Synthetic 10FN3-based mono- and bivalent inhibitors of MDM2/X function

Protein Eng Des Sel. 2018 Jul 1;31(7-8):301-312. doi: 10.1093/protein/gzy018.


Engineered non-antibody scaffold proteins constitute a rapidly growing technology for diagnostics and modulation/perturbation of protein function. Here, we describe the rapid and systematic development of high-affinity 10FN3 domain inhibitors of the MDM2 and MDMX proteins. These are often overexpressed in cancer and represent attractive drug targets. Using facile in vitro expression and pull-down assay methodology, numerous design iterations addressing insertion site(s) and spacer length were screened for optimal presentation of an MDM2/X dual peptide inhibitor in the 10FN3 scaffold. Lead inhibitors demonstrated robust, on-target cellular inhibition of MDM2/X leading to activation of the p53 tumor suppressor. Significant improvement to target engagement was observed by increasing valency within a single 10FN3 domain, which has not been demonstrated previously. We further established stable reporter cell lines with tunable expression of EGFP-fused 10FN3 domain inhibitors, and showed their intracellular location to be contingent on target engagement. Importantly, competitive inhibition of MDM2/X by small molecules and cell-penetrating peptides led to a readily observable phenotype, indicating significant potential of the developed platform as a robust tool for cell-based drug screening.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus
  • Amino Acid Sequence
  • Cell Line
  • Cell Nucleus / metabolism
  • Models, Molecular
  • Protein Domains
  • Proto-Oncogene Proteins c-mdm2 / immunology*
  • Single-Domain Antibodies / chemistry*
  • Single-Domain Antibodies / immunology*


  • Single-Domain Antibodies
  • Proto-Oncogene Proteins c-mdm2