Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 133, Issue 45, Pages 18280-18288Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ja2064389
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Funding
- National Cancer Institute [5U54CA119347 P.I.]
- Institute for Collaborative Biotechnologies (from the U.S. Army Research Office) [W911NF-09-D-0001]
- The Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore)
- Grand Duchy of Luxembourg via Institute for Systems Biology
- NRSF [1F32CA136150-01]
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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.
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