Engineering Archeal Surrogate Systems for the Development of Protein–Protein Interaction Inhibitors against Human RAD51

• DOI: 10.1016/j.jmb.2016.10.009
• Publication Date: 2016-10-11T23:10:57Z
• Authors: Tommaso Moschetti; Timothy Sharpe; Gerhard Fischer; May E. Marsh; Hong Kin Ng; Matthew Morgan; Duncan E. Scott; Tom L. Blundell; Ashok R. Venkitar...; John Skidmore; Chris Abell; Marko Hyvönen
• Keywords: Protein Engineering; Target protein

Protein-protein interactions (PPIs) are increasingly important targets for drug discovery. Efficient fragment-based discovery approaches to tackle PPIs often stymied by difficulties in the production of stable, unliganded target proteins. Here, we report an approach that exploits protein engineering "humanise" thermophilic archeal surrogate proteins as small-molecule inhibitor and exemplify this development inhibitors against PPI between recombinase RAD51 tumour suppressor BRCA2. As human has proved impossible produce a form is compatible with requirements discovery, have developed system using RadA from Pyrococcus furiosus. Using monomerised our starting point, adopted two parallel mutually instructive mimic enzyme: firstly mutating increase sequence identity BRC repeat binding sites, secondly generating chimeric archaeal protein. Both generate interact fourth affinity stoichiometry comparable RAD51. Stepwise humanisation also allowed us elucidate determinants repeats contributions key interacting residues interaction. These enabled biochemical biophysical assays ongoing programme they determine hundreds liganded structures support structure-guided design process, demonstrating feasibility advantages surrogates overcome handling