Abstract BACKGROUND: Targeting DNA repair pathways to selectively eliminate cancer cells has seen a huge success with multiple PARP1 inhibitors (PARPi) approved in different solid tumor indications. RAD51 is a key DNA repair gene with an important role in maintaining genomic stability by mediating homologous recombination repair (HRR) and promoting replication fork protection. Impairment of HRR reduces cell fitness in genetic backgrounds with high replication stress and sensitizes cancer cells to DNA-damaging agents and DNA Damage Response (DDR) inhibitors. METHODS: A drug discovery campaign was conducted to identify novel small-molecule inhibitors of BRCA2-RAD51 protein-protein interaction (PPI). Structure-based drug design was employed to develop multiple compound series. Biochemical potency was assessed using a Fluorescence Polarization (FP) assay and an ATPase assay to measure the DNA-induced activity of RAD51, which is reliant on RAD51 self-oligomerization. A cellular reporter assay to measure HRR proficiency and a high content imaging assay to measure RAD51 foci formation were developed to assess compounds target engagement. RESULTS: We have identified proprietary RAD51 PPI inhibitors, orally bioavailable, demonstrating single-digit nanomolar biochemical potency on RAD51 and anti-proliferative activity in a subset of cancer cell lines. These include cell lines resistant to cisplatin or PARP1 inhibitors with a sensitivity profile distinct from RAD51 genetic depletion (based on the analysis of DepMap dataset). The anti-proliferative potency correlates with biochemical potency, cellular target engagement and activation of DNA damage response, confirming the expected on-target mechanism of action. CONCLUSIONS: The novel mechanism of action, with potential for combination with DNA damaging agents and DDR inhibitors, positions Nerviano Medical Sciences’ RAD51 inhibitors as promising first-in-class candidates for further development across multiple cancer types resistant to standard of care. Citation Format: Alessandro Galbiati, Daniela Asa, Pietro Picconi, Annamaria Marra, Claudia Stellato, Patrizia Banfi, Liviana Cozzi, Fabio Gasparri, Frank Narjes, Claudia Perrera, Alessia Montagnoli. Highly potent inhibitors of RAD51, with first-in-class potential, targeting HR-proficient cancers abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4574.
Galbiati et al. (Fri,) studied this question.