Identification and subsequent development of RAD51-BRCA2 disruptors through a fragment-based approach

Protein–protein interactions (PPIs) play a vital role in nature, and are involved in various disease areas including infectious disease, neurodegenerative disease, and notably cancer. The PPI between RAD51 and BRCA2 plays a key role in homologous recombination (HR), and aids the repair of double stranded DNA breaks. Impairment of the HR repair mechanism through BRCA2 mutations can lead to the sensitization of cancer cells to therapeutic DNA-damaging agents. It has been suggested that disruption of the RAD51-BRCA2 PPI could induce an artificial ‘BRCAness’ in tumors, opening the door for treatment with PARP inhibitors, of which four have received regulatory approval in the past ten years for patients with tumors showing BRCA2 mutations. In the pursuit of RAD51-BRCA2 disruptors, and after an initial 19F NMR ligand binding screening of approximately 900 fluorinated fragments against full-length RAD51, ARN2037 was identified as a RAD51 binder. The activity of this compound was evaluated through biochemical and biophysical techniques (Elisa: EC50 > 350 µM and MST: KD 1.23 ± 0.13 mM), confirming its activity as a RAD51 binder able to inhibit HR at 400µM in BxPC3 cells (fully functional BRCA2 human primary pancreatic adenocarcinoma) (IC50: 369µM). ARN2037 was further able to interfere with the disassembly of RAD51 fibrils by BRC4, in a time-dependent manner. However, given the weak binding of ARN2037, a medicinal chemistry program was undertaken that culminated in the discovery of the hit compound ARN24922 which outperformed ARN2037 across biochemical and biophysical assays (Elisa: EC50 100 ± 20 µM and MST: KD 10.48 ± 3.92 µM). A similar improvement was seen in HR inhibition (IC50: 9.06 ± 3 µM). We hope to further study ARN24922 as a potential tool in better understanding the mechanisms and effects that occur as a result of the PPI disruption between RAD51-BRCA2.