Abstract
Synthetic lethality (SL) represents a new therapeutic approach for cancer treatment. DNA damage response offers the opportunity to exploit SL as proved by BRCA2 mutations that make cancer cells sensitive to PARP inhibitors (PARPi). Our team recently proposed the fully small-molecules-induced SL, which combines a small molecule inhibitor of RAD51-BRCA2 protein-protein interaction with PARPi olaparib to mimic the SL described above. Our previous studies led to ARN24089, which caused pancreatic cancer cell death in combination with olaparib, reproducing the SL paradigm. Based on these premises, the project will advance the studies on RAD51-BRCA2 small molecule inhibitors, performing structure-activity relationship studies and improving their pharmacokinetic properties, alongside the development of peptide and/or peptidomimetic inhibitors. The goal will be to evaluate their efficacy in pancreatic cancer models and validate the SL effect in vivo. A secondary goal will be to target RAD52, to explore triple synthetic lethality strategies by combining RAD52/RAD51/PARP inhibition. Operational units involved in the implementation of the project The project was implemented through the coordinated work of the operational units at the University of Siena (RU-SI), the University of Bologna (RU-BO), and the collaborating activities carried out with the University of Milan (RU-MI) network, including the Structural Biophysics Facility of IIT Genoa. The different units contributed complementary expertise in computational chemistry, medicinal chemistry, peptide chemistry, structural and biophysical characterization, cell biology, and functional omics. Description of the achievement of the objectives connected to the project and related outcomes The project achieved significant progress toward its scientific objectives. The University of Bologna further advanced the RAD51–BRCA2 workstream by characterizing compound ARN24089 and developing a new series of inhibitors, leading to the identification of ARN26912, a more selective and soluble compound. The University of Siena designed and optimized novel peptides and peptidomimetics targeting RAD52 oligomerization, identifying promising candidates for further development. The IIT Structural Biophysics Facility established an integrated assay platform to evaluate compounds generated by the project partners. In addition, a RAD52 knockout cellular model was generated and validated, providing a valuable tool for studying homologous recombination. Transcriptomic and cell-cycle analyses confirmed the relevance of the synthetic lethality approach in BRCA2-deficient pancreatic cancer models. These activities resulted in peer-reviewed publications, a PhD thesis, conference presentations, and the establishment of a strong multidisciplinary platform for future research and development. Detailed description of activities carried out by RU-BO The activities of RU-BO focused on the design, synthesis and biological evaluation of novel RAD51/RAD52 inhibitors aimed at exploiting SL as an innovative therapeutic strategy for pancreatic cancer. SL has proven to be a tactical paradigm for the rational design of synergistic anticancer drug combination. Our group recently published a comprehensive review on this topic (See Paper 1). In the context of SL, RU-BO and RU-MI recently proposed a novel anticancer drug discovery concept, termed “fully small molecule–induced SL,” which combines RAD51–BRCA2 disruptors with olaparib to simultaneously impair two DNA repair pathways, thereby reproducing the synthetic lethal phenotype observed in BRCA2-deficient tumors. We exploit this paradigm to target pancreatic cancer cells. Building upon our previously reported lead compound ARN24089 (35d in the paper 2), a dihydroquinolone pyrazoline-based derivative able to disrupt the RAD51-BRCA2 interaction, we performed an extensive characterization of its individual enantiomers. Following chromatographic separation of the racemic mixture and stereochemical assignment by circular dichroism, the two enantiomers, R-35d and S-35d, were evaluated using complementary biochemical and cellular approaches in 2D and 3D PDAC cultures. These studies demonstrated distinct pharmacological contributions of the stereoisomers and revealed a complex mechanism involving modulation of both RAD51-BRCA2 interaction and key DNA damage response kinases, including ATM, ATR, and DNA-PK. Collectively, these findings highlighted a previously unrecognized “within-pathway synthetic lethality” profile. Overall, the study underscores the potential of exploiting multi-target small molecules, opening new avenues for SL research and advancing personalized medicine strategies (See Paper 2). Despite its promising biological profile, ARN24089 displayed suboptimal aqueous solubility and unfavorable drug metabolism and pharmacokinetic (DM/PK) profile, limiting progression toward in vivo cancer model studies. To address these limitations, a structural optimization campaign was undertaken. Guided by molecular docking studies, a focused library of 30 analogues was synthesized and evaluated with the aim of improving physicochemical and biological properties. Despite this effort, none of the derivatives exceeded the parent compound in terms of biological activity or solubility (unpublished data). To identify alternative chemical scaffolds, a VS campaign targeting the RAD51/BRC4 Zone II interaction interface (PDB ID: 1N0W) was conducted using structure-based computational approaches. This effort led to identification of phenyl furan-carboxyquinoline ARN22142, which inhibited RAD51-BRC4 interaction in BRCA2-proficient BxPC-3 pancreatic cancer cells. While ARN22142 only moderately inhibited HR and generated additive effects in combination with olaparib, it exhibited significantly improved kinetic solubility in PBS and represented a suitable starting point for further optimization. Subsequent medicinal chemistry studies led to the development of new analogues and the identification of ARN26912 (19 in the paper 3), which emerged as a representative tool compound, overcoming the aqueous solubility limitations of previous inhibitors and displaying selectivity toward cancer over normal pancreatic cells. Computational point mutation analysis and NMR displacement assays supported its mechanism as a PPI inhibitor, in agreement with its biological activity as an HR inhibitor. In combination with olaparib, ARN26912 induced apoptosis through simultaneous disruption of complementary DNA repair mechanisms, consistent with the SL paradigm. Although further potency optimization is still required, this early-stage work provides a validated starting point for future structure refinement and expands the chemical biology toolkit for investigating RAD51–BRCA2 interaction in pancreatic cancer models (See paper 3). The second aim of the SyLAR project focused on identification of novel RAD52 inhibitors. RAD52 represents an attractive therapeutic target because cancer cells deficient in BRCA2-driven HR may become increasingly reliant on RAD52-mediated DNA repair mechanisms for survival. Consequently, simultaneous inhibition of RAD51, RAD52 and PARP could provide access to more complex SL strategies. Structural and computational analysis identified the RAD52 oligomerization interface as a promising targetable region. In particular, a previously unexploited binding cavity, designated pocket 36, emerged as the most suitable site for ligand discovery. Screening of an internal chemical library led to identification of a molecule capable of binding this region and disrupting key intermolecular interactions involved in RAD52 oligomer assembly, thereby providing initial validation of the target site. To further explore this opportunity, in collaboration with EPFL in Lausanne we generated and computationally screened a virtual library of ~500,000 dipeptides. The ten top candidates were synthesized, purified and characterized, including biotinylated analogues for binding studies. Biophysical screening (Microscale Thermophoresis, MST) performed in collaboration with IIT identified two dipeptides displaying binding to RAD52 at sub-100 µM concentrations. Further validation through biophysical and functional assays is underway. These results are part of the PhD thesis presented by Giovanni Ferrandi (see information below). Papers 1. Previtali, V. et al. New Horizons of Synthetic Lethality in Cancer: Current Development and Future Perspectives, «JOURNAL OF MEDICINAL CHEMISTRY», 2024, 1, 67, pp. 11488 – 11521. 2. Masi, M. et al., Investigating synthetic lethality and PARP inhibitor resistance in pancreatic cancer through enantiomer differential activity, «CELL DEATH DISCOVERY», 2025, 11, Article number: 106, pp. 1 – 23. 3. Ferrandi, G. et al. Targeting RAD51-BRCA2 Interaction to Enhance Synthetic Lethality with Olaparib in Pancreatic Cancer: Development of a Novel Phenyl Furan-Quinoline-Carboxylic Acid Series, «ACS MEDICINAL CHEMISTRY LETTERS», 2026, 17, pp. 520. PhD thesis PhD thesis presented by Giovanni Ferrandi, entitled “Design and synthesis of RAD51/RAD52 inhibitors to achieve synthetic lethality in combination with PARP inhibitors in pancreatic cancer.” PhD Program in Biotechnological, Biocomputational, Pharmaceutical and Pharmacological Sciences, University of Bologna (Cycle XXXVII, 2025). Oral presentations to national and international congresses - XXIX National Meeting on Medicinal Chemistry, Parma IT, Sept. 2025 Title: “Harnessing Phenylfuran-Carboxyquinoline Scaffold to Induce Synthetic Lethality in Pancreatic Cancer” - ACS Fall Meeting 2025, Washington DC, Aug. 2025 Title: “Harnessing phenylfuran-carboxyquinoline scaffold to induce synthetic lethality in pancreatic cancer” - SCI National Meeting, Milan IT, Aug. 2024 Title: “A journey through targeting BRCA2-RAD51 protein-protein interaction to establish synthetic lethality as paradigm for anticancer drug discovery
Dettagli del progetto
Responsabile scientifico: Marinella Roberti
Strutture Unibo coinvolte:
Dipartimento di Farmacia e Biotecnologie
Coordinatore:
Università degli Studi di MILANO(Italy)
Contributo totale Unibo: Euro (EUR) 65.354,00
Durata del progetto in mesi: 24
Data di inizio
18/10/2023
Data di fine:
28/02/2026
