Coiled-coil Technology for Regulating Intracellular Protein-protein Interactions

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This proposal will develop de novo designed coiled coils (CCs) as reagents that can (1) selectively inhibit cellular protein-protein interactions (PPIs), and (2) selectively degrade certain proteins in cells. As a proof of concept, we will target the BCL-2 family of apoptosis regulators. Next, to test the power of the approach, we will target the therapeutically underexplored eIFE/4G interaction. To do this, we assemble a multidisciplinary collaborative team across three research institutes and a biotech project partner. The research is organised through three interconnected work packages that deliver the necessary technical capabilities as follows:

WP1 - A CC design pipeline to target selectively many different PPIs: We will use computational methods to design CCs that recognise target proteins. The designs will be validated experimentally through (i) chemical synthesis, (ii) solution-phase biophysics (CD spectroscopy and analytical ultracentrifugation), (iii) binding assays (including: fluorescence anisotropy, isothermal titration calorimetry and surface plasmon resonance, and (iv) structural studies (X-ray crystallography). In this way, we will iterate and optimize the CC designs.

WP2 - Designing CCs that recruit E3 ubiquitin ligases: We will use the design pipeline developed in WP1 to deliver CCs that recognise a broad range of E3 ligases. These will be used in WP3 as adaptors to link target proteins to the ubiquitin machinery, thereby driving target degradation.

WP3 - Building hetero-bifunctional CCs for targeted degradation: We will use insights and reagents from WP1 and WP2 to design CC-based polyproxins; i.e., bi-specific scaffolds that bring a target protein and E3 ubiquitin ligase into mutual proximity to result in degradation of the former. Polyproxins will be (i) synthesized and characterized as in WP1, and (ii) transiently expressed using polyproxin-encoding plasmids to test the ability to inhibit the PPIs and to degrade the target proteins.