Coiled-coil Technology for Regulating Intracellular Protein-protein Interactions

Proteins are the workhorses of biology. Proteins rarely work alone, and they cooperative via so called protein-protein interactions. In this way, they form larger assemblies and networks of proteins. In turn, these provide frameworks for controlling all cellular processes that regulate life. Dysregulation of the underlying protein-protein interactions can result in disease. The scale of this framework of protein-protein interactions within a cell is enormous; it has been estimated to be around 650,000 different interactions. These present considerable opportunities for intervening in biological processes (a) to understand healthy cells better, and (b) to develop new therapeutics when subcellular mechanisms go wrong. There are different ways to do this. We propose a new approach that employs synthetic protein modules (i) to disrupt protein-protein interactions and (ii) to hijack endogenous cell machineries.

Synthetic chemical probes-such as small-molecule drugs-function by binding to a protein target within the body. This can be used to interfere with the target protein's function to help understand its biological role and as a starting point for drug discovery. Most chemical probes bind to well-defined pockets in proteins; this is analogous to a key fitting into a lock. By contrast, the design of probes to interfere with protein-protein interactions generally requires a fundamentally different type of association between the probe and one of the interacting target proteins; analogous to a hand gripping a ball. Thus, the development of effective probes that target protein-protein interactions raises new challenges that need to be met in future chemical biology and drug discovery. Two emerging approaches are promising for this, and we propose to combine them in this grant application.

The first is a synthetic-biology approach. This uses synthetic proteins called de novo coiled coils as scaffolds for building new protein-protein interactions from scratch. This is attractive because natural coiled-coil proteins exhibit an array of protein-recognition properties and we can design de novo coiled-coils with diverse structures thereby expanding their potential. The second involves the targeted destruction of cellular proteins; in essence, this is a search-and-destroy strategy that co-opts the cell's own waste-disposal machineries so as to block protein function or remove harmful proteins that cause disease.

The proposed research will develop new methodologies (i) to disrupt specified protein-protein interactions and (ii) to target certain proteins of interest for degradation. In this way, we will regulate specified processes in cells. To do this, we will not use conventional small molecules as the probes for intervening in the underlying protein-protein interactions. Rather, we will employ the synthetic coiled coils and adapt these to recognise the target proteins. In addition, for the second application, the coiled coils will be modified further to link the target protein to the cell's degradation pathways. Our aim is to deliver methods and reagents that will be of use to others in studying biological function and for developing new drugs to treat disease.

This work is necessarily interdisciplinary. Therefore, we bring together a team of computational and experimental chemists, biochemists and cell biologists to tackle it, and we partner with a biotech company to translate the work in timely and relevant manner.

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.