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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

Nature Chemistry volume 7, pages 455462 (2015) | Download Citation


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.

  • Compound C195H299N55O46S2

    Biotin - MSDVAIVKEGKY[Propargylglycine]KTWRPRYFLLKNDG

  • Compound C57H87N9O17S

    Biotin - PEG6 - yleaf

  • Compound C222H333N71O50S


  • Compound C75H104N9O15+

    Cy3 - PEG5 - yleaf

  • Compound C158H244N43O34S+

    Cy5 - 2-(2-(2-aminoethoxy)ethoxy)ethanol - sulfonylbenzyl - piperidine - yleaf - PEG5 - YGRKKRRQRRR

  • Compound C141H222N41O28+

    Cy5 - YGRKKRRQRRR - PEG5 - yleaf

  • Compound C62H92N10O18S

    Biotin - PEG6 - yleaf[propargylglycine]

  • Compound C105H154N24O29S

    Lys(N3) - yleaf - triazole - yksy - PEG5 - Biotin

  • Compound C133H197N29O41S

    Biotin - PEG5 - ivdae - triazole - yleaf - triazole - yksy

  • Compound C151H214N29O39+

    Cy3 - PEG5 -ivdae - triazole - yleaf - triazole - yksy

  • Compound C108H154N26O32

    ivdae - triazole - yleaf - triazole - yksy

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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.

Author information


  1. Department of Chemistry, California Institute of Technology, Pasadena, California 91125, USA

    • Kaycie M. Deyle
    • , Ying Qiao Hee
    • , Jeremy Work
    • , Michelle Wong
    • , Aiko Umeda
    • , Arundhati Nag
    • , Samir Das
    •  & James R. Heath
  2. Department of Applied Physics and Materials Science, California Institute of Technology, Pasadena, California 91125, USA

    • Blake Farrow
  3. Indi Molecular, Culver City, California 90230. USA

    • Bert Lai
  4. Department of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA

    • Steven W. Millward


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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.

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.

Corresponding author

Correspondence to James R. Heath.

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