A chemical biology probe identified through 19F NMR screening stimulates RAD51 oligomerization and disrupts BRC4 binding

The BRCA2–RAD51 interaction is essential to ensure high-fidelity repair of deleterious DNA double strand-breaks within Homologous Recombination (HR) [1,2]. BRCA2 engages RAD51 through eight repetitive sequences, named BRC-repeats, which mediate proper cytosolic RAD51 recruitment and promote its nuclear translocation [1,2]. Disruption of this crucial interaction impairs HR (BRCAness) and sensitizes cancer cells to PARP inhibitors (PARP-is), making it a compelling therapeutic target [3]. Indeed, small molecules capable of perturbing the BRCA2–RAD51 interface could mimic BRCAness [3]. In this condition, concomitant administration of PARP-is would synergistically result in cell death, potentially extending the synthetic lethality (SL) paradigm to BRCA2 wild type tumors [3]. To this end we performed a 19F NMR fragment-based screening using a recombinantly purified RAD51 WT, to identify novel hits capable of disrupting the RAD51-BRC4 interaction [4]. This approach led to the identification of a hit displaced by the BRC4 peptide, suggesting overlapping or allosterically coupled binding sites. Orthogonal biophysical and cellular assays highlighted that the fragment stabilized RAD51 fibrils, reducing both BRC4 driven depolymerization and DNA binding. AI driven docking with Boltz 2 suggested that the ligand may bind at the RAD51 protomer–protomer interface, overlapping the BRC4 binding site, as well as at the DNA binding site. Collectively, these results suggest that the identified hit may prevent RAD51 depolymerization through concomitant impairment of BRC4 binding and stabilization of a DNA bound state. This compound therefore represents a valuable chemical biology probe to investigate BRCA2 mediated recruitment and RAD51 DNA-binding mechanisms, as well as a tractable starting point for future medicinal chemistry efforts. Planned high resolution cryo EM studies will further validate the purported ligand binding mode and inform rational optimization toward improved mechanistic tools and therapeutic leads. References: [1] Rinaldi, F., Girotto, S. Current Opinion in Structural Biology 88 (2024) [2] Rinaldi, F., Girotto, S. Current Opinion in Structural Biology 95 (2025) [3] Previtali V.et al. Journal of Medicinal Chemistry 67 (2024) [4] Myers, S. H., et al. European Journal of Medicinal Chemistry 265 (2024)