Iterative in Situ Click Chemistry Assembles a Branched Capture Agent and Allosteric Inhibitor for Akt1
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.
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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Additional details
- PMCID
- PMC3651860
- Eprint ID
- 28663
- Resolver ID
- CaltechAUTHORS:20120105-084339625
- National Cancer Institute
- 5U54CA119347
- Army Research Office (ARO)
- W911NF-09-D-0001
- Agency for Science, Technology and Research (A*STAR)
- Grand Duchy of Luxembourg
- NRSF postdoctoral fellowship
- 1F32CA136150-01
- Created
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2012-01-05Created from EPrint's datestamp field
- Updated
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2021-11-09Created from EPrint's last_modified field