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 stable, synthetic capture agent that specifically binds a variant Akt1 protein wherein the variant Akt1 protein differs from wild-type Akt1 at one amino acid, wherein the capture agent comprises a designed anchor ligand, a designed secondary ligand, and optionally, a designed tertiary ligand, wherein the ligands specifically bind the variant Akt1.
2 . The capture agent of claim 1 , wherein the variant Akt1 has been mutated from a glutamate to a lysine at position 17.
3 . The capture agent of claim 1 , wherein the anchor ligand comprises an amino acid sequence yleaf.
4 . The capture agent of claim 1 , wherein the secondary ligand comprises the amino acid sequence selected from Table 4.
5 . The capture agent of claim 1 , wherein the secondary ligand comprises the amino acid sequence yksy.
6 . The capture agent of claim 1 , wherein the tertiary ligand comprises an amino acid sequence selected from Table 5.
7 . The capture agent of claim 1 , wherein the tertiary ligand comprises the amino acid sequence ivdae.
8 . 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).
9 . The capture agent of claim 1 , wherein the tertiary ligand is covalently bound to the anchor ligand.
10 . The capture agent of claim 1 , wherein the tertiary ligand is covalently bound to the anchor ligand.
11 . 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-PEG3, aminooxyacetate, 19 FB, 18 FB, 5-Carboxyfluorescein, and FITC-PEG3.
12 . (canceled)
13 . The capture agent of claim 1 , wherein the capture agent further comprises a cell penetrating peptide.
14 . The capture agent of claim 13 , wherein the cell penetrating peptide is HIV-TAT.
15 . The capture agent of claim 1 having the structure:
16 . (canceled)
17 . (canceled)
18 . A method for detecting E17K Akt1 in a biological sample, comprising the step of contacting the biological sample with the capture agent of claim 1 .
19 . (canceled)
20 . (canceled)
21 . (canceled)
22 . (canceled)
23 . (canceled)
24 . (canceled)
25 . (canceled)
26 . (canceled)
27 . (canceled)
28 . (canceled)
29 . (canceled)
30 . The capture agent of claim 1 , wherein the Akt protein is E17K Akt1.
31 . (canceled)
32 . 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 the capture agent of claim 1 .
33 . The method of claim 32 , wherein the capture agent is also linked to a cell penetrating peptide.
34 . The method of claim 33 , wherein the cell penetrating peptide is HIV-TAT.
35 - 44 . (canceled)