Engineering mono- and multi-valent inhibitors on a modular scaffold.

Area of Science:

• Protein engineering
• Synthetic biology
• Cancer therapeutics

Background:

• Consensus-designed tetratricopeptide repeat proteins (CTPRs) offer a stable, modular scaffold.
• Human tankyrase proteins (hTNKS) are key regulators of Wnt signaling and are implicated in cancer.
• Short linear motifs (SLiMs) are crucial for protein-protein interactions.

Purpose of the Study:

• To develop a versatile CTPR-based platform for creating synthetic binding proteins.
• To engineer CTPR constructs capable of inhibiting hTNKS and modulating Wnt signaling.
• To explore the impact of multivalency and geometric arrangement on binding and inhibition efficacy.

Main Methods:

• Grafting SLiMs onto CTPR scaffolds to create specific binders.
• Assembling mono-valent and multi-valent CTPR-hTNKS inhibitors with varying geometries.
• Assessing protein stability, folding, and in vitro/cellular inhibition of hTNKS activity.
• Utilizing nanoparticle encapsulation for intracellular delivery of CTPR proteins.

Main Results:

• Designed synthetic CTPRs demonstrated stability, correct folding, and effective binding to hTNKS.
• Engineered CTPR constructs successfully inhibited hTNKS cellular activity, downregulating Wnt signaling.
• Multivalent CTPR-hTNKS interactions formed large, visualizable macromolecular assemblies.
• Nanoparticle-delivered multivalent CTPRs exhibited superior Wnt pathway inhibition compared to small molecule inhibitors.

Conclusions:

• The CTPR platform is a powerful tool for assembling synthetic binding molecules with programmable geometries and built-in multivalency.
• Engineered CTPR proteins show significant therapeutic potential for inhibiting Wnt signaling in cancer.
• This approach offers a promising strategy for developing novel cancer therapeutics targeting pathways resistant to conventional drugs.