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

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• 作者：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 ; James R. Heath
• 刊名：Journal of the American Chemical Society
• 出版年：2011
• 出版时间：November 16, 2011
• 年：2011
• 卷：133
• 期：45
• 页码：18280-18288
• 全文大小：965K
• 年卷期：v.133,no.45(November 16, 2011)
• ISSN：1520-5126

文摘

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.