A fragment identified by 19F NMR stabilizes RAD51 fibrils and antagonizes BRC4 binding

Rationale: The interface between BRCA2’s BRC repeats and RAD51 is an intriguing cancer drug target because it is essential for homologous recombination (HR) mediated DNA repair. Indeed, the disruption of this crucial interaction impairs the HR process and synthetizes cancer cells to PARP inhibitors (PARP-is). Therefore, developing ligands that perturb BRCA2–RAD51 binding, could mimic a BRCAness condition, in which the concomitant administration of PARP-is would synergistically result in cell death thus generalizing the synthetic lethality (SL) paradigm also to BRCA2 nonmutated patients. Methods: Full-length human RAD51 was expressed and purified and its propensity to form self-assembled fibrils was characterized. Orthogonal biophysical assays were performed to validate binding and assess the effects of ligands identified through a 19F-NMR fragment screening on RAD51 oligomeric state and stability. Finally, AI-driven docking using Boltz-2 was performed to generate models supporting the biophysical observations. Results: 19F-NMR led to the identification of a fragment that binds RAD51 and is competitively displaced by the BRC4 peptide, indicating overlapping or allosterically coupled binding sites [1]. Dynamic light scattering, mass photometry and negative staining transmission electron microscopy (NS-TEM) clearly show that the fragment promotes RAD51 fibrils stabilization and reduces the depolymerization promoted by BRC4, the fourth BRC-repeat. Intriguingly, AI-generated models place the fragment at a protomer–protomer interface, partially overlapping with the ATP binding pocket. Conclusions: Biophysical data show that the identified hit stabilizes RAD51 oligomers and interferes with BRC4 binding. Beyond serving as a starting point for medicinal chemistry campaigns [1], this compound also represent a promising chemical-biology tool to probe RAD51 oligomerization, BRCA2-mediated recruitment and BRC4 competition in cellular and biochemical assays. Based on modeling data, the ligand appears to engage a functional protomer-protomer interface hotspot on RAD51. Planned high-resolution structural studies (Cryo-EM) will validate the binding mode of the hit and enable the rational design of derivatives for both mechanistic studies and therapeutic lead optimization. References: [1] Myers, Samuel H., et al. European Journal of Medicinal Chemistry 265 (2024): 116114.