S -Y Lau, J W Siau, R M Sobota, C -I Wang, P Zhong, D P Lane, F J Ghadessy, Synthetic 10FN3-based mono- and bivalent inhibitors of MDM2/X function, Protein Engineering, Design and Selection, Volume 31, Issue 7-8, July-August 2018, Pages 301–312, https://doi.org/10.1093/protein/gzy018
Abstract:
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.
License type:
http://creativecommons.org/licenses/by-nc/4.0/
Funding Info:
This work was supported by the National Research Foundation [NRF2013-THE001-070]; Institute of Molecular and Cell Biology at the Agency for Science, Technology and Research [core fund]; Biomedical Research Council at the Agency for Science, Technology and Research [Young Investigator Grant YIG 2015]; and the National Medical Research Council MS-CETSA platform grant [MOHIAFCAT2/004/2015 to R.M.S.].