Interrogation and engineering of RAD51:BRC repeat interactions

The maintenance of genetic information is a fundamental function of every organism. The DNA in every human cell endures thousands of lesions per day, and eukaryotes have developed a sophisticated response to tackle this damage. Double strand breaks (DSBs) are the most toxic type of DNA damage, potentially leading to chromosomal rearrangements and tumorigenesis. Homologous recombination (HR) is a repair pathway that uses a homologous template to faithfully repair the DSBs. Central to HR is a recombinase protein RAD51, which forms a nucleoprotein filament with the broken ends, and allows efficient homology search. RAD51 is a promising therapeutic target in oncology. BRCA2 is a crucial mediator of RAD51 function and interacts with RAD51 through a set of 8 BRC repeats. A BRC repeat consists of two modules, an FxxA and an LFDE module, each containing hot-spot residues that mediate binding. In this work, I set out to utilise these for the pharmacological targeting of human RAD51. First, using biophysical methods, I investigated a novel, high-affinity chimeric repeat BRC8-2 composed of FxxA and LFDE modules from different natural repeats. I determined the X-ray crystallographic structure of its complex, which allowed me to uncover the determinants of high affinity binding. I applied these findings to the design of novel macrocyclic peptide inhibitors of RAD51. A method for cysteine stapling of recombinantly produced peptides was optimised to yield the correct product of high purity. The optimised stapling methodology provides a promising general approach for recombinant production and evaluation of cysteine-stapled peptides. Peptides were rationally designed in a structure-guided manner, and a variety of stapling architectures were evaluated for binding. The resulting purified molecules exhibit high potency and are stable in serum. Further crystallographic studies shed light on how the stapling moiety affects the peptide binding mode. The derived peptides serve as novel modalities for targeting Rad51 protein-protein interactions and can inform subsequent development of therapeutic drugs. In addition to the human proteins, I investigated the BRC:RAD51 interactions in orthologs from Leishmania infantum, the causative agent of leishmaniasis. Using crystallography and biophysical methods, I uncovered novel features of BRC repeat binding. The presented work expands our understanding of the structural determinants of homologous recombination.MRC DT

Poxviruses and paramyxoviruses use a conserved mechanism of STAT1 antagonism to inhibit interferon signaling.

The induction of interferon (IFN)-stimulated genes by STATs is a critical host defense mechanism against virus infection. Here, we report that a highly expressed poxvirus protein, 018, inhibits IFN-induced signaling by binding to the SH2 domain of STAT1, thereby preventing the association of STAT1 with an activated IFN receptor. Despite encoding other inhibitors of IFN-induced signaling, a poxvirus mutant lacking 018 was attenuated in mice. The 2.0 Å crystal structure of the 018:STAT1 complex reveals a phosphotyrosine-independent mode of 018 binding to the SH2 domain of STAT1. Moreover, the STAT1-binding motif of 018 shows similarity to the STAT1-binding proteins from Nipah virus, which, similar to 018, block the association of STAT1 with an IFN receptor. Overall, these results uncover a conserved mechanism of STAT1 antagonism that is employed independently by distinct virus families

Peptidomimetic plasmepsin inhibitors with potent anti-malarial activity and selectivity against cathepsin D.

Following up the open initiative of anti-malarial drug discovery, a GlaxoSmithKline (GSK) phenotypic screening hit was developed to generate hydroxyethylamine based plasmepsin (Plm) inhibitors exhibiting growth inhibition of the malaria parasite Plasmodium falciparum at nanomolar concentrations. Lead optimization studies were performed with the aim of improving Plm inhibition selectivity versus the related human aspartic protease cathepsin D (Cat D). Optimization studies were performed using Plm IV as a readily accessible model protein, the inhibition of which correlates with anti-malarial activity. Guided by sequence alignment of Plms and Cat D, selectivity-inducing structural motifs were modified in the S3 and S4 sub-pocket occupying substituents of the hydroxyethylamine inhibitors. This resulted in potent anti-malarials with an up to 50-fold Plm IV/Cat D selectivity factor. More detailed investigation of the mechanism of action of the selected compounds revealed that they inhibit maturation of the P. falciparum subtilisin-like protease SUB1, and also inhibit parasite egress from erythrocytes. Our results indicate that the anti-malarial activity of the compounds is linked to inhibition of the SUB1 maturase plasmepsin subtype Plm X

Divergent binding mode for a protozoan BRC repeat to RAD51.

Interaction of BRCA2 through ca. 30 amino acid residue motifs, BRC repeats, with RAD51 is a conserved feature of the double-strand DNA break repair by homologous recombination in eukaryotes. In humans the binding of the eight BRC repeats is defined by two sequence motifs, FxxA and LFDE, interacting with distinct sites on RAD51. Little is known of the interaction of BRC repeats in other species, especially in protozoans, where variable number of BRC repeats are found in BRCA2 proteins. Here, we have studied in detail the interactions of the two BRC repeats in Leishmania infantum BRCA2 with RAD51. We show LiBRC1 is a high-affinity repeat and determine the crystal structure of its complex with LiRAD51. Using truncation mutagenesis of the LiBRC1 repeat, we demonstrate that high affinity binding is maintained in the absence of an LFDE-like motif and suggest compensatory structural features. These observations point towards a divergent evolution of BRC repeats, where a common FxxA-binding ancestor evolved additional contacts for affinity maturation and fine-tuning

Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats.

Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended β-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.BBSRC (BB/K013629/1), Cancer Research UK (C7905/A25715), EPSRC (grants EP/L015889/1 and EP/H018301/1), the Wellcome Trust (grants 3-3249/Z/16/Z and 089703/Z/09/Z), MRC (grants MR/K015850/1 and MR/K02292X/1), ERC, H2020 Marie-Curi

A recombinant approach for stapled peptide discovery yields inhibitors of the RAD51 recombinase

Acknowledgements: We are grateful to Dr Laurens Lindenburg for the gift of BRC4-fluorescein. We thank the Biophysical and X-ray crystallographic facilities (Department of Biochemistry, University of Cambridge) for access to instrumentation and support. We thank Diamond Light Source for access to beamlines i03 and i04 (proposals mx18548, mx25402). We thank Dr Peter Martin and the Institute of Cancer Research Light Microscopy Facility for support. TP was supported by MRC DTP. The Spring Laboratory acknowledges support from the EPSRC, BBSRC and MRC. PZ-V and JAD were supported by CRUK (C7905/A25715).An approach for stapled peptide preparation in small scale using recombinant expression of peptide–protein fusions in bacteria. We use this approach to design binders of RAD51, characterise their interaction and demonstrate activity in cells.</jats:p

Fragment-Based Discovery of 2‑Aminoquinazolin-4(3<i>H</i>)‑ones As Novel Class Nonpeptidomimetic Inhibitors of the Plasmepsins I, II, and IV

2-Aminoquinazolin-4­(3<i>H</i>)-ones were identified as a novel class of malaria digestive vacuole plasmepsin inhibitors by using NMR-based fragment screening against Plm II. Initial fragment hit optimization led to a submicromolar inhibitor, which was cocrystallized with Plm II to produce an X-ray structure of the complex. The structure showed that 2-aminoquinazolin-4­(3<i>H</i>)-ones bind to the open flap conformation of the enzyme and provided clues to target the flap pocket. Further improvement in potency was achieved via introduction of hydrophobic substituents occupying the flap pocket. Most of the 2-aminoquinazolin-4­(3<i>H</i>)-one based inhibitors show a similar activity against digestive Plms I, II, and IV and >10-fold selectivity versus CatD, although varying the flap pocket substituent led to one Plm IV selective inhibitor. In cell-based assays, the compounds show growth inhibition of Plasmodium falciparum 3D7 with IC₅₀ ∼ 1 μM. Together, these results suggest 2-aminoquinazolin-4­(3<i>H</i>)-ones as perspective leads for future development of an antimalarial agent