A Fragment Based Approach to Targeting the RAD51:BRCA2 Protein-Protein InteractionAnthony G. Coyne, Duncan E. Scott, Chiara R. Valenzano, John Skidmore, Tara Pukala, Chris Abell

Department of Chemistry, University of Cambridge, CB2 1EW, UKMay 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 091058Translation 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.

ObjectiveUse 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 RepeatsBRC1 HSFGGSFRTASNKEIBRC2 EVGFRGFYSAHGTKLBRC3 ETSDTFFQTASGKNIBRC4 EPTLLGFHTASGKKVBRC6 EVGPPAFRIASGKIVBRC7 ANTCGIFSTASGKSVBRC8 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 ScreeningThermal Shift Screening

Secondary ScreeningNMR Spectroscopy

X-Ray Crystallography Binding AffinityIsothermal Calorimetry (ITC)

Molecular DesignFragment Analoguing, Docking

Chemical SynthesisFragment Growing, Fragment Linking

Iterative Development Cycle

Target Protein Thermal Shift Screening NMR: STD (Saturated Transfer Difference) Isothermal Titration Calorimetry (ITC)

RAD51-BRCA2Binding site

PfRadAGreen: Identical ResiduesRed: Different Residues

PfRadA MutantGreen: Identical ResiduesRed: Different Residues

Fragment Library

OHN

ON

N

N

O

HO

CF3

N

O

O

O

HN

OO

OO

OH

O

HN

NH2NO

HO

N

S

1338 Fragments

0

5000

10000

15000

20000

25 30 35 40 45 50 55

Temperature (oC)

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 >1oC 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

1H 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 M KD = 2.6 M

Fragment/Peptide Hybrid 1 Fragment/Peptide Hybrid 2

FATA 280 MFNTA 630 MFPTA No binding

WHTA 95 M FHTG 1000 M

FHPA 110 MFHAA 500 MFHTA 280 M

The fragments and peptides were merged to give compounds that had significantly greater potency than the natural FXXA peptide

sequence

KLVPMGFTTATEFHQRLVPMGFVTAADFHMKLVPMGFTTATEFHQKLVPLGFLSARTFYQAANLGTFMRADEYLK

HsRad51

ScRad51

GgRad51

DmRad51

PfRadA