Using a fragment-based approach to target protein-protein interactions.
The ability to identify inhibitors of protein-protein interactions represents a major challenge in modern drug discovery and in the development of tools for chemical biology. In recent years, fragment-based approaches have emerged as a new methodology in drug discovery; however, few examples of small molecules that are active against chemotherapeutic targets have been published. Herein, we describe the fragment-based approach of targeting the interaction between the tumour suppressor BRCA2 and the recombination enzyme RAD51; it makes use of a screening pipeline of biophysical techniques that we expect to be more generally applicable to similar targets. Disruption of this interaction in vivo is hypothesised to give rise to cellular hypersensitivity to radiation and genotoxic drugs. We have used protein engineering to create a monomeric form of RAD51 by humanising a thermostable archaeal orthologue, RadA, and used this protein for fragment screening. The initial fragment hits were thoroughly validated biophysically by isothermal titration calorimetry (ITC) and NMR techniques and observed by X-ray crystallography to bind in a shallow surface pocket that is occupied in the native complex by the side chain of a phenylalanine from the conserved FxxA interaction motif found in BRCA2. This represents the first report of fragments or any small molecule binding at this protein-protein interaction site.
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
Duncan E
DE
Scott
Matthias T
MT
Ehebauer
Tara
T
Pukala
May
M
Marsh
Tom L
TL
Blundell
Ashok R
AR
Venkitaraman
Chris
C
Abell
Marko
M
Hyvönen
D001105:Archaea / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism; D024682:BRCA2 Protein / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002151:Calorimetry; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D015202:Protein Engineering; D060066:Protein Interaction Maps; D017434:Protein Structure, Tertiary; D051135:Rad51 Recombinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D054852:Small Molecule Libraries / Q000737:chemistry / Q000378:metabolism
D019843:Archaeal Proteins; D024682:BRCA2 Protein; D004268:DNA-Binding Proteins; C117252:RadA protein, archaeal; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D051135:Rad51 Recombinase