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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

Deyle, Kaycie M. and Farrow, Blake and Hee, Ying Qiao and Work, Jeremy and Wong, Michelle and Lai, Bert and Umeda, Aiko and Millward, Steven W. and Nag, Arundhati and Das, Samir and Heath, James R. (2015) A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1. Nature Chemistry, 7 (5). pp. 455-462. ISSN 1755-4330. PMCID PMC4408887. http://resolver.caltech.edu/CaltechAUTHORS:20150217-102335573

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Abstract

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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http://dx.doi.org/10.1038/nchem.2223DOIArticle
https://www.nature.com/nchem/journal/v7/n5/full/nchem.2223.htmlPublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4408887/PubMed CentralArticle
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ORCID:
AuthorORCID
Heath, James R.0000-0001-5356-4385
Additional Information:© 2015 Macmillan Publishers Limited. Received 17 September 2014 Accepted 27 February 2015 Published online 13 April 2015. 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 contributions: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.
Funders:
Funding AgencyGrant Number
Army Research Office (ARO)W911NF-09-0001
National Cancer Institute5U54 CA119347
Defense Advanced Research Projects Agency (DARPA)HR0011-11-2-0006
Jean Perkins FoundationUNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
PubMed Central ID:PMC4408887
Record Number:CaltechAUTHORS:20150217-102335573
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20150217-102335573
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:54865
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:15 Apr 2015 18:22
Last Modified:25 Apr 2017 03:57

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