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

被引:40
作者
Millward, Steven W. [1 ]
Henning, Ryan K. [1 ]
Kwong, Gabriel A. [2 ]
Pitram, Suresh [3 ]
Agnew, Heather D. [3 ]
Deyle, Kaycie M. [1 ]
Nag, Arundhati [1 ]
Hein, Jason [4 ,5 ]
Lee, Su Seong [6 ]
Lim, Jaehong [6 ]
Pfeilsticker, Jessica A. [1 ]
Sharpless, K. Barry [4 ,5 ]
Heath, James R. [1 ]
机构
[1] CALTECH, Div Chem & Chem Engn, Nanosyst Biol Canc Ctr, Pasadena, CA 91125 USA
[2] MIT, Div Hlth Sci & Technol, Cambridge, MA 02139 USA
[3] Integrated Diagnost, Culver City, CA 90230 USA
[4] Scripps Res Inst, Dept Chem, La Jolla, CA 92037 USA
[5] Scripps Res Inst, Skaggs Inst Chem Biol, La Jolla, CA 92037 USA
[6] Inst Bioengn & Nanotechnol, Singapore 138669, Singapore
关键词
SYNTHETIC PEPTIDE LIBRARY; TERMINAL ALKYNES; SMALL MOLECULES; PHAGE DISPLAY; HUMAN CANCER; KINASE; BINDING; SPECIFICITY; RECOGNITION; DISCOVERY;
D O I
10.1021/ja2064389
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
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.
引用
收藏
页码:18280 / 18288
页数:9
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