Ligands that can bind selectively to proteins with single amino-acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wild type (WT). However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical synthetic epitope-targeting strategy that we used to discover a 5-mer peptide with selectivity for the E17K-transforming point mutation in the pleckstrin homology domain of the Akt1 oncoprotein. A fragment of Akt1 that contained the E17K mutation and an I19[propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that clicked covalently onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to the WT, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) substrate.
Biotin - MSDVAIVKEGKY[Propargylglycine]KTWRPRYFLLKNDG
Biotin - PEG6 - yleaf
Cy3 - PEG5 - yleaf
Cy5 - 2-(2-(2-aminoethoxy)ethoxy)ethanol - sulfonylbenzyl - piperidine - yleaf - PEG5 - YGRKKRRQRRR
Cy5 - YGRKKRRQRRR - PEG5 - yleaf
Biotin - PEG6 - yleaf[propargylglycine]
Lys(N3) - yleaf - triazole - yksy - PEG5 - Biotin
Biotin - PEG5 - ivdae - triazole - yleaf - triazole - yksy
Cy3 - PEG5 -ivdae - triazole - yleaf - triazole - yksy
ivdae - triazole - yleaf - triazole - yksy
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Protein Data Bank
This work was supported by the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from the US Army Research Office, the National Cancer Institute through grant 5U54 CA119347, the Defense Advanced Research Projects Agency through Cooperative Agreement No. HR0011-11-2-0006 and the Jean Perkins Foundation. B.F. is supported by a Howard Hughes Medical Institute International Student Research Fellowship. We gratefully acknowledge assistance and resources from F. Rusnak, J. Zhou and the Protein and Peptide Mass Analysis Laboratory, M. Shahgholi and the Mass Spectrometry Lab, J. Vielmetter and the Protein Expression Center, and the Beckman Institute Biological Imaging Center.
About this article
Scientific Reports (2017)