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Iterative in Situ Click Chemistry Assembles a Branched Capture Agent and Allosteric Inhibitor for Akt1

Millward, Steven W. and Henning, Ryan K. and Kwong, Gabriel A. and Pitram, Suresh and Agnew, Heather D. and Deyle, Kaycie M. and Nag, Arundhati and Hein, Jason and Lee, Su Seong and Lim, Jaehong and Pfeilsticker, Jessica A. and Sharpless, K. Barry and Heath, James R. (2011) Iterative in Situ Click Chemistry Assembles a Branched Capture Agent and Allosteric Inhibitor for Akt1. Journal of the American Chemical Society, 133 (45). pp. 18280-18288. ISSN 0002-7863. PMCID PMC3651860. https://resolver.caltech.edu/CaltechAUTHORS:20120105-084339625

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Abstract

We describe the use of iterative in situ click chemistry to design an Akt-specific branched peptide triligand that is a drop-in replacement for monoclonal antibodies in multiple biochemical assays. Each peptide module in the branched structure makes unique contributions to affinity and/or specificity resulting in a 200 nM affinity ligand that efficiently immunoprecipitates Akt from cancer cell lysates and labels Akt in fixed cells. Our use of a small molecule to preinhibit Akt prior to screening resulted in low micromolar inhibitory potency and an allosteric mode of inhibition, which is evidenced through a series of competitive enzyme kinetic assays. To demonstrate the efficiency and selectivity of the protein-templated in situ click reaction, we developed a novel QPCR-based methodology that enabled a quantitative assessment of its yield. These results point to the potential for iterative in situ click chemistry to generate potent, synthetically accessible antibody replacements with novel inhibitory properties.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/ja2064389DOIArticle
http://pubs.acs.org/doi/abs/10.1021/ja2064389PublisherArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651860/PubMed CentralArticle
ORCID:
AuthorORCID
Sharpless, K. Barry0000-0001-6051-1599
Heath, James R.0000-0001-5356-4385
Additional Information:© 2011 American Chemical Society. Received: July 11, 2011. Publication Date (Web): September 30, 2011. This work was supported by the National Cancer Institute grant No. 5U54CA119347 (J.R.H., P.I.), the Institute for Collaborative Biotechnologies (contract no. W911NF-09-D-0001 from the U.S. Army Research Office), The Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore), and the Grand Duchy of Luxembourg via a subcontract from the Institute for Systems Biology. S.W.M. acknowledges support from an NRSF postdoctoral fellowship 1F32CA136150-01. We thank Prof. Carl Parker for the generous use of his equipment and expertise. The Akt1-S473E plasmid was a gift from Dr. Shoshana Klein (The Hebrew University of Jerusalem). Expression of the Akt1-S473E was carried out by the Protein Expression Center at Caltech.
Funders:
Funding AgencyGrant Number
National Cancer Institute5U54CA119347
Army Research Office (ARO)W911NF-09-D-0001
Agency for Science, Technology and Research (A*STAR)UNSPECIFIED
Grand Duchy of LuxembourgUNSPECIFIED
NRSF postdoctoral fellowship1F32CA136150-01
Issue or Number:45
PubMed Central ID:PMC3651860
Record Number:CaltechAUTHORS:20120105-084339625
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20120105-084339625
Official Citation:Iterative in Situ Click Chemistry Assembles a Branched Capture Agent and Allosteric Inhibitor for Akt1 Steven W. Millward, Ryan K. Henning, Gabriel A. Kwong, Suresh Pitram, Heather D. Agnew, Kaycie M. Deyle, Arundhati Nag, Jason Hein, Su Seong Lee, Jaehong Lim, Jessica A. Pfeilsticker, K. Barry Sharpless, and James R. Heath Journal of the American Chemical Society 2011 133 (45), 18280-18288
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:28663
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:05 Jan 2012 17:00
Last Modified:09 Mar 2020 13:19

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