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

Protein Engineering, Design & Selection : PEDS
S-Y LauFarid J Ghadessy

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 develope...Continue Reading

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Citations

Feb 23, 2021·The Journal of Biological Chemistry·Peter G ChandlerAshley M Buckle
May 14, 2021·Scientific Reports·Sharon Min Qi CheeFarid J Ghadessy

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Methods Mentioned

BETA
ubiquitination
PCR
immunoprecipitation
pull-down
transfection
fluorescence
pull-downs
fluorescence assisted cell sorting
fluorescence microscopy
confocal microscopy

Software Mentioned

ImageJ
Mascot
Sequest HT
Summit
GraphPad
Prism
MOP3
Proteome Discoverer

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