Mutant Akt-specific capture agents, compositions, and methods of using and making
The present application provides stable peptide-based Akt capture agents and methods of use as detection and diagnosis agents and in the treatment of diseases and disorders. The application further provides methods of manufacturing Akt capture agents using iterative on-bead in situ click chemistry.
1. A capture agent that binds a variant Akt1 protein, wherein the variant Akt1 protein has been mutated from a glutamate to a lysine at position 17, wherein the capture agent comprises an anchor ligand, a secondary ligand, and optionally, a tertiary ligand, wherein the anchor ligand specifically binds the variant Akt1,
wherein the anchor ligand comprises an amino acid sequence yleaf (SEQ ID NO: 43),
wherein the secondary ligand is identified by contacting an anchor ligand selection block and a plurality of first candidate peptides with the variant Akt1 protein, wherein (a) the anchor ligand selection block comprises the anchor ligand and an azido group and the first candidate peptides each comprise a peptide and an alkynyl group or (b) the anchor ligand selection block comprises the anchor ligand and an alkynyl group and the first candidate peptides each comprise a peptide and an azido group, wherein the candidate peptides comprise five amino acids each randomly selected from 18 D-stereoisomers of the natural amino acids, minus Cysteine and Methionine, whereby a biligand is formed by a disubstituted 1,2,3-triazole linkage being formed between the anchor ligand selection block and one of the first candidate peptides by the azido group or alkynyl group of the anchor ligand selection block and the alkynyl group or azido group of the one of the first candidate peptides being brought in close proximity by binding to the variant Akt1 protein, wherein the candidate peptide with which the triazole linkage is formed with the anchor ligand selection block is identified as the secondary ligand of the capture agent,
wherein the tertiary ligand is identified by contacting a biligand selection block and a plurality of second candidate peptides with the variant Akt1 protein, wherein (a) the biligand selection block comprises the biligand and an azido group and the second candidate peptides each comprise a peptide and an alkynyl group or (b) the biligand selection block comprises the biligand and an alkynyl group and the second candidate peptides each comprise a peptide and an azido group, wherein the candidate peptides comprise five amino acids each randomly selected from 18 D-stereoisomers of the natural amino acids, minus Cysteine and Methionine, whereby a triligand is formed by a disubstituted 1,2,3-triazole linkage being formed between the biligand selection block and one of the second candidate peptides by the azido group or alkynyl group of the biligand selection block and the alkynyl group or azido group of the one of the second candidate peptides being brought in close proximity by binding to the variant Akt1 protein, wherein the candidate peptide with which the triazole linkage is formed with the biligand selection block is identified as the tertiary ligand of the capture agent.
2. The capture agent of claim 1 , wherein the secondary ligand comprises the amino acid sequence selected from hwpr (SEQ ID NO: 61), nvyl (SEQ ID NO: 59), hyrw (SEQ ID NO: 55), rdyr (SEQ ID NO: 75), ynyk (SEQ ID NO: 74), yktw (SEQ ID NO: 88), srfy (SEQ ID NO: 73), yksy (SEQ ID NO: 76), yysr (SEQ ID NO: 71), rhws (SEQ ID NO: 66), pwwr (SEQ ID NO: 89), nfry (SEQ ID NO: 90), ywrl (SEQ ID NO: 57), ywkG (SEQ ID NO: 56), ayly (SEQ ID NO: 63), hwrw (SEQ ID NO: 91), nwrl (SEQ ID NO: 53), aarw (SEQ ID NO: 60), Grwy (SEQ ID NO: 72), wfri (SEQ ID NO: 58), rpyy (SEQ ID NO: 65), vwfr (SEQ ID NO: 70).
3. The capture agent of claim 1 , wherein the secondary ligand comprises the amino acid sequence yksy (SEQ ID NO: 76).
4. The capture agent of claim 1 , wherein the tertiary ligand comprises an amino acid sequence selected from Glm, iryrn (SEQ ID NO: 92), and ivdae SEQ ID NO: 78.
5. The capture agent of claim 1 , wherein the tertiary ligand comprises the amino acid sequence ivdae (SEQ ID NO: 78).
6. The capture agent of claim 1 , wherein the anchor ligand and secondary ligand are linked together via a 1,4-substituted-1,2,3-triazole residue (Tz4) or via a 1,5-substituted-1,2,3-triazole residue (Tz5).
7. The capture agent of claim 1 , wherein the tertiary ligand is covalently bound to the secondary ligand.
8. The capture agent of claim 1 , wherein the capture agent is labeled with a label selected from the group consisting of biotin, copper-DOTA, biotin-PEG 3 , aminooxyacetate, 19 FB, 18 FB, 5-Carboxyfluorescein, and FITC-PEG3.
9. The capture agent of claim 1 , wherein the capture agent is labeled with the detectable moiety consisting of 64 Cu DOTA, 68 Ga DOTA, 68 Ga NOTA, 18 F, Al 18 F NOTA, 64 Cu, 68 Ga, 89 Zr, 124 I, 86 Y, 94m Tc, 110m In, C and 76 Br.
10. The capture agent of claim 1 , wherein the capture agent further comprises a cell penetrating peptide.
11. The capture agent of claim 10 , wherein the cell penetrating peptide is HIV-TAT.
12. The capture agent of claim 1 having the structure:
13. A composition comprising two or more capture agents of claim 1 .
14. A method of monitoring treatment of a cancer associated with increased E17K Akt1 expression in a subject, comprising the steps of:
(a) contacting a first biological sample from the subject with one or more capture agents of claim 1 , wherein each capture agent is linked to a detectable moiety;
(b) detecting the moiety linked to the capture agent, wherein the capture agent is bound to E17K Akt1;
(c) administering a treatment for the cancer associated with increased E17K Akt1 expression to the subject;
(d) contacting a second biological sample from the subject one or more capture agents of claim 1 , wherein each capture agent is linked to a detectable moiety; and
(e) detecting the moiety linked to the capture agent, wherein the capture agent is bound to E17K Akt1;
(f) comparing the level of moiety detected in step (b) with the level of moiety detected in step (d);
wherein, if less of the moiety is detected in step (e) than in step (b), the treatment is improving cancer in the subject.
15. A method of detecting E17K Akt1 in a biological sample, comprising the steps of:
(a) contacting the sample with a capture agent of claim 1 , wherein the capture agent is linked to a detectable moiety; and
(b) detecting the moiety linked to the capture agent, wherein the capture agent is bound to E17K Akt1; and
wherein detection of the moiety indicates the presence of E17K Akt1 in the subject.
16. A multiplex capture agent comprising two or more capture agents of claim 1 , wherein the multiplex capture agent binds E17K Akt1.
17. The capture agent of claim 1 , wherein the Akt protein is E17K Akt1.
18. The capture agent of claim 1 , wherein the capture agent further comprises a tag that comprises the amino acid sequence ALAPYIP (SEQ ID NO: 93).
19. A method of treating a cancer associated with increased E17K Akt1 expression and/or activity in a subject in need thereof, comprising administering a therapeutically effective amount of a capture agent of claim 1 .
20. A method of inhibiting E17K Akt1 activity in a subject comprising administering to the subject a capture agent of claim 1 .
21. A method of imaging a cancer associated with increased E17K Akt1 expression and/or activity in a subject in need thereof, comprising administering an effective amount of a capture agent of claim 1 .
22. A method of synthesizing a capture agent to a target peptide comprising:
(a) modifying a target peptide to include an azido group or an alkynyl group;
(b) preparing a plurality of candidate peptides to select an anchor ligand for the target peptide, the plurality of peptides comprising an azido group, or an alkynyl group, if the target peptide comprises an alkynyl group, or an azido group, respectively;
(c) contacting the plurality of candidate peptides to select the anchor ligand with the target peptide; and
(d) selecting the capture agent biligand that has an affinity with the target protein.