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Published November 16, 2011 | Accepted Version + Supplemental Material
Journal Article Open

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


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 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.

Attached Files

Accepted Version - nihms334474.pdf

Supplemental Material - ja2064389_si_001.pdf


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