Targeting the FN3K–Nrf2 axis: Discovery and pre-clinical evaluation of novel inhibitors for breast cancer therapy

Introduction: Fructosamine-3-kinase (FN3K), a deglycation enzyme implicated in redox regulation through the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, has emerged as a novel therapeutic target in breast cancer. Elevated FN3K activity enhances antioxidant defenses, promoting cancer cell survival and resistance to therapies. Pharmacological inhibition of FN3K may sensitize tumors to oxidative stress. Objective: This study aimed to identify and validate potent FN3K inhibitors through structure-based virtual screening (SBVS) and in vitro evaluation in breast cancer models. Methods: A homology-modeled FN3K structure was generated and validated using SWISS-MODEL, PROCHECK, and QMEAN. Compound libraries, including the Food and Drug Administration (FDA)-approved kinase inhibitors, World Health Organization (WHO) essential medicines, and anti-breast cancer agents, were screened using Schrödinger’s Glide module. Top-ranked compounds were prioritized based on binding affinity, molecular interactions, absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling. In vitro validation in MCF-7, BT-474, T-47D, and Vero cell lines included MTT cytotoxicity assay and evaluation of FN3K and Nrf2 expression through quantitative PCR (qPCR) and Western blotting. Statistical analyses were performed to assess the significance of observed effects. Results: Oxaliplatin, lansoprazole, and capivasertib exhibited strong binding affinities (Glide scores: −9.2 to −8.1 kcal/mol) and selective cytotoxicity in breast cancer cell lines (IC50: 90 – 110 μg/mL). qPCR analysis revealed >99% downregulation of FN3K, accompanied by significant suppression of Nrf2 in cancer cells. Minimal modulation was observed in Vero cells, indicating tumor selectivity. Western blotting further corroborated the downregulation of FN3K and Nrf2 at the protein level. Molecular dynamics (MD) simulations validated the binding stability of the lead small molecules, reinforcing their potential as effective inhibitors. Conclusion: The integrated in silico and in vitro analysis supports FN3K as a viable therapeutic target in breast cancer. Oxaliplatin, lansoprazole, and capivasertib demonstrated strong FN3K inhibition and modulation of tumor redox homeostasis, suggesting their potential for further pre-clinical development as novel anti-cancer agents targeting metabolic adaptability.