Final Report Summary - BRCA2-RAD51 (The use of fragment-based drug discovery to develop novel small molecules that modulate the BRCA2-RAD51 interaction for the treatment of cancer)
The aim of the project is to develop a small molecule that disrupts the protein-protein interaction between the human recombinase RAD51, and the hub protein BRCA2. This interaction is essential for DNA repair by homologous DNA recombination (HDR), a key cellular pathway involved in the resistance of cancer cells to ionizing radiation and radio-mimetic drugs. BRCA2 binds RAD51 by eight highly conserved BRC peptide repeats that include two tetrameric amino acid motifs, i.e. FxxA at the N-terminus and LFDE at the C-terminus of BRC repeat, which contribute to most of the binding. The two tetrameric sequences interact at two distinct sites in RAD51 which thus potentially constitute two separate suitable hot-spots, this suggest that a molecule by binding to either of this two sites can effectively disrupt this crucial protein-protein interaction. While it is understood that the FxxA sequence at the N-terminus of the BRC repeats blocks RAD51 oligomerisation by mimicking the self-association motif of RAD51, the role of the C-terminus LFDE motif and the significance of its interaction at the Velcro site on RAD51 is still to be clarified. Our objective, at first, was to develop inhibitors that specifically target the Velcro pocket by using fragment-based drug discovery methods.
To apply this methodology we expressed and purified a surrogate monomeric form of RAD51 and we carried out a thermal shift and ligand-based NMR screening of our in-house fragment library against this protein. Fragment hits were then tested for binding in competition with two half peptides, i.e. N-terminal oligomerisation FxxA-half peptide and C-terminal Velcro LFDE-half peptide, generated by the dissection of the fourth repeat (BRC-4) of BRCA2 that naturally binds RAD51. Unfortunately all the fragment hits were displaced by the FxxA-half peptide suggesting a competition for the common oligomerisation site on RAD51 and no fragments indicate binding to the Velcro site.
The failure to find binder to the site of interest, led us to adopt an alternative strategy to pursue our project goal.
The crystal structure of the RAD51-BRC4 complex revealed that the portion of BRC-4 that binds to the Velcro-site is helical (Figure 1B). This suggested that the BRCA2-RAD51 complex might be targeted by alpha-helix mimetics, specifically the hydrocarbon-stapled alpha-helical peptide approach established by Verdine and co-workers. A stapled peptide is a peptide that while conserving the natural amino acids crucial for interacting with the target protein, includes two un-natural amino acids that when chemically modified, form an all-carbon linker called staple. The staple by stabilising the alpha helix conformation brings an increase in affinity. The native C-terminal Velcro LFDE-half peptide binds very weakly to both monomeric surrogate RAD51 and human oligomeric RAD51, therefore the stapled peptide approach was particularly attractive.
Six stapled peptides (SPs) were designed by incorporation of a non-natural amino acid at neighbouring position (i and i+4, as well as i and i+7) along one face of the alpha-helix in place of native amino acids that showed no interaction with the RAD51 protein as indicated by the BRC4-RAD51 crystal structure and corroborated by alanine scanning and stapled scanning of the interaction. The stapled peptides were prepared, characterised and they all showed an increase of helicity compared to the native Velcro peptide. When we tested the stapled peptide for binding to RAD51, three out of six SP bound to human RAD51 and displaced BRC-4 indicating that we develop indeed molecules that dirrupt RAD51-BRCA2 interaction. In particular on stapled peptide, namely SP3, had a 100 fold higher affinity to human RAD51 than the native Velcro peptide. Interestingly none of the stapled peptides bound to the monomeric surrogate RAD51.
We developed the first stapled peptide to disrupt RAD51-BRCA2 interaction by specifically blocking the Velcro site; no other ligands are known to interact to RAD51 at the proposed target site. Moreover we had in our hand for the first time, the chemical tool to investigate the effect on the oligomerisation of RAD51 when RAD51-BRCA2 interaction is inhibited at this site. To further investigate the stapled peptide binding mode we carried out native mass spectrometry experiments of human RAD51 co-incubated with SP3. The results orthogonally confirmed the formation of a complex between multimeric form of RAD51 and SP3 while showing no evidence of a monomeric RAD51:SP3 complex. In particular, RAD51 dimer binds one SP3 while RAD51 trimer binds two SP3 peptides. Furthermore dynamic light scattering experiments showed that when SP3 is incubated with human oligomeric RAD51, the protein preserves its oligomeric distribution. This body of experimental evidences corroborated also by preliminary structural information, suggested that SP3 binds at the interface between two monomer of RAD51 and that the hydrocarbon chain of the staple directly contributes to the interaction with the protein.
In conclusion we developed the first inhibitor specific for the Velcro site of human RAD51 which modulate BRCA2-RAD51 complex formation and we investigated its binding mode. Stapled peptides, by combining the intrinsic specificity of the natural protein binder partner with advantageous chemo-physical properties conferred by the staple (i.e. cell permeability and resistance to proteolysis), are an emerging new class of drugs to target protein-protein interaction. We expected that the Velcro specific stapled peptide SP3 will have the stapled peptide advantageous properties to be used as chemical tools to uncover different mechanistic aspects of the HDR process in cells. The development of the stapled peptide has a wider socio-economic impact because, by proving the druggability of the Velcro site, opens the door for the discovery and elaboration of novel therapeutics in combination with existing cancer treatments.
To apply this methodology we expressed and purified a surrogate monomeric form of RAD51 and we carried out a thermal shift and ligand-based NMR screening of our in-house fragment library against this protein. Fragment hits were then tested for binding in competition with two half peptides, i.e. N-terminal oligomerisation FxxA-half peptide and C-terminal Velcro LFDE-half peptide, generated by the dissection of the fourth repeat (BRC-4) of BRCA2 that naturally binds RAD51. Unfortunately all the fragment hits were displaced by the FxxA-half peptide suggesting a competition for the common oligomerisation site on RAD51 and no fragments indicate binding to the Velcro site.
The failure to find binder to the site of interest, led us to adopt an alternative strategy to pursue our project goal.
The crystal structure of the RAD51-BRC4 complex revealed that the portion of BRC-4 that binds to the Velcro-site is helical (Figure 1B). This suggested that the BRCA2-RAD51 complex might be targeted by alpha-helix mimetics, specifically the hydrocarbon-stapled alpha-helical peptide approach established by Verdine and co-workers. A stapled peptide is a peptide that while conserving the natural amino acids crucial for interacting with the target protein, includes two un-natural amino acids that when chemically modified, form an all-carbon linker called staple. The staple by stabilising the alpha helix conformation brings an increase in affinity. The native C-terminal Velcro LFDE-half peptide binds very weakly to both monomeric surrogate RAD51 and human oligomeric RAD51, therefore the stapled peptide approach was particularly attractive.
Six stapled peptides (SPs) were designed by incorporation of a non-natural amino acid at neighbouring position (i and i+4, as well as i and i+7) along one face of the alpha-helix in place of native amino acids that showed no interaction with the RAD51 protein as indicated by the BRC4-RAD51 crystal structure and corroborated by alanine scanning and stapled scanning of the interaction. The stapled peptides were prepared, characterised and they all showed an increase of helicity compared to the native Velcro peptide. When we tested the stapled peptide for binding to RAD51, three out of six SP bound to human RAD51 and displaced BRC-4 indicating that we develop indeed molecules that dirrupt RAD51-BRCA2 interaction. In particular on stapled peptide, namely SP3, had a 100 fold higher affinity to human RAD51 than the native Velcro peptide. Interestingly none of the stapled peptides bound to the monomeric surrogate RAD51.
We developed the first stapled peptide to disrupt RAD51-BRCA2 interaction by specifically blocking the Velcro site; no other ligands are known to interact to RAD51 at the proposed target site. Moreover we had in our hand for the first time, the chemical tool to investigate the effect on the oligomerisation of RAD51 when RAD51-BRCA2 interaction is inhibited at this site. To further investigate the stapled peptide binding mode we carried out native mass spectrometry experiments of human RAD51 co-incubated with SP3. The results orthogonally confirmed the formation of a complex between multimeric form of RAD51 and SP3 while showing no evidence of a monomeric RAD51:SP3 complex. In particular, RAD51 dimer binds one SP3 while RAD51 trimer binds two SP3 peptides. Furthermore dynamic light scattering experiments showed that when SP3 is incubated with human oligomeric RAD51, the protein preserves its oligomeric distribution. This body of experimental evidences corroborated also by preliminary structural information, suggested that SP3 binds at the interface between two monomer of RAD51 and that the hydrocarbon chain of the staple directly contributes to the interaction with the protein.
In conclusion we developed the first inhibitor specific for the Velcro site of human RAD51 which modulate BRCA2-RAD51 complex formation and we investigated its binding mode. Stapled peptides, by combining the intrinsic specificity of the natural protein binder partner with advantageous chemo-physical properties conferred by the staple (i.e. cell permeability and resistance to proteolysis), are an emerging new class of drugs to target protein-protein interaction. We expected that the Velcro specific stapled peptide SP3 will have the stapled peptide advantageous properties to be used as chemical tools to uncover different mechanistic aspects of the HDR process in cells. The development of the stapled peptide has a wider socio-economic impact because, by proving the druggability of the Velcro site, opens the door for the discovery and elaboration of novel therapeutics in combination with existing cancer treatments.
