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 stable, synthetic capture agent that specifically binds Akt, wherein the capture agent comprises an anchor ligand, a secondary ligand, and optionally a tertiary ligand,
wherein the capture agent binds to a non-ATP and non-peptide substrate binding site of Akt and 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-CONH2 (SEQ ID NO: 17),
wherein Az8 represents and azide amino acid having the structure:
2. The capture agent of claim 1 , wherein said secondary ligand comprises the peptide sequence Pra-FWFLRG-CONH 2 (SEQ ID NO: 18),
wherein Pra represents propargylglycine.
3. The capture agent of claim 1 , wherein the capture agent comprises a tertiary ligand and wherein said tertiary ligand comprises the peptide sequence Ac-C8-RHERI-CONH 2 (SEQ ID NO: 19),
wherein Ac represents an acetyl moiety, and
wherein C8 represents an eight carbon alkyl chain.
4. The capture agent of claim 1 , wherein said capture agent has the structure:
5. The capture agent according to claim 1 , 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).
6. A method of detecting Akt in a biological sample comprising measuring the presence or absence of Akt in the sample with the Akt capture agent of claim 1 .
7. The capture agent of claim 1 , wherein the capture agent is stable in storage as a lyophilized powder.
8. The capture agent of claim 1 , wherein the capture agent is stable in storage at a temperature of about −80° C. to about 40° C.
9. The capture agent of claim 1 , wherein the capture agent is stable in storage at room temperature.
10. The capture agent of claim 1 , wherein the capture agent is stable in serum for at least 24 hours.
11. The capture agent of claim 1 , wherein the capture agent is stable at a pH in the range of about 3 to about 8.
12. The capture agent of claim 1 , 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,
wherein DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.