Repurposing FDA-Approved Drugs as Potential Inhibitors of Feline Infectious Peritonitis Virus 3CL pro : An Integrated In Silico and In Vitro Study with Synergistic Combination Analysis

Feline infectious peritonitis (FIP), caused by feline coronavirus (FCoV) remains a critical therapeutic challenge with no universally approved treatments. We employed a drug repurposing strategy targeting the viral main protease (3CLpro), essential for FCoV replication. Virtual screening of FDA-approved drugs identified 15 candidates, with three─saquinavir (antiretroviral), lumacaftor (CFTR corrector), and gliquidone (antidiabetic)─demonstrating notable antiviral activity. Postentry antiviral assays (cytopathic effect reduction, immunoperoxidase staining, RT-qPCR) yielded EC50 values of 0.25–0.48 μM (saquinavir), 30.32–58.49 μM (lumacaftor), and 48.85–51.28 μM (gliquidone). We validated 3CLpro inhibition using an intracellular dual-luciferase reporter assay designed to simultaneously account for compound cell penetration and metabolic stability. This confirmed specific protease inhibition with IC50 values of 23.70 ± 1.38 μM (saquinavir) and 38.68 ± 1.59 μM (lumacaftor), and 82.52 ± 1.50 μM (gliquidone). Synergy analysis with established anti-FIP agents (GC376, remdesivir, GS-441524, and molnupiravir) using SynergyFinder 3.0 revealed strong synergistic interactions: saquinavir + GC376 (mean ZIP score: 54.59), lumacaftor + GC376 (mean ZIP: 21.44), and gliquidone + remdesivir (mean ZIP: 24.13). Notably, optimal synergistic doses achieved 0.97–2.03 log10 viral suppression using only 20–50% of individual drug EC50 values, concurrently modulating viral-induced cytokine expression (TNF-α, IFN-β, and IL-6) in CRFK cells (p < 0.05). These rational combinations enable substantial dose reduction, offering practical strategies to improve treatment accessibility, reduce costs, and minimize adverse effects. This study establishes a framework for repurposing FDA-approved drugs in FIP therapy and supports translational evaluation of these combination regimens.