title: 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.
authors.affiliation: Nanosystems Biology Cancer Center, Division of Chemistry and Chemical Engineering, MC-127-72, California Institute of Technology, Pasadena, California 91125, United States.
authors.forename: Steven W
authors.initials: SW
authors.lastname: Millward
authors.forename: Ryan K
authors.initials: RK
authors.lastname: Henning
authors.forename: Gabriel A
authors.initials: GA
authors.lastname: Kwong
authors.forename: Suresh
authors.initials: S
authors.lastname: Pitram
authors.forename: Heather D
authors.initials: HD
authors.lastname: Agnew
authors.forename: Kaycie M
authors.initials: KM
authors.lastname: Deyle
authors.forename: Arundhati
authors.initials: A
authors.lastname: Nag
authors.forename: Jason
authors.initials: J
authors.lastname: Hein
authors.forename: Su Seong
authors.initials: SS
authors.lastname: Lee
authors.forename: Jaehong
authors.initials: J
authors.lastname: Lim
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Pfeilsticker
authors.forename: K Barry
authors.initials: KB
authors.lastname: Sharpless
authors.forename: James R
authors.initials: JR
authors.lastname: Heath
mesh_terms: D000495:Allosteric Site / Q000187:drug effects*; D057930:Click Chemistry; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors*; D013329:Structure-Activity Relationship
chemical_list: D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt