title: Engineering Archeal Surrogate Systems for the Development of Protein-Protein Interaction Inhibitors against Human RAD51.
abstract: Protein-protein interactions (PPIs) are increasingly important targets for drug discovery. Efficient fragment-based drug discovery approaches to tackle PPIs are often stymied by difficulties in the production of stable, unliganded target proteins. Here, we report an approach that exploits protein engineering to "humanise" thermophilic archeal surrogate proteins as targets for small-molecule inhibitor discovery and to exemplify this approach in the development of inhibitors against the PPI between the recombinase RAD51 and tumour suppressor BRCA2. As human RAD51 has proved impossible to produce in a form that is compatible with the requirements of fragment-based drug discovery, we have developed a surrogate protein system using RadA from Pyrococcus furiosus. Using a monomerised RadA as our starting point, we have adopted two parallel and mutually instructive approaches to mimic the human enzyme: firstly by mutating RadA to increase sequence identity with RAD51 in the BRC repeat binding sites, and secondly by generating a chimeric archaeal human protein. Both approaches generate proteins that interact with a fourth BRC repeat with affinity and stoichiometry comparable to human RAD51. Stepwise humanisation has also allowed us to elucidate the determinants of RAD51 binding to BRC repeats and the contributions of key interacting residues to this interaction. These surrogate proteins have enabled the development of biochemical and biophysical assays in our ongoing fragment-based small-molecule inhibitor programme and they have allowed us to determine hundreds of liganded structures in support of our structure-guided design process, demonstrating the feasibility and advantages of using archeal surrogates to overcome difficulties in handling human proteins.
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
authors.forename: Tommaso
authors.initials: T
authors.lastname: Moschetti
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
authors.forename: Timothy
authors.initials: T
authors.lastname: Sharpe
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Fischer
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
authors.forename: May E
authors.initials: ME
authors.lastname: Marsh
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
authors.forename: Hong Kin
authors.initials: HK
authors.lastname: Ng
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
authors.forename: Matthew
authors.initials: M
authors.lastname: Morgan
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: Duncan E
authors.initials: DE
authors.lastname: Scott
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
authors.forename: Tom L
authors.initials: TL
authors.lastname: Blundell
authors.affiliation: Medical Research Council Cancer Unit, University of Cambridge, Hills Road, Cambridge CB2 0XZ, UK.
authors.forename: Ashok
authors.initials: A
authors.lastname: R Venkitaraman
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: John
authors.initials: J
authors.lastname: Skidmore
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: Chris
authors.initials: C
authors.lastname: Abell
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK. Electronic address: mh256@cam.ac.uk.
authors.forename: Marko
authors.initials: M
authors.lastname: Hyvönen
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D024682:BRCA2 Protein / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D055808:Drug Discovery / Q000379:methods; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D006801:Humans; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects*; D015202:Protein Engineering / Q000379:methods*; D019711:Pyrococcus / Q000201:enzymology; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D024682:BRCA2 Protein; C551750:BRCA2 protein, human; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D050505:Mutant Proteins; C117252:RadA protein, archaeal; D011994:Recombinant Proteins; C495153:RAD51 protein, human; D051135:Rad51 Recombinase
keywords: fragment-based drug discovery; humanisation; protein engineering; recombinase; surrogate system