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

Journal name:
Nature Chemistry
Year published:
Published online


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.

At a glance


  1. Epitope design strategy for the in situ click screen.
    Figure 1: Epitope design strategy for the in situ click screen.

    The full Akt1 PH domain protein (grey) contains an E17K mutation (blue). To focus a chemical library screen on the region that surrounds this mutation, only a portion of the PH domain (pink) that contained the E17K mutation was synthesized as a separate peptide epitope (1). Chemical synthesis of the epitope allowed the substitution of the isoleucine at residue 19 (yellow), the most proximal side chain to the E17K mutation, with an alkyne-containing Pra amino acid. This substitution focused the azide-library screening directly to the site of the mutation. This in situ click-screen format allows the determination of peptides that bind in close proximity to the E17K mutation. Triazoles formed between an azide-containing library peptide and the alkyne-substituted epitope must be catalysed by a molecular recognition event, which indicates that the library peptide binds strongly near the mutation.

  2. Anchor-ligand structure and characterization.
    Figure 2: Anchor-ligand structure and characterization.

    a, The peptide anchor ligand, yleaf, was discovered using the epitope-targeting technique. The different sets of R groups indicate the modifications that were made to perform the various experiments described. b, Point ELISA for the specificity obtained by immobilizing the biotinylated yleaf anchor ligand 2 onto a Neutravidin plate, and then incubating with a six His-tagged epitope 3. This assay demonstrates the preference of the biotinylated yleaf anchor ligand for the E17K mutant epitope when it is immobilized on a surface. c, Binding curves of the yleaf anchor ligand 4 to the full-length WT or E17K mutant Akt1 proteins obtained in solution by FP. A saturation point for the WT ligand was not reached because of the prohibitively high protein concentration required. d, Full binding curves of the yleaf anchor ligand 4 to the WT or E17K mutant epitopes. These FP curves demonstrate the significant preference of the yleaf ligand for the E17K mutation in both the full protein assay (c) and the epitope peptide assay (d). All the assays were performed in triplicate and averaged. Error bars indicate the s.d.

  3. Ligand-directed labelling experiments.
    Figure 3: Ligand-directed labelling experiments.

    a, Schematic of the ligand-directed labelling reaction. The yleaf anchor ligand 5 (red) binds to the PH domain (grey), and a nucleophilic amino acid (Nu) on the protein surface attacks the tosyl group, transferring the dye (pink star) onto the protein near the anchor-binding site. The protein was then digested and analysed via MALDI-TOF MS to discover the fragments that increased in mass by the weight of the dye. This experiment was designed to locate areas of the protein that are in close proximity to the bound yleaf anchor. b, Fluorescent-gel image of the GST–Akt1–PH domain prior to trypsin digestion. Lane 1 is the ladder, lane 2 is unlabelled protein and lane 3 shows fluorescently labelled protein (and excess fluorescent ligand at the bottom), which confirms that the reaction has taken place. c, This PyMOL-assembled fusion protein highlights the PH domain (blue) and the 33-mer epitope fragment used for screening (pink). Sites that contain a label on the GST–Akt1–PH domain, as identified by MS analysis, are shown in cyan. These results indicate that the yleaf anchor binds only in the area of the epitope, and does not have off-target binding interactions with any other part of this large protein.

  4. Images of the Cy5–yleaf–PEG5–Tat peptide ligand in cells.
    Figure 4: Images of the Cy5–yleaf–PEG5–Tat peptide ligand in cells.

    a, The PH domain was fused to GFP to visualize the expression. Cy5–yleaf–PEG5–Tat was added to live cells expressing the protein, washed extensively and imaged. The first panel shows the spatial map of GFP expression in green. The second panel shows the spatial map of the dye-labelled yleaf ligand and the third panel shows DAPI (4,6-diamidino-2-phenylindole)-stained nuclei. The r values indicate the Pearson correlation coefficient between the GFP protein and the Cy5 anchor. As demonstrated by the difference in both the Cy5 signal and the Pearson correlation coefficients, the anchor ligand is retained in cells that express the E17K mutant significantly more than in the WT cells, which indicates ligand binding and selectivity even in a complex cellular environment. Scale bars, 50 μm. b, Zoomed-in image of cells expressing the E17K mutant protein with the anchor ligand showing a significant Cy5 signal in the areas of the cells containing GFP-tagged protein. Scale bar, 10 μm. c, Schematic of the experimental design for the imaging experiments. The HEK-293T cells were transfected to express the GST-tagged WT or E17K mutant protein, and then incubated with the CPP–anchor–Cy5 peptide 6. These cells were then incubated for one hour in media, fixed and imaged.

  5. The PCC agent triligand development strategy, structure and inhibition data.
    Figure 5: The PCC agent triligand development strategy, structure and inhibition data.

    a, The yleaf anchor ligand appended with a C-terminal alkyne 2 was screened against an azide-terminated library in the presence of the full-length PH domain to identify a biligand. The biligand 8 was then appended with an N-terminal azide and screened against an alkyne library to identify a triligand. b, The final triligand structure 11 colour-coded to highlight each segment. c, FP assays demonstrate the triligand 10 affinity for the WT (blue) and E17K (red) full-length proteins, which indicates that the E17K mutant selectivity is retained by the triligand. All the assays were performed in triplicate and averaged. Error bars indicate the s.d. d, Inhibition assays were performed with a PIP3-coated resin that was incubated with constant concentrations of the E17K mutant protein and each ligand. The blank measures the binding of the E17K PH domain to PIP3 with no ligand present. The decreased binding of the protein in the presence of the biligand 8 and triligand 9 indicates inhibition. e, Expanded inhibition assay with varying concentrations of triligand 9 (indicated here as the molar ratio with respect to the protein) incubated with WT or E17K proteins. The only slight reduction in WT binding indicates little inhibition of this protein, whereas significant inhibition is shown against the E17K variant.


11 compounds View all compounds
  1. Biotin - MSDVAIVKEGKY[Propargylglycine]KTWRPRYFLLKNDG
    Compound 1 Biotin - MSDVAIVKEGKY[Propargylglycine]KTWRPRYFLLKNDG
  2. Biotin - PEG6 - yleaf
    Compound 2 Biotin - PEG6 - yleaf
  4. Cy3 - PEG5 - yleaf
    Compound 4 Cy3 - PEG5 - yleaf
  5. Cy5 - 2-(2-(2-aminoethoxy)ethoxy)ethanol - sulfonylbenzyl - piperidine - yleaf - PEG5 - YGRKKRRQRRR
    Compound 5 Cy5 - 2-(2-(2-aminoethoxy)ethoxy)ethanol - sulfonylbenzyl - piperidine - yleaf - PEG5 - YGRKKRRQRRR
  6. Cy5 - YGRKKRRQRRR - PEG5 - yleaf
    Compound 6 Cy5 - YGRKKRRQRRR - PEG5 - yleaf
  7. Biotin - PEG6 - yleaf[propargylglycine]
    Compound 7 Biotin - PEG6 - yleaf[propargylglycine]
  8. Lys(N3) - yleaf - triazole - yksy - PEG5 - Biotin
    Compound 8 Lys(N3) - yleaf - triazole - yksy - PEG5 - Biotin
  9. Biotin - PEG5 - ivdae - triazole - yleaf - triazole - yksy
    Compound 9 Biotin - PEG5 - ivdae - triazole - yleaf - triazole - yksy
  10. Cy3 - PEG5 -ivdae - triazole - yleaf - triazole - yksy
    Compound 10 Cy3 - PEG5 -ivdae - triazole - yleaf - triazole - yksy
  11. ivdae - triazole - yleaf - triazole - yksy
    Compound 11 ivdae - triazole - yleaf - triazole - yksy

Accession codes

Primary accessions

Protein Data Bank


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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


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 financial 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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