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A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1


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.

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Figure 1: Epitope design strategy for the in situ click screen.
Figure 2: Anchor-ligand structure and characterization.
Figure 3: Ligand-directed labelling experiments.
Figure 4: Images of the Cy5–yleaf–PEG5–Tat peptide ligand in cells.
Figure 5: The PCC agent triligand development strategy, structure and inhibition data.

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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.

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K.M.D., B.F. and J.R.H. designed the project and wrote the manuscript. K.M.D., B.F., Y.Q.H., J.W. and M.W. carried out the experiments. B.L. carried out the MS analysis. A.U. and S.W.M. designed and helped execute the protein expression and cell culture work. A.N. and S.D. helped develop the epitope-targeting strategies. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to James R. Heath.

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Competing interests

J.R.H. is a founder and board member of Indi Molecular. Indi Molecular is seeking to commercialize the PCC-agent technology. B.L. is an employee of Indi Molecular. K.M.D. has consulted for Indi Molecular. The patent ‘Multi-ligand capture agents and related compositions, methods and systems’ (Patent WO2009155420 A1) by H. Agnew et al. was published 23 December 2009.

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Deyle, K., Farrow, B., Qiao Hee, Y. et al. A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1. Nature Chem 7, 455–462 (2015).

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