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 http://resolver.caltech.edu/CaltechAUTHORS:20120105-084339625
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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.
|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 ResearchOffice), 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.|
|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.|
|Deposited By:||Ruth Sustaita|
|Deposited On:||05 Jan 2012 17:00|
|Last Modified:||26 Dec 2012 14:40|
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