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

• Published in: Journal of the American Chemical Society Vol. 133; no. 45; pp. 18280 - 18288
• Main Authors: Millward, Steven W, Henning, Ryan K, Kwong, Gabriel A, Pitram, Suresh, Agnew, Heather D, Deyle, Kaycie M, Nag, Arundhati, Hein, Jason, Lee, Su Seong, Lim, Jaehong, Pfeilsticker, Jessica A, Sharpless, K. Barry, Heath, James R
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 16-11-2011
• Subjects: Allosteric Site - drug effects; Click Chemistry; Protein Kinase Inhibitors - chemistry; Protein Kinase Inhibitors - pharmacology; Proto-Oncogene Proteins c-akt - antagonists & inhibitors; Structure-Activity Relationship
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja2064389

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.