This document describes a fragment-based approach to target the protein-protein interaction between RAD51 and BRCA2 for cancer treatment. Key points: 1) RAD51 and BRCA2 interact through the BRC repeats of BRCA2, which is important for DNA repair. Inhibiting this interaction could block RAD51 activity in tumor cells. 2) The researchers used fragment screening, biophysical techniques like NMR and X-ray crystallography, and chemical elaboration to discover fragments that bind the RAD51-BRCA2 interaction site. 3) Hits were found that bound with low mM affinity. Further optimization through growing and linking fragments yielded compounds with μM affinity, outperforming the natural

1. A Fragment Based Approach to Targeting the RAD51:BRCA2 Protein-Protein Interaction
Anthony G. Coyne, Duncan E. Scott, Chiara R. Valenzano, John Skidmore, Tara Pukala, Chris Abell
Department of Chemistry, University of Cambridge, CB2 1EW, UK
May Marsh, Tim Sharpe, Matthias Ehebauer, Luca Pellegrini, Tom L. Blundell , Marko Hyvonen
Department of Biochemistry, University of Cambridge, CB2 1GA, UK.
David Huggins, Nicola-Jane Francis, Grahame McKenzie Ashok Venkitaraman
Cambridge Molecular Therapeutics Programme, Hutchison/MRC Research Centre, University of Cambridge, CB2 0XZ, UK.
(1) RAD51: BRCA2 (2) RAD51 Interaction
(4) Preliminary Results: From Fragments to Hybrid Peptides
(3) Fragment-Based Methodology
(5) Conclusions and Future Work Acknowledgements
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Seeding Drug Discovery Initiative: GR 091058
Translation Award: GR 080083
 The breast cancer susceptibility protein BRCA2 controls
the function of RAD51, a recombinase enzyme in pathways
for DNA repair by homologus recombination.
 BRCA2 binds RAD51 through eight BRC repeats and an
overexpression of the BRC4 repeat in cells prevents
formation of RAD51-ssDNA nucleofilament and results in
RAD51 diffusion and failure to repair DNA breaks
 The disruption on the RAD51-BRCA2 binding interface by
small molecule inhibitors is expected to block RAD51
activity.
Objective
Use fragment-based drug discovery as a tool to develop
small molecule inhibitors that specifically modulate the
RAD51-BRCA2 interaction in tumor cells
 Human RAD51 (HsRAD51) is one part of a broad
class of recombinases comprising structural and
functional homologues in every species (RecA in
E.coli and RadA in Archea)
 The BRC repeats disrupt the oligomeric RAD51
form by binding at the FXXA and LFDE hot spots
 The FXXA repeat is conserved through each of the
BRC repeats but also in other homologues
 The aim of this research is to target the FXXA
binding region where both the BRC repeats and the
N-terminal of RAD51 binds
 The aim is to see whether these binding sites can
be targeted using the fragment-based approach
RAD51-BRC4 Interaction
RAD51-RAD51 Interaction
BRC Repeats
BRC1 HSFGGSFRTASNKEI
BRC2 EVGFRGFYSAHGTKL
BRC3 ETSDTFFQTASGKNI
BRC4 EPTLLGFHTASGKKV
BRC6 EVGPPAFRIASGKIV
BRC7 ANTCGIFSTASGKSV
BRC8 SSAFSGFSTASGKQV
RAD51
RAD51-RAD51 Filament
(S. Cerevisiae (PDB Code: 1szp))a
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RAD51-BRC4 showing the FXXA and
LFDE hot-spot binding regions
(HsRAD51 (PDB Code: 1now)b
LFDE
FXXA
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(a) Rice, P.A. et al, Nat. Struct. Mol. Biol, 2004, 11, 791-796
(b) Pellegrini L.. et al, Nature, 2002, 420, 287-293
Target Protein Fragment Library
Primary Screening
Thermal Shift Screening
Secondary Screening
NMR Spectroscopy
X-Ray Crystallography
Binding Affinity
Isothermal Calorimetry (ITC)
Molecular Design
Fragment Analoguing, Docking
Chemical Synthesis
Fragment Growing, Fragment Linking
Iterative Development Cycle
Target Protein Thermal Shift Screening NMR: STD (Saturated Transfer Difference) Isothermal Titration Calorimetry (ITC)
RAD51-BRCA2
Binding site
PfRadA
Green: Identical Residues
Red: Different Residues
PfRadA Mutant
Green: Identical Residues
Red: Different Residues
Fragment Library
OH
N
O
N
N
N
O
HO
CF3
N
O
O
O
HN
O
O
O
O
OH
O
H
N
NH2N
O
HO
N
S
1338 Fragments
0
5000
10000
15000
20000
25 30 35 40 45 50 55
Temperature (o
C)
 The unfolding temperature TM is
monitored for the protein and any
fragment binding observed causes a
increase in TM of the protein
 Where a fragment gives a ∆TM >1o
C
this is defined as a hit by thermal shift
 The Nuclear Overhauser Enhancement (NOE) on the fragment is
measured. This is the direct transfer of magnetization from the
methyl groups on the protein to the fragment
 A known displacer (fragment/peptide) is added and this is used to
determine where the fragment is binding on the protein
1
H NMR Fragment
No Protein
Protein+ Fragment
Protein+ Fragment +
Displacer
 ITC measures the heat released or
absorbed during ligand binding to the protein.
 The binding constant (KB), n (stoichiometry)
∆H and ∆S can all be calculated from this
experiment.
Fragment (mM) Elaborated Fragment (nM)
ITC KD 0.56 mM to 2mM
email: agc40@cam.ac.uk
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Tetrapeptide SAR (KD ITC)Fragment Hits (X-Ray Crystal Structure)
Merging Fragment and
Peptide
Phe Pocket
Ala Pocket
Fragment: KD = 1.4 mM Peptide: KD = 0.28 mM
 We have applied a range of biophysical techniques in order to discover and validate fragments against RAD51
 Iterative cycles of chemical elaboration guided by X-ray crystallography is currently in progress
 The most potent compounds will be evaluated in cellular assays and information from successful compounds will be fed
back to the design and synthesis process
KD = 5.9 µM KD = 2.6 µM
Fragment/Peptide Hybrid 1 Fragment/Peptide Hybrid 2
FATA 280 µM
FNTA 630 µM
FPTA No binding
WHTA 95 µM FHTG 1000 µM
FHPA 110 µM
FHAA 500 µMFHTA 280 µM
The fragments and peptides were merged to give compounds that
had significantly greater potency than the natural FXXA peptide
sequence
KLVPMGFTTATEFHQ
RLVPMGFVTAADFHM
KLVPMGFTTATEFHQ
KLVPLGFLSARTFYQ
AANLGTFMRADEYLK
HsRad51
ScRad51
GgRad51
DmRad51
PfRadA