Akt-specific capture agents, compositions, and methods of using and making
The present application provides stable peptide-based Akt capture agents and the use thereof as detection, diagnosis, and treatment agents. The application further provides novel methods of developing stable peptide-based capture agents, including Akt capture agents, using iterative on-bead in situ click chemistry.
1. A method of imaging at least one permeabilized cancer cell associated with increased Akt expression and/or activity in vitro, comprising administering a detectable amount of a stable, synthetic triligand capture agent that specifically binds Akt to a permeabilized cancer cell in vitro,
wherein the capture agent comprises an anchor ligand, a secondary ligand, and a tertiary ligand, and the capture agent binds to a non-ATP and non-peptide substrate binding site of Akt,
wherein the capture agent comprises a label,
wherein the capture agent is capable of allosterically inhibiting Akt activity upon binding to the binding site,
wherein said anchor ligand comprises the peptide sequence Az8-VFYRLGY-CONH 2 (SEQ ID NO: 17), and
wherein Az8 represents and azide amino acid having the structure:
to the at least one permeabilized cancer cell and detecting the label on the capture agent thereby imaging the at least one permeabilized cancer cell in vitro.
2. The method of claim 1 , wherein the capture agent is stable in storage as a lyophilized powder.
3. The method of claim 1 , wherein the capture agent is stable in storage at a temperature of about −80° C. to about 40° C.
4. The method of claim 3 , wherein the capture agent is stable in storage at room temperature.
5. The method of claim 3 , wherein the capture agent is stable in serum for at least 24 hours.
6. The method of claim 3 , wherein the capture agent is stable at a pH in the range of about 3 to about 8.
7. The method of claim 3 , wherein the capture agent is labeled with 64 Cu DOTA, 68 Ga DOTA, 18 F, 64 Cu, 68 Ga, 89 Zr, 124 I, 86 Y, 94m Tc, 110m In, 11 C or 76 Br.
8. The method of claim 3 , wherein said secondary ligand comprises the peptide sequence Pra-FWFLRG-CONH 2 (SEQ ID NO: 18).
9. The method of claim 3 , wherein said tertiary ligand comprises the peptide sequence Ac-C8-RHERI-CONH 2 (SEQ ID NO: 19).
10. The method of claim 3 , wherein said capture agent has the structure:
11. The method according to claim 3 , wherein the linkage between one or more of the anchor ligand, secondary ligand, and tertiary ligand comprises a 1,4-substituted-1,2,3-triazole residue (Tz4).
12. A method of detecting Akt in a biological sample using an immunoassay, wherein the immunoassay utilizes a stable, synthetic triligand capture agent that specifically binds Akt,
wherein the capture agent comprises an anchor ligand, a secondary ligand, and a tertiary ligand, and the capture agent binds to a non-ATP and non-peptide substrate binding site of Akt,
wherein the capture agent comprises a label,
wherein the capture agent is capable of allosterically inhibiting Akt activity upon binding to the binding site,
wherein said anchor ligand comprises the peptide sequence Az8-VFYRLGY-CONH 2 (SEQ ID NO: 17), and
wherein Az8 represents and azide amino acid having the structure:
and wherein said capture agent replaces an antibody or its equivalent in the immunoassay.
13. The method of claim 12 , wherein the immunoassay is selected from the group of Western blot, pull-down assay, dot blot, and ELISA.