title: Double Strain-Promoted Macrocyclization for the Rapid Selection of Cell-Active Stapled Peptides.
abstract: Peptide stapling is a method for designing macrocyclic alpha-helical inhibitors of protein-protein interactions. However, obtaining a cell-active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain-promoted azide-alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell-active stapled peptides. As a proof of concept, MDM2-binding peptides were stapled in parallel, directly in cell culture medium in 96-well plates, and simultaneously evaluated in a p53 reporter assay. This in situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α-Helicity was confirmed by a crystal structure of the MDM2-peptide complex. This work introduces in situ stapling as a versatile biocompatible technique with many other potential high-throughput biological applications.
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Rd, Cambridge CB2 1EW (UK).
authors.forename: Yu Heng
authors.initials: YH
authors.lastname: Lau
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Rd, Cambridge CB2 1EW (UK).
authors.forename: Yuteng
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Rd, Cambridge CB2 1GA (UK).
authors.forename: Maxim
authors.initials: M
authors.lastname: Rossmann
authors.affiliation: p53Lab, A*STAR, 8A Biomedical Grove, #06-04/05 Neuros/Immunos, Singapore 138648 (Singapore).
authors.forename: Ban Xiong
authors.initials: BX
authors.lastname: Tan
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Rd, Cambridge CB2 1EW (UK).
authors.forename: Peterson
authors.initials: P
authors.lastname: de Andrade
authors.affiliation: Bioinformatics Institute, A*STAR, 30 Biopolis St, #07-01 Matrix, Singapore 138671 (Singapore).
authors.forename: Yaw Sing
authors.initials: YS
authors.lastname: Tan
authors.affiliation: Bioinformatics Institute, A*STAR, 30 Biopolis St, #07-01 Matrix, Singapore 138671 (Singapore).|Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543.|School of Biological Sciences, Nanyang Technological University, 50 Nanyang Drive, Singapore 637551.
authors.forename: Chandra
authors.initials: C
authors.lastname: Verma
authors.affiliation: Hutchison/MRC Research Centre, Hills Rd, Cambridge CB2 0XZ (UK).
authors.forename: Grahame J
authors.initials: GJ
authors.lastname: McKenzie
authors.affiliation: Hutchison/MRC Research Centre, Hills Rd, Cambridge CB2 0XZ (UK).
authors.forename: Ashok R
authors.initials: AR
authors.lastname: Venkitaraman
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Rd, Cambridge CB2 1GA (UK).
authors.forename: Marko
authors.initials: M
authors.lastname: Hyvönen
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Rd, Cambridge CB2 1EW (UK). spring@ch.cam.ac.uk.
authors.forename: David R
authors.initials: DR
authors.lastname: Spring
mesh_terms: D003470:Culture Media; D047028:Macrocyclic Compounds / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry
chemical_list: D003470:Culture Media; D047028:Macrocyclic Compounds; D010455:Peptides; D051736:Proto-Oncogene Proteins c-mdm2
keywords: bioorthogonal chemistry; click chemistry; macrocycles; peptide stapling; peptides