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

Area of Science:

• Biotechnology
• Molecular Biology
• Drug Discovery

Background:

• Engineered non-antibody scaffold proteins offer versatile tools for diagnostics and protein function modulation.
• MDM2 and MDMX proteins are frequently overexpressed in cancer, making them significant therapeutic targets.

Purpose of the Study:

• To rapidly develop high-affinity 10FN3 domain inhibitors targeting both MDM2 and MDMX proteins.
• To optimize inhibitor design for enhanced target engagement and cellular activity.
• To establish a cell-based screening platform for drug discovery.

Main Methods:

• Facile in vitro expression and pull-down assays were used to screen 10FN3 scaffold design iterations.
• Valency within the 10FN3 domain was increased to improve target engagement.
• Stable reporter cell lines expressing EGFP-fused inhibitors were established.

Main Results:

• High-affinity dual inhibitors of MDM2/X were successfully developed using the 10FN3 scaffold.
• On-target cellular inhibition led to the activation of the p53 tumor suppressor.
• Increased valency within a single domain significantly improved target engagement.

Conclusions:

• The developed 10FN3 domain inhibitors effectively target MDM2/X, leading to p53 activation.
• The platform demonstrates potential as a robust tool for cell-based drug screening.
• This work highlights the potential of engineered scaffolds for cancer therapeutics and diagnostics.