTGAGGGCGATGCCACCTACGGCAAGCTGAC CCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGA CCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCA GCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCT TCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGA CACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAAC ATCCTGGGGCACAAGCTGGAGTACAACTTGCTGCATAAGTTTTCTCAAGAACAGAT CGGCGAAAACATTGTGTGCAGGGTCATTTGTACCACGGGTCAAATTCCCATCCGAG ATTTGTCAGCTGATATTTCACAAGTGCTTAAGGAAAAACGATCCATAAAGAAAGTT 4 175727263.1TGGACATTTGGTAGAAACCCAGCCTGTGACTATCATTTAGGAAACATTTCAAGACT GTCAAATAAGCATTTCCAAATACTACTAGGAGAAGACGGTAACCTTTTATTGAATG ACATTTCCACTAATGGGACCTGGTTAAATGGGCAAAAAGTCGAGAAGAACAGCAA TCAGTTACTGTCTCAAGGTGATGAAATAACCGTTGGTGTAGGCGTGGAATCAGATA TTTTATCTCTGGTCATTTTCATAAACGACAAATTTAAGCAGTGCCTCGAGCAGAAC AAAGTTGATCGCTAA SEQ ID NO:13: ATGCTGAGAAGGCACACAGTCGAGGACGCAGTCGGCGGCACCGGCGGCAGCGAG CTCAGCTTCTGCAACGTCTATATCAAGGCCGACAAGCAGAAGAACGGCATCAAGG CGAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCTACCA CTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC TACCTGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGCGCGATCACA TGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTG TACAAGGGCGGTACCGGAGGGAGCATGGTGAGCAAGGGCGAGGAGCTGTTCACC GGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCA GCGTGTCCGGCGAGGGTGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGA CCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTC TTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCTTCTTCAAGGA CGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTG AACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGC ACAAGCTGGAGTACAACTTGCTGCATAAGTTTTCTCAAGAACAGATCGGCGAAAA CATTGTGTGCAGGGTCATTTGTACCACGGGTCAAATTCCCATCCGAGATTTGTCAG CTGATATTTCACAAGTGCTTAAGGAAAAACGATCCATAAAGAAAGTTTGGACATTT GGTAGAAACCCAGCCTGTGACTATCATTTAGGAAACATTTCAAGACTGTCAAATA AGCATTTCCAAATACTACTAGGAGAAGACGGTAACCTTTTATTGAATGACATTTCC ACTAATGGGACCTGGTTAAATGGGCAAAAAGTCGAGAAGAACAGCAATCAGTTAC TGTCTCAAGGTGATGAAATAACCGTTGGTGTAGGCGTGGAATCAGATATTTTATCT CTGGTCATTTTCATAAACGACAAATTTAAGCAGTGCCTCGAGCAGAACAAAGTTG ATCGCTAA SEQ ID NO:14: ATGCCCGCCCTGGTGCGGAGGCACACCCTCGAGGATCGTAGTGGCGGCACCGGCG GCAGCGAGCTCAGCTTCTGCAACGTCTATATCAAGGCCGACAAGCAGAAGAACGG CATCAAGGCGAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTC GCCTACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCG ACAACCACTACCTGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGCG CGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGG ACGAGCTGTACAAGGGCGGTACCGGAGGGAGCATGGTGAGCAAGGGCGAGGAGC TGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCA 5 175727263.1CAAGTTCAGCGTGTCCGGCGAGGGTGAGGGCGATGCCACCTACGGCAAGCTGACC CTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGAC CACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAG CACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCTT CTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGAC ACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACA TCCTGGGGCACAAGCTGGAGTACAACTTGCTGCATAAGTTTTCTCAAGAACAGATC GGCGAAAACATTGTGTGCAGGGTCATTTGTACCACGGGTCAAATTCCCATCCGAG ATTTGTCAGCTGATATTTCACAAGTGCTTAAGGAAAAACGATCCATAAAGAAAGTT TGGACATTTGGTAGAAACCCAGCCTGTGACTATCATTTAGGAAACATTTCAAGACT GTCAAATAAGCATTTCCAAATACTACTAGGAGAAGACGGTAACCTTTTATTGAATG ACATTTCCACTAATGGGACCTGGTTAAATGGGCAAAAAGTCGAGAAGAACAGCAA TCAGTTACTGTCTCAAGGTGATGAAATAACCGTTGGTGTAGGCGTGGAATCAGATA TTTTATCTCTGGTCATTTTCATAAACGACAAATTTAAGCAGTGCCTCGAGCAGAAC AAAGTTGATCGCTAA SEQ ID NO:15: ATGCATCGTCAGGAAACGGTTGACGATCTCAAAGGCGGCACCGGCGGCAGCGAGC TCAGCTTCTGCAACGTCTATATCAAGGCCGACAAGCAGAAGAACGGCATCAAGGC GAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCTACCAC TACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACT ACCTGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGCGCGATCACAT GGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGT ACAAGGGCGGTACCGGAGGGAGCATGGTGAGCAAGGGCGAGGAGCTGTTCACCG GGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAG CGTGTCCGGCGAGGGTGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTC ATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGAC CTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCT TCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCTTCTTCAAGGAC GACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGA ACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCA CAAGCTGGAGTACAACTTGCTGCATAAGTTTTCTCAAGAACAGATCGGCGAAAAC ATTGTGTGCAGGGTCATTTGTACCACGGGTCAAATTCCCATCCGAGATTTGTCAGC TGATATTTCACAAGTGCTTAAGGAAAAACGATCCATAAAGAAAGTTTGGACATTT GGTAGAAACCCAGCCTGTGACTATCATTTAGGAAACATTTCAAGACTGTCAAATA AGCATTTCCAAATACTACTAGGAGAAGACGGTAACCTTTTATTGAATGACATTTCC ACTAATGGGACCTGGTTAAATGGGCAAAAAGTCGAGAAGAACAGCAATCAGTTAC TGTCTCAAGGTGATGAAATAACCGTTGGTGTAGGCGTGGAATCAGATATTTTATCT CTGGTCATTTTCATAAACGACAAATTTAAGCAGTGCCTCGAGCAGAACAAAGTTG ATCGCTAA SEQ ID NO:16: 6 175727263.1ATGCATAGACAAGAGACCGTTGAAGACTTGAAGGGCGGCACCGGCGGCAGCGAG CTCAGCTTCTGCAACGTCTATATCAAGGCCGACAAGCAGAAGAACGGCATCAAGG CGAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCTACCA CTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC TACCTGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGCGCGATCACA TGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTG TACAAGGGCGGTACCGGAGGGAGCATGGTGAGCAAGGGCGAGGAGCTGTTCACC GGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCA GCGTGTCCGGCGAGGGTGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGA CCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTC TTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCTTCTTCAAGGA CGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTG AACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGC ACAAGCTGGAGTACAACTTGCTGCATAAGTTTTCTCAAGAACAGATCGGCGAAAA CATTGTGTGCAGGGTCATTTGTACCACGGGTCAAATTCCCATCCGAGATTTGTCAG CTGATATTTCACAAGTGCTTAAGGAAAAACGATCCATAAAGAAAGTTTGGACATTT GGTAGAAACCCAGCCTGTGACTATCATTTAGGAAACATTTCAAGACTGTCAAATA AGCATTTCCAAATACTACTAGGAGAAGACGGTAACCTTTTATTGAATGACATTTCC ACTAATGGGACCTGGTTAAATGGGCAAAAAGTCGAGAAGAACAGCAATCAGTTAC TGTCTCAAGGTGATGAAATAACCGTTGGTGTAGGCGTGGAATCAGATATTTTATCT CTGGTCATTTTCATAAACGACAAATTTAAGCAGTGCCTCGAGCAGAACAAAGTTG ATCGCTAA SEQ ID NO:17: ATGCCCGAAGGCTACATCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACT ACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGA GCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAG TACAACTTGCTGCATAAGTTTTCTCAAGAACAGATCGGCGAAAACATTGTGTGCAG GGTCATTTGTACCACGGGTCAAATTCCCATCCGAGATTTGTCAGCTGATATTTCAC AAGTGCTTAAGGAAAAACGATCCATAAAGAAAGTTTGGACATTTGGTAGAAACCC AGCCTGTGACTATCATTTAGGAAACATTTCAAGACTGTCAAATAAGCATTTCCAAA TACTACTAGGAGAAGACGGTAACCTTTTATTGAATGACATTTCCACTAATGGGACC TGGTTAAATGGGCAAAAAGTCGAGAAGAACAGCAATCAGTTACTGTCTCAAGGTG ATGAAATAACCGTTGGTGTAGGCGTGGAATCAGATATTTTATCTCTGGTCATTTTC ATAAACGACAAATTTAAGCAGTGCCTCGAGCAGAACAAAGTTGATCGCTAA SEQ ID NO:18: ATGTCTAGCCTGGCCAGACAGCGGACCCTGGAAGACGAGGGCGGAACAGGCGGC AGCGAGCTGTCCTTCTGCAACGTCTATATCAAGGCCGACAAGCAGAAGAACGGCA TCAAGGCGAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGC 7 175727263.1CTACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGAC AACCACTACCTGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGCGCG ATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGAC GAGCTGTACAAGGGCGGTACCGGAGGGAGCATGGTGAGCAAGGGCGAGGAGCTG TTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACA AGTTCAGCGTGTCCGGCGAGGGTGAGGGCGATGCCACCTACGGCAAGCTGACCCT GAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCA CCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCA CGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCTTCT TCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACA CCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACAT CCTGGGGCACAAGCTGGAGTACAACTTGCTGCATAAGTTTTCTCAAGAACAGATC GGCGAAAACATTGTGTGCAGGGTCATTTGTACCACGGGTCAAATTCCCATCCGAG ATTTGTCAGCTGATATTTCACAAGTGCTTAAGGAAAAACGATCCATAAAGAAAGTT TGGACATTTGGTAGAAACCCAGCCTGTGACTATCATTTAGGAAACATTTCAAGACT GTCAAATAAGCATTTCCAAATACTACTAGGAGAAGACGGTAACCTTTTATTGAATG ACATTTCCACTAATGGGACCTGGTTAAATGGGCAAAAAGTCGAGAAGAACAGCAA TCAGTTACTGTCTCAAGGTGATGAAATAACCGTTGGTGTAGGCGTGGAATCAGATA TTTTATCTCTGGTCATTTTCATAAACGACAAATTTAAGCAGTGCCTCGAGCAGAAC AAAGTTGATCGCTAA SEQ ID NO:19: ATGTCTAGCCTGGCCAGACAGCGGACCCTGGAAGGCGGAACAGGCGGCAGCGAG CTGTCCTTCTGCAACGTCTATATCAAGGCCGACAAGCAGAAGAACGGCATCAAGG CGAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCTACCA CTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC TACCTGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGCGCGATCACA TGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTG TACAAGGGCGGTACCGGAGGGAGCATGGTGAGCAAGGGCGAGGAGCTGTTCACC GGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCA GCGTGTCCGGCGAGGGTGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGA CCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTC TTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCTTCTTCAAGGA CGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTG AACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGC ACAAGCTGGAGTACAACTTGCTGCATAAGTTTTCTCAAGAACAGATCGGCGAAAA CATTGTGTGCAGGGTCATTTGTACCACGGGTCAAATTCCCATCCGAGATTTGTCAG CTGATATTTCACAAGTGCTTAAGGAAAAACGATCCATAAAGAAAGTTTGGACATTT GGTAGAAACCCAGCCTGTGACTATCATTTAGGAAACATTTCAAGACTGTCAAATA AGCATTTCCAAATACTACTAGGAGAAGACGGTAACCTTTTATTGAATGACATTTCC ACTAATGGGACCTGGTTAAATGGGCAAAAAGTCGAGAAGAACAGCAATCAGTTAC TGTCTCAAGGTGATGAAATAACCGTTGGTGTAGGCGTGGAATCAGATATTTTATCT 8 175727263.1CTGGTCATTTTCATAAACGACAAATTTAAGCAGTGCCTCGAGCAGAACAAAGTTG ATCGCTAA SEQ ID NO:20: ATGTCTAGCCTGGCCAGACAGCGGACACTGGAAGACGAGGAAGGCACCGGCGGA TCCGAGCTGAGCTTCTGCAACGTCTATATCAAGGCCGACAAGCAGAAGAACGGCA TCAAGGCGAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGC CTACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGAC AACCACTACCTGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGCGCG ATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGAC GAGCTGTACAAGGGCGGTACCGGAGGGAGCATGGTGAGCAAGGGCGAGGAGCTG TTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACA AGTTCAGCGTGTCCGGCGAGGGTGAGGGCGATGCCACCTACGGCAAGCTGACCCT GAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCA CCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCA CGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCTTCT TCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACA CCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACAT CCTGGGGCACAAGCTGGAGTACAACTTGCTGCATAAGTTTTCTCAAGAACAGATC GGCGAAAACATTGTGTGCAGGGTCATTTGTACCACGGGTCAAATTCCCATCCGAG ATTTGTCAGCTGATATTTCACAAGTGCTTAAGGAAAAACGATCCATAAAGAAAGTT TGGACATTTGGTAGAAACCCAGCCTGTGACTATCATTTAGGAAACATTTCAAGACT GTCAAATAAGCATTTCCAAATACTACTAGGAGAAGACGGTAACCTTTTATTGAATG ACATTTCCACTAATGGGACCTGGTTAAATGGGCAAAAAGTCGAGAAGAACAGCAA TCAGTTACTGTCTCAAGGTGATGAAATAACCGTTGGTGTAGGCGTGGAATCAGATA TTTTATCTCTGGTCATTTTCATAAACGACAAATTTAAGCAGTGCCTCGAGCAGAAC AAAGTTGATCGCTAA SEQ ID NO:21: ATGTCTAGCCTGGCCAGACAGCGGACACTGGAAGACGAGGAAACCGGCGGCAGC GAGCTGTCCTTCTGCAACGTCTATATCAAGGCCGACAAGCAGAAGAACGGCATCA AGGCGAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCTA CCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAAC CACTACCTGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGCGCGATC ACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGA GCTGTACAAGGGCGGTACCGGAGGGAGCATGGTGAGCAAGGGCGAGGAGCTGTT CACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAG TTCAGCGTGTCCGGCGAGGGTGAGGGCGATGCCACCTACGGCAAGCTGACCCTGA AGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACC CTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACG ACTTCTTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCTTCTTC 9 175727263.1AAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACC CTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCC TGGGGCACAAGCTGGAGTACAACTTGCTGCATAAGTTTTCTCAAGAACAGATCGG CGAAAACATTGTGTGCAGGGTCATTTGTACCACGGGTCAAATTCCCATCCGAGATT TGTCAGCTGATATTTCACAAGTGCTTAAGGAAAAACGATCCATAAAGAAAGTTTG GACATTTGGTAGAAACCCAGCCTGTGACTATCATTTAGGAAACATTTCAAGACTGT CAAATAAGCATTTCCAAATACTACTAGGAGAAGACGGTAACCTTTTATTGAATGAC ATTTCCACTAATGGGACCTGGTTAAATGGGCAAAAAGTCGAGAAGAACAGCAATC AGTTACTGTCTCAAGGTGATGAAATAACCGTTGGTGTAGGCGTGGAATCAGATATT TTATCTCTGGTCATTTTCATAAACGACAAATTTAAGCAGTGCCTCGAGCAGAACAA AGTTGATCGCTAA SEQ ID NO:22: ATGTCTAGCCTGGCCAGACAGCGGACCCTGGAAGACGAGGAAGGCAGCGAGCTGT CCTTCTGCAACGTCTATATCAAGGCCGACAAGCAGAAGAACGGCATCAAGGCGAA CTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCTACCACTAC CAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACC TGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGCGCGATCACATGGT CCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACA AGGGCGGTACCGGAGGGAGCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGG TGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGT GTCCGGCGAGGGTGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATC TGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTA CGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCA AGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCTTCTTCAAGGACGA CGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAAC CGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACA AGCTGGAGTACAACTTGCTGCATAAGTTTTCTCAAGAACAGATCGGCGAAAACAT TGTGTGCAGGGTCATTTGTACCACGGGTCAAATTCCCATCCGAGATTTGTCAGCTG ATATTTCACAAGTGCTTAAGGAAAAACGATCCATAAAGAAAGTTTGGACATTTGG TAGAAACCCAGCCTGTGACTATCATTTAGGAAACATTTCAAGACTGTCAAATAAGC ATTTCCAAATACTACTAGGAGAAGACGGTAACCTTTTATTGAATGACATTTCCACT AATGGGACCTGGTTAAATGGGCAAAAAGTCGAGAAGAACAGCAATCAGTTACTGT 10 175727263.1CTCAAGGTGATGAAATAACCGTTGGTGTAGGCGTGGAATCAGATATTTTATCTCTG GTCATTTTCATAAACGACAAATTTAAGCAGTGCCTCGAGCAGAACAAAGTTGATC GCTAA

[0011] In a further aspect, a biosensor is provided (which may suitably comprise: i) an enzyme substrate, ii) a detectably labelled protein, and iii)a phospho-amino acid binding protein) as disclosed herein and be encoded by a nucleic acid sequence having at least 70, 80, 90, 95 or 99 % sequence identity to any of SEQ ID NOS: 12-22.

[0012] In further aspects, methods of identifying modulators of kinases are provided which suitably comprise: a) contacting a biosensor or a cell expressing a biosensor, with one or more candidate agents, wherein the biosensor comprises an enzyme substrate comprising one or more peptides having at least 70% sequence identity to any of SEQ ID NOS: 1-11, a detectably labelled protein, and a phospho-amino acid binding protein; b) assaying for the presence or absence of a signal from the detectable label; and, c) identifying one or more modulators of kinases. In aspects, a signal from the detectable label is identified as an activator of the kinase. In aspects, the absence of a signal from the detectable label is identified as an inhibitor of the kinase.

[0013] Definitions

[0014] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, and biochemistry).

[0015] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0016] As used herein, the term “about” in the context of a numerical value or range means ±10% of the numerical value or range recited or claimed, unless the context requires a more limited range. 11 175727263.1

[0017] In the descriptions above and in the claims, phrases such as “at least one of” or “one or more of” may occur followed by a conjunctive list of elements or features. The term “and / or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and / or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items. For example, the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and / or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” In addition, use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible.

[0018] As used herein, the term “agent” or “candidate agent” is meant to encompass any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent, or biological agent capable of preventing, ameliorating, or treating a dysfunction or other medical condition. The term includes small molecule compounds, antisense oligonucleotides, siRNA reagents, antibodies, antibody fragments bearing epitope recognition sites, such as Fab, Fab’, F(ab’)2fragments, Fv fragments, single chain antibodies, antibody mimetics (such as DARPins, affibody molecules, affilins, affitins, anticalins, avimers, fynomers, Kunitz domain peptides and monobodies), peptoids, aptamers; enzymes, peptides organic or inorganic molecules, natural or synthetic compounds and the like. An agent can be assayed in accordance with the methods of the disclosure at any stage during clinical trials, during pre-trial testing, or following FDA-approval.

[0019] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements--or, as appropriate, 12 175727263.1equivalents thereof--and that other elements can be included and still fall within the scope / definition of the defined item, composition, apparatus, method, process, system, etc.

[0020] As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0021] A “derivative” polypeptide or peptide is one that is modified, for example, by glycosylation, pegylation, phosphorylation, sulfation, reduction / alkylation, acylation, chemical coupling, or mild formalin treatment. A derivative may also be modified to contain a detectable label, either directly or indirectly, including, but not limited to, a radioisotope, fluorescent, and enzyme label.

[0022] The term “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity over a specified region, e.g., of an entire polypeptide sequence or an individual domain thereof), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection. Such sequences that are at least about 80% identical are said to be “substantially identical.” In some embodiments, two sequences are 100% identical. In certain embodiments, two sequences are 100% identical over the entire length of one of the sequences (e.g., the shorter of the two sequences where the sequences have different lengths). In various embodiments, identity may refer to the complement of a test sequence. In some embodiments, the identity exists over a region that is at least about 10 to about 100, about 20 to about 75, about 30 to about 50 amino acids or nucleotides in length. In certain embodiments, the identity exists over a region that is at least about 50 amino acids in length, or more preferably over a region that is 100 to 500, 100 to 200, 150 to 200, 175 to 200, 175 to 225, 175 to 250, 200 to 225, 200 to 250 or more amino acids in length.

[0023] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. In various embodiments, when using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence 13 175727263.1coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0024] A “comparison window” refers to a segment of any one of the number of contiguous positions (e.g., least about 10 to about 100, about 20 to about 75, about 30 to about 50, 100 to 500, 100 to 200, 150 to 200, 175 to 200, 175 to 225, 175 to 250, 200 to 225, 200 to 250) in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. In various embodiments, a comparison window is the entire length of one or both of two aligned sequences. In some embodiments, two sequences being compared comprise different lengths, and the comparison window is the entire length of the longer or the shorter of the two sequences. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math.2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds.1995 supplement)).

[0025] In various embodiments, an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.215:403-410 (1990), respectively. BLAST and BLAST 2.0 may be used, with the parameters described herein, to determine percent sequence identity for nucleic acids and proteins. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information, as known in the art. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the 14 175727263.1query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0026] A “label” or a “detectable label” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radio labeled molecules fluorophores, radiochemical, luminescent compounds, electron- dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, radioactive compounds, non-radioactive compounds, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a label into the peptide.

[0027] The term “ligand,” includes any compound, composition or molecule capable of specifically or substantially specifically (that is with limited cross-reactivity) binding another compound or molecule, which, in the case of immune-recognition contains an epitope. In many instances, the ligands are antibodies, such as polyclonal or monoclonal antibodies. “Ligands” also include derivatives or analogs of antibodies, including without limitation: Fv fragments; single chain Fv (scFv) fragments; Fab' fragments; F(ab')2fragments; humanized antibodies and 15 175727263.1antibody fragments; camelized antibodies and antibody fragments; and multivalent versions of the foregoing. Multivalent binding reagents also may be used, as appropriate, including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((scFv)fragments), diabodies, tribodies or tetrabodies, which typically are covalently linked or otherwise stabilized (i.e., leucine zipper or helix stabilized) scFv fragments. “Ligands” also include peptoids, peptide or nucleic acid aptamers, or antibody mimetics such as DARPins, affibody molecules, affilins, affitins, anticalins, avimers, fynomers, Kunitz domain peptides and monobodies.

[0028] As used herein, “modulate,” “modulates” or “modulation” refers to enhancement (e.g., an increase) or inhibition (e.g., diminished, reduced or suppressed) of the specified activity or expression of a gene, polynucleotides, oligonucleotides, proteins, polypeptides, peptides or combinations thereof. Accordingly, a “modulator” enhances or inhibits expression, function or activity of gene, polynucleotides, oligonucleotides, etc.

[0029] The term “enhancement,” “enhance,” “enhances,” or “enhancing” refers to an increase in the specified parameter (e.g., at least about a 1.1-fold, 1.25-fold, 1.5-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold or more increase) and / or an increase in the specified activity of at least about 5%, 10%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%.

[0030] The term “inhibit,” “diminish,” “reduce” or “suppress” refers to a decrease in the specified parameter (e.g., at least about a 1.1-fold, 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold or more increase) and / or a decrease or reduction in the specified activity of at least about 5%, 10%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%. These terms are intended to be relative to a reference or control.

[0031] The term “fluorophore” includes any compound, composition or molecule capable of emitting light in response to irradiation. In many instances, fluorophores emit light in the visible region of light. In other instances, the fluorophores can emit light in the non- visible regions of light, such as ultraviolet, near-ultraviolet, near-infrared, and infrared. For example and without limitation, examples of fluorophores include: quantum dots; 16 175727263.1nanoparticles; fluorescent proteins, such as green fluorescent protein and yellow fluorescent protein; heme-based proteins or derivatives thereof; carbocyanine-based chromophores, such as IRDye 800CW, Cy 3, and Cy 5; coumarin-based chromophores, such as (7-diethylamino-3- (4'-maleimidylphenyl)-4-methylcoumarin) (CPM); fluorine-based chromophores, such as fluorescein, fluorescein isothiocyanate (FITC); and numerous ALEXA FLUORTMchromophores and ALEXA FLUORTMbioconjugates, which absorb in the visible and near- infrared spectra. The emission from the fluorophores can be detected by any number of methods, including but not limited to, fluorescence spectroscopy, fluorescence microscopy, fluorimeters, fluorescent plate readers, infrared scanner analysis, laser scanning confocal microscopy, automated confocal nanoscanning, laser spectrophotometers, fluorescent-activated cell sorters (FACS), image-based analyzers and fluorescent scanners (e.g., gel / membrane scanners).

[0032] As used herein, the term “chromophore” refers to a substituent which, with another chromophore, can be used for energy transfer (e.g., FRET assay).

[0033] The term “chemiluminescent compound” includes any compound, composition or molecule capable of emitting light in response to a chemical reaction. A “bioluminescent compound” refers to a naturally occurring form of a chemiluminescent compound. Examples of chemiluminescent compounds include: lucigenin, luminol. Examples of bioluminescent compounds include: luciferins, coelenterazines. The emission from chemiluminescent compounds can be detected by luminometers or scanning spectrometers.

[0034] The term “luminescent component” or “luminescent compound” as used herein refers to a component capable of absorbing energy, such as electrical (e.g., electro- luminescence), chemical (e.g., chemi-luminescence) or acoustic energy and then emitting at least some fraction of that energy as light over time. The term “component” as used herein includes discrete compounds, molecules, bioluminescent proteins and macro-molecular complexes or mixtures of luminescent and non-luminescent compounds or molecules that act to cause the emission of light.

[0035] The term “radiochemical” is intended to encompass any organic, inorganic or organometallic compound comprising a covalently-attached radioactive isotope, any inorganic 17 175727263.1radioactive ionic solution (e.g., Na[18F]F ionic solution), or any radioactive gas (e.g., [11C]CO2), particularly including radioactive molecular imaging probes intended for administration to a patient (e.g., by inhalation, ingestion, or intravenous injection) for tissue imaging purposes, which are also referred to in the art as radiopharmaceuticals, radiotracers, or radioligands. The compounds could also be readily adapted for synthesis of any radioactive compound comprising a radionuclide, including radiochemicals useful in other imaging systems, such as single photon emission computed tomography (SPECT).

[0036] The term “sample” as used herein refers to a biological sample obtained for the purpose of evaluation in vitro. With regard to the methods disclosed herein, the sample or patient sample preferably may comprise any body fluid or tissue. In some embodiments, the bodily fluid includes, but is not limited to, blood, plasma, serum, lymph, breast milk, saliva, mucous, semen, vaginal secretions, cellular extracts, inflammatory fluids, cerebrospinal fluid, feces, vitreous humor, or urine obtained from the subject. In some aspects, the sample is a composite panel of at least two of a blood sample, a plasma sample, a serum sample, and a urine sample. In exemplary aspects, the sample comprises blood or a fraction thereof (e.g., plasma, serum, fraction obtained via leukopheresis). Preferred samples are whole blood, serum, plasma, or urine. A sample can also be a partially purified fraction of a tissue or bodily fluid.

[0037] Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee. 18 175727263.1

[0039] FIG.1 is a schematic of a present biosensor system.

[0040] FIG.2 is a schematic of one approach for identifying an enzyme substrate peptide useful in the present biosensors.

[0041] FIG.3 depicts an analysis system as show in the Examples which follow.

[0042] FIG.4 shows FHA1 screens (left and right panels) of the Examples which follow.

[0043] FIG.5 shows dynamic range and signal:noise ratio values of enzyme substrate peptides. Dynamic range is quantified as the ratio of peak fluorescent to baseline (Fmax / F0). The signal:noise factor for each enzyme substrate peptide as measured as the ratio between the maximum-fold signal increase to the standard deviation of fluorescence across baseline measurements.

[0044] FIG.6 shows IC50 value for and enzyme substrate peptide. K562 cells were infected with lentivirus to stably express constitutively active enzyme substrate peptide and plated at different densities. Ruxolitinib, a CaMKII inhibitor, was then added to generate IC50 curves for both CaMKAR and the enzyme substrate peptide.

[0045] FIGS.7-11 shows data and results of Example 4 which follows. DETAILED DESCRIPTION

[0046] The Ca2+and Calmodulin dependent protein kinase II (CaMKII) is a known driver of heart injury, promoting heart failure, arrhythmias and death. There is great interest to further understand CaMKII signaling in the heart and to make CaMKII inhibitors to prevent heart injury and death. Both of these goals have been hindered by the lack of tools for measuring CaMKII activity with precise temporal and subcellular resolution in living cells.

[0047] Calcium / calmodulin-dependent protein kinase II (CaMKII) is a calcium signal transducer in cardiomyocytes and a known driver of heart failure and arrythmias. 19 175727263.1

[0048] As disclosed in PCT / US2023 / 016315 (WO2023 / 183625), CaMKAR has been developed, a circularly permutated GFP- (cpGFP) based biosensor that comprises a CaMKII target peptide and an FHA1 domain, which binds to phosphorylated amino acids.

[0049] We now enhance CaMKAR’s sensing of CaMKII activity by providing new peptide sequences (see, e.g., SEQ ID NOS.1-11 and sequences having at least 70% identity to one of those SEQ ID NOS.1-11) that in preferred aspects can improve affinity for CaMKII and / or binding to the FHA1 domain.

[0050] In one protocol, an empirically validated dataset of CaMKII substrates were filtered for candidates with appropriate sensitivity and specificity for CaMKII that also had phosphoresidues and surrounding sequences known to be favored by FHA1. With these criteria, 19 CaMKAR-derived plasmids were generated. We tested Dynamic range (Fmax / F0) of these CaMKAR derivatives was tested, in preferred aspects in HEK293T cells using fluorescence microscopy following treatment with ionomycin, a calcium ionophore. Multiple derivatives demonstrated dynamic range greater than CaMKAR, with one plasmid achieving an Fmax / F0 of 2.36 compared to 1.6 with CaMKAR. Secondary modifications were made to that preferred sequence to identify optimal length of the linker regions. We created 8 variants that altered the length of the substrate-cpGFP linker. Five of these linker modifications further improved the molecule, including a new sensor that achieved an Fmax / F0 of 3.1.

[0051] Biosensors

[0052] In one aspect, the present disclosure is based in part on the discovery of a CaMKAR (CaMKII Activity Reporter) a novel, genetically encoded fluorescent biosensor that reports CaMKII activity in living cells and in vitro. The biosensor is comprised of an amino acid sequence containing a CaMKII substrate, a fluorescent protein, and a phospho-amino acid binding protein. When CaMKII is active, it phosphorylates the substrate, which causes the fluorescent protein to become detectably brighter. The biosensor signal is reported as a ratio collected at two wavelengths (405; which decreases in active state, and 488: which increases). The results show that this phosphorylation is due to CaMKII activity and triggers increased fluorescence that is measurable by microscopy, plate reader, and flow cytometry. The biosensor sensitively reports CaMKII activity using pharmacological (Ionomycin-mediated Ca2+overload) and genetic (CaMKIIT287D, constitutively active mutation) activators of 20 175727263.1CaMKII. The instantly described biosensor has vastly superior dynamic range, signal-to-noise ratio, and activation kinetics compared to currently available sensors.

[0053] Accordingly, in certain embodiments, the synthetic biosensor comprises: an enzyme substrate, a detectably labelled protein, and a phospho-amino acid binding protein. In certain embodiments, the enzyme substrate is a kinase. In certain embodiments, the kinase is calcium / calmodulin-dependent protein kinase II (CaMKII). In certain embodiments, the CaMKII substrate comprises an amino acid sequence having at least about 70% sequence identity to one or more of SEQ ID NOS.1-11. In certain embodiments, the CaMKII substrate comprises an amino acid sequence having at least about 80, 90 or 95% sequence identity to one or more of SEQ ID NOS.1-11. In certain embodiments, the CaMKII substrate comprises the amino acid sequence of any one or more of SEQ ID NOS.1-11.

[0054] In certain embodiments, a calcium / calmodulin-dependent protein kinase II (CaMKII) substrate comprises an amino acid sequence having at least about 70% sequence identity to one or more of SEQ ID NOS.1-11. In certain embodiments, the CaMKII substrate comprises an amino acid sequence having at least about 80, 90 or 95 % sequence identity to one or more of SEQ ID NOS.1-11. In certain embodiments, the CaMKII substrate comprises the amino acid sequence of any one of SEQ ID NOS.1-11.

[0055] In certain embodiments, the components are linked together to provide a unimolecular biosensor. In certain embodiments, the components are covalently attached by a linker, preferably a flexible polypeptide linker. In an embodiment, the flexible polypeptide linker has a length corresponding to the length of a random amino acid sequence of about 50 to about 500-1000 amino acids, for example corresponding to the length of a random amino acid sequence of about 100 to about 400-500 amino acids, preferably about 200-400 amino acids, for example about 300. In a further embodiment, the flexible linker comprises a random amino acid sequence of about 50 to about 500-1000 amino acids, for example a random amino acid sequence of about 100 to about 400-500 amino acids, preferably a random amino acid sequence of about 200-400 amino acids, for example about 300 amino acids. Methods for designing flexible amino acid linkers, and more specifically linkers with minimal globularity and maximal disorder, are known in the art. This may be achieved, for example, using the 21 175727263.1Globplot 2.3 program. The sequence may be further optimized to eliminate putative aggregation hotspots, localization domains, and / or interaction and phosphorylation motifs.

[0056] In certain embodiments, the biosensor comprises one or more amino acid variants. The variant as used herein refers to a protein / polypeptide having has an identity or similarity of at least 60% with a reference (e.g., native) sequence and retains a desired activity thereof, for example the capacity to bind to a target protein and / or to translocation to a cellular compartment. In further embodiments, the variant has a similarity or identity of at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% with a reference (e.g., native) sequence and retains a desired activity thereof. “Similarity” and “identity” refers to sequence similarity / identity between two polypeptide molecules. The similarity or identity can be determined by comparing each position in the aligned sequences. A degree of similarity or identity between amino acid sequences is a function of the number of matching or identical amino acids at positions shared by the sequences. Optimal alignment of sequences for comparisons of similarity or identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol.48: 443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sd. USA 85: 2444, and the computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.). Sequence similarity or identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol.215: 403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information web site (ncbi.nlm.nih.gov / ).

[0057] The term “amino acid” as used herein refers to naturally occurring and synthetic α, β, γ, and δ amino acids, and includes but is not limited to, amino acids found in proteins, i.e. glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine. Alternatively, the amino acid can be a derivative of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl, 22 175727263.1histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleucinyl, β-prolinyl, β-phenylalaninyl, β- tryptophanyl, β-methioninyl, β-glycinyl, β-serinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β- asparaginyl, β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl or β-histidinyl. When the term amino acid is used, it is considered to be a specific and independent disclosure of each of the esters of α, β, γ, and δ glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine in the D and L-configurations.

[0058] Accordingly, the amino acids in the biosensors can include natural and synthetic amino acid substitutions.

[0059] Detectable Label: Any fluorescent polypeptide (also referred to herein as a fluorescent label) well known in the art is suitable for use as a domain of the subject biosensor polypeptides of the present invention. A suitable fluorescent polypeptide will be one that can be expressed in a desired host cell, such as a mammalian cell, and will readily provide a detectable signal that can be assessed qualitatively (positive / negative) and quantitatively (comparative degree of fluorescence). Exemplary fluorescent polypeptides include, but are not limited to, yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), GFP, mRFP, RFP (tdimer2), HCRED, etc., or any mutant (e.g., fluorescent proteins modified to provide for enhanced fluorescence or a shifted emission spectrum), analog, or derivative thereof. Further suitable fluorescent polypeptides, as well as specific examples of those listed herein, are provided in the art and are well known.

[0060] In some embodiments where multiple biosensor polypeptides are present in a cell, the fluorescent polypeptide of the biosensor is selected so that each biosensor polypeptide in the cell has a detectably different emission spectrum. For example, in such embodiments, the G1BP fluorescent label, the SBP fluorescent label, and the MBP fluorescent label, when present in the same cell, are designed to have detectably distinct emission spectra to facilitate detection of a distinct signal from each biosensor (e.g., through use of different filters in the imaging system).

[0061] In certain embodiments, the label may be radioactive. Some examples of useful radioactive labels include32P,125I,131I, and3H. Use of radioactive labels have been described in U.K.2,034,323, U.S. Pat. No.4,358,535, and U.S. Pat. No.4,302,204. Some examples of 23 175727263.1non-radioactive labels include enzymes, chromophores, atoms and molecules detectable by electron microscopy, and metal ions detectable by their magnetic properties.

[0062] Some useful enzymatic labels include enzymes that cause a detectable change in a substrate. Some useful enzymes and their substrates include, for example, horseradish peroxidase (pyrogallol and o-phenylenediamine), β-galactosidase (fluorescein β-D- galactopyranoside), and alkaline phosphatase (5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium). The use of enzymatic labels has been described in U.K.2,019,404, EP 63,879, and by Rotman, Proc. Natl. Acad. Sci. USA, 47, 1981-1991 (1961).

[0063] Useful chromophores include, for example, fluorescent, chemiluminescent, and bioluminescent molecules, as well as dyes. Some specific chromophores useful in the present disclosure include, for example, fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone, luminol.

[0064] The labels may be conjugated to the biosensor by methods that are well known in the art. The labels may be directly attached through a functional group on the probe. The probe either contains or can be caused to contain such a functional group. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate. Alternatively, labels such as enzymes and chromophores may be conjugated to the antibodies or nucleotides by means of coupling agents, such as dialdehydes, carbodiimides, dimaleimides, and the like.

[0065] In certain embodiment, the biosensors of the disclosure can be used for imaging. In imaging uses, the complexes are labeled so that they can be detected outside the body. Typical labels are radioisotopes, usually ones with short half-lives. The usual imaging radioisotopes, such as123I,124I,125I,131I,99mTC,186Re,188Re,64Cu,67Cu,212Bi,213Bi,67Ga,90Y,111In,18F,3H,14C,35S or32P can be used. Nuclear magnetic resonance (NMR) imaging enhancers, such as gadolinium-153, can also be used to label the complex for detection by NMR. Methods and reagents for performing the labeling, either in the polynucleotide or in the protein moiety, are considered known in the art.

[0066] Reporter genes useful in the present disclosure include acetohydroxy acid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red 24 175727263.1fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline. Methods to determine modulation of a reporter gene are well known in the art, and include, but are not limited to, fluorometric methods (e.g. fluorescence spectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy), antibiotic resistance determination.

[0067] Localization Sequence: The term “localization sequence” refers to a biomolecule, such as a polypeptide or peptide, which, when attached to the synthetic biosensor embodied herein (as a fusion protein, for example), targets them to a particular compartment, organelle or localization within the cell, such as for example the plasma membrane (or a particular subdomain of the plasma membrane, such as lipid rafts), the endosomes (e.g. early and / or late endosomes), the lysosomes, the phagosomes, the ribosomes, the mitochondria, the endoplasmic reticulum, the Golgi apparatus, the nucleus, etc. Peptides that target proteins to specific compartment, organelle or localization within the cell are known in the art and include endoplasmic reticulum (ER) signal peptide or ER-retrieval sequence, nuclear localization signal (NLS) peptide, and mitochondrial localization signal (MLS) peptide, for example.

[0068] In certain embodiments, the localization sequence is a plasma membrane (PM) targeting sequence. Any localization sequence capable of recruiting the biosensor to the PM may be used in the biosensors. The biosensor may thus be fused to any protein found at the plasma membrane (e.g., receptors or any other protein found at the PM), or fragments thereof. Examples or localization sequences include peptides / polypeptides comprising a signal sequence for protein lipidation / fatty acid acylation, such as myristoylation, palmitoylation and prenylation, as well as polybasic domains. Several proteins are known to be myristoylated, palmitoylated and / or prenylated (e.g., protein kinases and phosphatases such as Yes, Fyn, Lyn, Lck, Hck, Fgr, G, proteins, nitric oxide synthase, ADP-ribosylation factors (ARFs), calcium binding proteins and membrane or cytoskeleton-associated structural proteins such as MARCKS (see, e.g., Wright et al., J Chem Biol. March 2010; 3(1): 19-35; Alcart-Ramos et al., Biochimica et Biophysica Acta (BBA)--Biomembranes, Volume 1808, Issue 12, December 25 175727263.12011, Pages 2981-2994), and thus the myristoylation, palmitoylation and prenylation signal sequences from any of these proteins may be used in the biosensor. In certain embodiments, the myristoylation and / or palmitoylation sequence is from the Lyn kinase.

[0069] In certain embodiments, the localization sequence comprises a cytokine sequence which can bind to its receptor, e.g., IL-2 or comprises a receptor sequence which binds to the cytokine. In other embodiments, the localization sequence can be an aptamer or binding fragment of an antibody, e.g., scFv.

[0070] In certain embodiments, the localization sequence is an endosomal targeting moiety. Several endosomal targeting moieties / markers are known in the art and include the Rab family of proteins (RAB4, RAB5, RAB7, RAB9 and RAB11), mannose 6-phosphate receptor (M6PR), caveolin-1 and -2, transferrin and its receptor, clathrin, as well as proteins comprising a FYVE domain such as early endosome autoantigen 1 (EEA1), Rabenosyn-5, Smad anchor for receptor activation (SARA), Vps27p and Endofin. Some markers are more specific to early endosomes (e.g., RAB4, Transferrin and its receptor, and proteins comprising a FYVE domain), others are more specific to late endosomes (e.g., RAB7, RAB9, and M6PR) and others are more specific to recycling endosomes (e.g., RAB11, RAB4). Thus, these proteins or suitable fragments thereof may be fused to the synthetic biosensor embodied herein to link / target them to an endosomal localization.

[0071] In certain embodiments, the localization sequence is a lysosomal targeting moiety, such as for example LAMP1 and LAMP2. Thus, these proteins or suitable fragments thereof may be fused to the synthetic biosensor embodied herein to link / target them to a lysosomal localization.

[0072] In certain embodiments, the localization sequence is a peroxisomal targeting moiety, such as for example PMP70, PXMP2 and Catalase. Thus, these proteins or suitable fragments thereof may be fused to the synthetic biosensor embodied herein to link / target them to a peroxisomal localization.

[0073] In certain embodiments, the localization sequence is an autophagosomal targeting moiety, such as for example ATG (AuTophaGy related) family proteins (ATG4, ATG5, ATG16, ATG12, see Lamb et al., Nature Reviews Molecular Cell Biology 14, 759-774 26 175727263.1(2013)), LC3A / B and SQSTMI / p62. Thus, these proteins or suitable fragments thereof may be fused to the synthetic biosensor embodied herein to link / target them to an autophagosomal localization.

[0074] In certain embodiments, the localization sequence is a ribosome targeting moiety. Several endosomal targeting moieties / markers are known in the art and include the Ribosomal Proteins (L7a, S3 and S6). Thus, these proteins or suitable fragments thereof may be fused to the synthetic biosensor embodied herein to link / target them to a ribosomal localization.

[0075] In certain embodiments, the localization sequence is an endoplasmic reticulum (ER) targeting moiety. Several ER targeting moieties / markers are known in the art and include ERp72, ERp29, Protein disulphide isomerase (PDI), HSP70 family proteins such as GRP78 (HSPA5), GRP94 (HSP90B1) and GRP58 (PDIA3), Calnexin and Calreticulin. Thus, these proteins or suitable fragments thereof may be fused to the synthetic biosensor embodied herein to link / target them to an ER localization.

[0076] In certain embodiments, the localization sequence is a Golgi targeting moiety. Several Golgi targeting moieties / markers are known in the art and include eNOS (e.g., the N- terminal portion thereof, J. Liu et al., Biochemistry, 35 (1996), pp.13277-13281), GM130, Golgin-97, the 58K protein, Trans-Golgi network membrane protein 2 (TGOLN2), TGN46, TGN38, Mannosidase 2, Syntaxin 6, GM130 (GOLGA2), Golgin-160, Membrin (GS27), GS28, Coatomer proteins, Rbet1 and RCAS1. Thus, these proteins or suitable fragments thereof may be fused to the synthetic biosensor embodied herein to link / target them to a Golgi apparatus localization.

[0077] In certain embodiments, the localization sequence is a mitochondria targeting moiety. Several mitochondria targeting moieties / markers are known in the art and include AIF, COX IV, Cytochrome C, hexokinase I, SOD1, SDHA, Pyruvate dehydrogenase, VDAC, TOMM22, UCP1, UCP2, UCP3, PHB1 Galpha12 (or the N-terminal portion thereof; Andreeva et al., FASEB J.2008 August; 22(8):2821-31. Epub 2008 Mar.26), a protein of the BcI-family member or a fragment thereof (Mossalam et al., Mol Pharm.2012 May 7; 9(5): 1449-1458). Thus, these proteins or suitable fragments thereof may be fused to the synthetic biosensor 27 175727263.1embodied herein to link / target them to a mitochondrial localization. The nuclear targeting moiety may also comprise a mitochondrial targeting signal, which is a 10-70 amino acid long peptide that directs newly synthesized proteins to the mitochondria. It is found at the N- terminus and consists of an alternating pattern of hydrophobic and positively charged amino acids to form an amphipathic helix. Mitochondrial targeting signals can contain additional signals that subsequently target the protein to different regions of the mitochondria, such as the mitochondrial matrix.

[0078] In certain embodiments, the localization sequence is a nuclear targeting moiety. Several nuclear targeting moieties / markers are known in the art and include Lamin A / C, Nudeoporins (NUP), ASHL2, ESET, Histones, LSD1, DNA repair enzymes such as PARP, and P84 / THOC1. Thus, these proteins or suitable fragments thereof may be fused to The synthetic biosensor embodied herein to link / target them to a nuclear localization. The nuclear targeting moiety may also comprises a nuclear localization signal or sequence (NLS), which is an amino acid sequence that tags a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. The best characterized transport signal is the classical NLS (cNLS) for nuclear protein import, which consists of either one (monopartite) or two (bipartite) stretches of basic amino acids. Monopartite cNLSs are exemplified by the SV40 large T antigen NLS and bipartite cNLSs are exemplified by the nucleoplasmin NLS.

[0079] In certain embodiments, the localization sequence is a nuclear export sequence (NES). NES is a short amino acid sequence (typically 4 hydrophobic residues) in a protein that targets it for export from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport. The sequence of such NES may be for example LxxxLxxLxL, where “L” is a hydrophobic residue (often leucine) and “x” is any other amino acid. In proteins that are translocated from cytosol to nucleus (such as ERK or MDM2), a decrease in the BRET signal is detected using an NES moiety.

[0080] In certain embodiments, the localization sequence is a cytoskeleton targeting moiety, for example actin or a fragment thereof, or a protein comprising an actin-binding domain (ABD), such as the N-terminal F-actin binding domain of Inositol-1,4,5-trisphosphate- 28 175727263.13-kinase-A (ITPKA) (Johnson and Schell, Mol. Biol. Cell Dec.15, 2009 vol.20 no.245166- 5180).

[0081] The localization sequence can be fused or linked to the N- and / or C-termini of the biosensor. Other domains or linkers may be present at the N-terminal, C-terminal or within the components of the biosensor. In embodiments, the synthetic biosensor embodied herein may be covalently linked to the localization sequence either directly (e.g., through a peptide bond) or “indirectly” via a suitable linker moiety, e.g., a linker of one or more amino acids (e.g., a polyglycine linker) or another type of chemical linker (e.g., a carbohydrate linker, a lipid linker, a fatty acid linker, a polyether linker, PEG, etc. In an embodiment, one or more additional domain(s) may be inserted before (N-terminal), between or after (C-terminal) the components of the biosensor. In certain embodiments, the linker comprises about 4 to about 50 amino acids, about 4 to about 40, 30 or 20 amino acids, or about 5 to about 15 amino acids, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

[0082] Vectors: The present disclosure provides a nucleic acid encoding the biosensor. In certain embodiments, the nucleic acid is present in a vector / plasmid, in a further embodiment an expression vector / plasmid. Such vectors comprise a nucleic acid sequence capable of encoding the above-defined first and / or second component(s) operably linked to one or more transcriptional regulatory sequence(s), such as promoters, enhancers and / or other regulatory sequences.

[0083] The term “vector” refers to a nucleic acid molecule, which is capable of transporting another nucleic acid to which it has been linked. One type of vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Preferred vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors”. A recombinant expression vector of the present invention can be constructed by standard techniques known to one of ordinary skill in the art and found, for example, in Sambrook et al. (1989) in Molecular Cloning: A Laboratory Manual. A variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments and can be readily determined by persons skilled in the art. The vectors of the present invention may also contain 29 175727263.1other sequence elements to facilitate vector propagation and selection in bacteria and host cells. In addition, the vectors of the present invention may comprise a sequence of nucleotides for one or more restriction endonuclease sites. Coding sequences such as for selectable markers and reporter genes are well known to persons skilled in the art.

[0084] A recombinant expression vector comprising a nucleic acid sequence of the present invention may be introduced into a cell (a host cell), which may include a living cell capable of expressing the protein coding region from the defined recombinant expression vector. The living cell may include both a cultured cell and a cell within a living organism. Accordingly, the invention also provides host cells containing the recombinant expression vectors of the invention. The terms “cell”, “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0085] Vector DNA can be introduced into cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection” refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can for example be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory manuals. “Transcriptional regulatory sequence / element” is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals which induce or control transcription of protein coding sequences with which they are operably linked. A first nucleic acid sequence is “operably-linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences. Generally, operably-linked DNA sequences are contiguous 30 175727263.1and, where necessary to join two protein coding regions, in reading frame. However, since for example enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous.

[0086] Host Cells: In another aspect, the present disclosure provides a cell comprising or expressing the biosensors embodied herein. In an embodiment, the cell has been transfected or transformed with a nucleic acid encoding the above-defined first and / or second component(s). The disclosure further provides a recombinant expression system, vectors and cells, such as those described above, for the expression of the biosensors, using for example culture media and reagents well known in the art. The cell may be any cell capable of expressing the first and second component(s) defined above. Suitable host cells and methods for expression of proteins are well known in the art. Any cell capable of expressing the component(s) defined above may be used. For example, eukaryotic host cells such as mammalian cells may be used (e.g., rodent cells such as mouse, rat and hamster cell lines, human cells / cell lines). In another embodiment, the above-mentioned cell is a human cell line, for example an embryonic kidney cell line (e.g., HEK293 or HEK293T cells).

[0087] Methods Of Use

[0088] In certain embodiments, biosensor embodied herein is used in an assay, such as for example a high-throughput screening assays for assessing or identifying modulators of kinases. Accordingly, in certain embodiments, a method of identifying modulators of kinases comprises contacting a biosensor or a cell expressing a biosensor, with one or more candidate agents, wherein the biosensor comprises an enzyme substrate, a detectably labelled protein, and a phospho-amino acid binding protein; assaying for the presence or absence of a signal from the detectable label; and, identifying modulators of kinases. In certain embodiments, a signal from the detectable label is identified as an activator of the kinase. In certain embodiments, the absence of a signal from the detectable label is identified as an inhibitor of the kinase. In certain embodiments, the kinase is calcium / calmodulin-dependent protein kinase II (CaMKII). In certain embodiments, the CaMKII comprises an amino acid sequence having at least about sequence identity to one or more of SEQ ID NOS.1-11.

[0089] Kits 31 175727263.1

[0090] The present disclosure provides for kits. As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and / or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. As used herein, the term “fragmented kit” refers to a delivery systems comprising two or more separate containers that each contain a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides. The term “fragmented kit” is intended to encompass kits containing Analyte specific reagents (ASR's) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.

[0091] Accordingly, in certain embodiments, a kit comprises a synthetic biosensor embodied herein. In certain embodiments, a kit comprises an expression vector encoding a synthetic biosensor embodied herein. In certain embodiments, a kit comprises a host cell comprising a synthetic biosensor embodied herein or vector encoding a synthetic biosensor embodied herein. In certain embodiments, a kit comprises a peptide comprising one or more of SEQ ID NOS.1-11. EXAMPLES Example 1 Novel CaMKII biosensor enables identification of potent CaMKII inhibitors.

[0092] Ca2+ / Calmodulin-dependent protein kinase II (CaMKII) is a highly validated cause or contributor to major cardiac illnesses, including heart failure, myocardial infarction, 32 175727263.1and arrhythmias. Excessive CaMKII activity causes intracellular Ca2+dysregulation, inflammation, maladaptive transcription, and cell death. Yet, there are currently no approved CaMKII inhibiting therapies. Thus, development of safe and effective CaMKII inhibitors is a translational priority.

[0093] We sought to identify and optimize a CaMKII substrate peptide that improves sensing of CaMKII activity by CaMKAR; this can be achieved via either improved binding to FHA1 (Phosphorylated amino acid binding domain in CaMKAR), improved affinity for CaMKII as a substrate, or both.

[0094] Method: To identify peptide sequences that could improve CaMKAR performance (either via improved substrate affinity for CaMKII and / or improved binding to the FHA1 domain), we identified desirable sequences within an empirically validated dataset of CaMKII substrates (PMID: 36631611). We filtered this dataset to reveal peptides that: have a kinase activity percentile above 50 for all 4 CaMKII isoforms (to enrich for sensitive peptides), had an average kinase selectivity rank across all 4 CaMKII of 95 or better (to enrich for peptides specific to CaMKII), have threonine (T) as the phosphoresidue (the preferred phosphoresidue of FHA1, PMID: 11106755), lack cysteine (C) or tryptophan (W) in the first or second positions after the phosphoresidue (C or W at positions pT+1 or +2 are unfavored by FHA1, PMID: 11106755), have aspartic acid (D) in the third position after the phosphoresidue (preferred residue at the pT+3 position by FHA1, PMID: 11106755). This yielded 17 peptides (the original CaMKAR substrate was also part of this subset but was removed). Additionally, we included 2 modified versions of the original CaMKAR substrate sequence: one which substituted C at position pT+3 for D, and the second which replaced positions pT+2 and +3 for E and D, both in an effort to create a more favorable peptide for FHA1 binding (PMID: 11106755). These 19 peptides comprised our primary screen.

[0095] Secondary screen: Peptide P8 was identified as the most promising candidate due to improved dynamic range compared to CaMKAR. Secondary modifications were made to this peptide to identify optimal distance from the CaMKAR linker and orientation relative to FHA1. We created 4 variants that truncated the original peptide from its C-terminus by 1 amino acid and 4 variants that truncated the substrate-CaMKAR linker from its N-terminus by 1 amino acid. 33 175727263.1

[0096] Example 2 CaMKAR-based screen of drugs in clinical use

[0097] Next, the clinically-approved pharmacopeia was canvassed for drugs that are safe for human pharmacotherapy and potent CaMKII inhibitors. Due to its high throughput tractability, CaMKAR is uniquely configured to address this task. For screening, a stable line of human K562 cells were first created that co-express CaMKAR and CaMKIIδCA: K562CaMKII-CaMKAR. K562 cells were chosen because they grow at high density and remain viable after expression of active CaMKII .

[0098] Example 3:

[0099] FIG.6 shows IC50 value for and enzyme substrate peptide. K562 cells were infected with lentivirus to stably express constitutively active enzyme substrate peptide and plated at different densities. Ruxolitinib, a CaMKII inhibitor, was then added to generate IC50 curves for both CaMKAR and the enzyme substrate peptide. [000100] Example 4: Screening protocol [000101] Ca2+ / calmodulin-dependent protein kinase II (CaMKII) hyperactivity is proven to contribute to the pathogenesis of cardiovascular diseases,including atrial fibrillation. There is currently no curative treatment for atrial fibrillation, and medical therapies often have serious side effects. Studies have shown the therapeutic benefit of CaMKII inhibition; however, there is no FDA-approved CaMKII inhibitor drug. Reyes Gaido OE et al., Annu Rev Pharmacol Toxicol.2023 Jan 20;63:249-272. doi: 10.1146 / annurev-pharmtox-051421-111814. Epub 2022 Aug 16. PMID: 35973713; PMCID: PMC11019858. We have previously designed a fluorescent CaMKII activity reporter (CaMKAR) to conduct a drug repurposing screen of FDA approved drugs. From this initial screen, ruxolitinib emerged as a previously unrecognized CaMKII inhibitor due to its targeting of the ATP-binding site. Reyes Gaido et al., Sci. Transl. Med.15,eabq7839(2023).DOI:10.1126 / scitranslmed.abq7839. [000102] This Example provides a high throughput screen that includes use of commercial library of 24,000 “Hinge Binders,” which target the ATP-binding domain essential for kinase function. Hinge Binders Library. (n.d.). Enamine. https: / / enamine.net / compound- libraries / targetedlibraries / kinase-library / hinge-binders-library / [000103] K562 cells were infected with lentivirus containingdynamic range. The 34 175727263.1noted re-assembled hinge-binder drug library was used, with DMSO and ruxolitinib as controls. CaMKAR fluorescence was measured at 488 nm and 405 nm for ratiometric quantification of CaMKII activity. Fluorescence ratios were standardized using the DMSO control, with significant candidates selected based on a Bonferroni-adjusted p-value threshold. Outliers were removed according to the 405 nm signal to account for the expected stability. [000104] The average 488 / 405 ratio for DMSO was 2.77 compared to ruxolitinib of 1.85. After standardization of CaMKII inhibition, 181 drugs were selected as initial candidates. Once outliers were removed, 38 drugs remained as candidates for secondary testing. The average 488 / 405 ratio for the 38 candidates was 2.21. Results are shown in FIGS.7-11. In FIG.11, the values shown starting at about -5 at the bottom of the y axis and progressing in order vertically are as follows: Ruxolitinib, Z5, Z3, Z6, Z2, Z1, Z4. [000105] This expanded high-throughput screen successfully demonstrated consistent ruxolitinib and DMSO 488 / 405 ratios across different plates and days as well as identified candidate drugs with potential CaMKII inhibitory activity. These results provide a proof of concept for high throughput drug screens to identify kinase inhibitors. Data from the drug screen can also inform computation optimization of designed CaMKII inhibitors. Subsequent steps include iterating between computational in silico development of CaMKII inhibitors and data acquisition through in vitro and cellular inhibitory assays. With candidates that display potent inhibition, physiological experiments can be performed trialing anti-arrhythmic and ischemic protective properties of candidates. OTHER EMBODIMENTS

[0001] From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. All citations to sequences, patents and publications in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. 35 175727263.1

Claims

What is claimed:

1. A synthetic biosensor comprising: an enzyme substrate comprising one or more peptides having at least 70% sequence identity to any of SEQ ID NOS: 1-11; a detectably labelled protein; and a phospho-amino acid binding protein.

2. The synthetic biosensor of claim 1, wherein the enzyme substrate comprising one or more peptides having at least 80% sequence identity to any of SEQ ID NOS: 1-11.

3. The synthetic biosensor of claim 1, wherein the enzyme substrate comprising one or more peptides having at least 90% sequence identity to any of SEQ ID NOS: 1-11.

4. The synthetic biosensor of claim 1, wherein the enzyme substrate comprising one or more peptides having at least 95% sequence identity to any of SEQ ID NOS: 1-11.

5. The synthetic biosensor of claim 1, wherein the enzyme substrate comprising one or more peptides having SEQ ID NOS: 1-11.

6. The synthetic biosensor of any one of claims 1 through 5, wherein the detectably labelled protein comprises a radio labeled molecule, a fluorophore, a radiochemical, a luminescent compound, an electron-dense reagent, an enzyme, biotin, a radioactive compound, a non-radioactive compound, digoxigenin or a hapten.

7. The synthetic biosensor of claim 6, wherein the detectably labelled protein comprises a fluorophore. 36 175727263.

18. The synthetic biosensor of any one of claims 1 to 7, further comprising an intracellular or extracellular localization sequence.

9. The synthetic biosensor of claim 8, wherein the localization sequence is fused to the biosensor.

10. The synthetic biosensor of claim 8, wherein the localization sequence is covalently linked through a flexible linker.

11. The synthetic biosensor of claim 10, wherein the flexible linker is a polypeptide comprising at least five amino acids.

12. An expression vector encoding a biosensor comprising: an enzyme substrate comprising one or more peptides having at least 70% sequence identity to any of SEQ ID NOS: 1-11; a detectably labelled protein, and a phospho-amino acid binding protein.

13. The expression vector of claim 12, wherein the enzyme substrate comprise an amino acid sequence having at least about 80% sequence identity to any of SEQ ID NOS: 1-11.

14. The expression vector of claim 12, wherein the enzyme substrate comprise an amino acid sequence having at least about 90% sequence identity to any of SEQ ID NOS: 1-11.

15. The expression vector of claim 12, wherein the enzyme substrate comprise an amino acid sequence having at least about 95% sequence identity to any of SEQ ID NOS: 1-11. 37 175727263.

116. The expression vector of claim 12, wherein the enzyme substrate comprise an amino acid of any of SEQ ID NOS: 1-11.

17. The expression vector of any of claims 12 to 16, further comprising an intracellular or extracellular localization sequence.

18. A host cell comprising the synthetic biosensor of any one of claims 1 through 11 or the expression vector of any one of claims 12 through 17.

19. A method of identifying modulators of kinases comprising: contacting a biosensor or a cell expressing a biosensor, with one or more candidate agents, wherein the biosensor comprises i) an enzyme substrate comprising one or more peptides having at least 70% sequence identity to any of SEQ ID NOS: 1-11, ii) a detectably labelled protein, and iii) a phospho-amino acid binding protein; assaying for the presence or absence of a signal from the detectable label; and, identifying modulators of kinases.

20. The method of claim 19, wherein a signal from the detectable label is identified as an activator of the kinase.

21. The method of claim 19 or 20, wherein the absence of a signal from the detectable label is identified as an inhibitor of the kinase.

22. The method of any one of claims 19 to 21, wherein the enzyme substrate comprises an amino acid sequence having at least about 80% sequence identity to any of SEQ ID NOS: 1-11.

23. The method of any one of claims 19 to 21, wherein the enzyme substrate comprises an amino acid sequence having at least about 90% sequence identity to any of SEQ ID NOS: 1-11. 38 175727263.

124. The method of any one of claims 19 to 21, wherein the enzyme substrate comprises an amino acid sequence having at least about 95% sequence identity to any of SEQ ID NOS: 1-11.

25. The method of claims 19 to 21, wherein the enzyme substrate comprises an amino acid sequence of any of SEQ ID NOS: 1-11.

26. The method of any one of claims 19 through 25, wherein the detectably labelled protein comprises a radio labeled molecule, a fluorophore, a radiochemical, a luminescent compound, an electron-dense reagent, an enzyme, biotin, a radioactive compound, a non-radioactive compound, digoxigenin or a hapten.

27. The method of claim 26, wherein the detectably labelled protein comprises a fluorophore.

28. A calcium / calmodulin-dependent protein kinase II (CaMKII) substrate comprising an amino acid sequence having at least about 70% sequence identity to any of SEQ ID NOS: 1-11.

29. The CaMKII substrate of claim 28, wherein the CaMKII substrate comprises an amino acid sequence having at least about 80% sequence identity to any of SEQ ID NOS: 1-11.

30. The CaMKII substrate of claim 28, wherein the CaMKII substrate comprises an amino acid sequence having at least about 90% sequence identity to any of SEQ ID NOS: 1-11.

31. The CaMKII substrate of claim 28, wherein the CaMKII substrate comprises an amino acid sequence having at least about 95% sequence identity to any of SEQ ID NOS: 1-11.

32. The CaMKII substrate of claim 33, wherein the CaMKII substrate comprises the amino acid sequence of any of SEQ ID NOS: 1-11. 39 175727263.

133. The CaMKII substrate of claim 28 to 32, further comprising a localization sequence.

34. The CaMKII substrate of claim 33, wherein the localization sequence is an intracellular or extracellular localization sequence.

35. The CaMKII substrate of claim 34, wherein the extracellular localization sequence comprises a ligand or receptor sequence.

36. The CaMKII substrate of claim 34 or 35, wherein the localization sequence is covalently linked through a flexible linker.

37. The CaMKII substrate of claim 36, wherein the flexible linker is a polypeptide comprising at least five amino acids.

38. The CaMKII substrate of any one of claims 38 through 37, further comprising one or more amino acids at the N-terminus, the C-terminus or the combination thereof.

39. The CaMKII substrate of claim 38, further comprising one or more amino acid analogs.

40. A kit comprising the synthetic biosensor of any one of claims 1 through 11, the expression vector of any one of claims 12 through 17, the host cell of claim 18 or the calcium / calmodulin-dependent protein kinase II (CaMKII) substrate of any one of claims 19 through 39.

41. A synthetic biosensor encoded by a sequence having at least 70% sequence identity to any of SEQ ID NOS: 12-22.

42. A synthetic biosensor encoded by a sequence having at least 80%, 90%, 95, 98% or 99% sequence identity to any of SEQ ID NOS: 12-22. 40 175727263.

143. A method of identifying modulators of kinases comprising: contacting a biosensor or a cell expressing a biosensor, with one or more candidate agents, wherein the biosensor comprises an enzyme substrate comprising one or more peptides having at least 70% sequence identity to any of SEQ ID NOS: 1-11, a detectably labelled protein, and a phospho-amino acid binding protein; assaying for the presence or absence of a signal from the detectable label; and, identifying modulators of kinases.

44. The method of claim 43, wherein a signal from the detectable label is identified as an activator of the kinase.

45. The method of claim 43 or 44, wherein the absence of a signal from the detectable label is identified as an inhibitor of the kinase. 41 175727263.1

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