FDA-approved IMPDH inhibitors synergize with ribavirin to inhibit respiratory syncytial virus by interfering with purine <i>de novo</i> synthesis

ABSTRACTRespiratory syncytial virus (RSV), a leading cause of severe lower respiratory tract infections in pediatric and elderly populations, presents an urgent need for improved therapeutic interventions. Although ribavirin (RBV) is an FDA-approved antiviral for treating RSV, its clinical application is constrained by dose-dependent toxicity and limited therapeutic efficacy. Here, we developed a high-throughput system to screen FDA-approved drugs that enhance RBV's antiviral efficacy. Unlike conventional combination therapies, this approach does not require both components to demonstrate direct antiviral activity; rather, it enhances antiviral efficacy by targeting cellular pathways, even when the synergistic agents exhibit minimal antiviral effects as monotherapies. We identified mycophenolate mofetil (MMF) and its active metabolite, mycophenolic acid (MPA), clinically approved inhibitors of inosine monophosphate dehydrogenase (IMPDH), which significantly enhance the antiviral efficacy of RBV against RSV. Mechanistically, through IMPDH knockdown and exogenous GTP supplementation, we propose that MMF and MPA reduce intracellular GTP levels by inhibiting IMPDH activity, thereby enhancing RBV incorporation and improving its antiviral efficacy. Our studies show that IMPDH inhibitors and RBV work synergistically to combat both RSV A/B genotypes and human metapneumovirus (hMPV) <i>in vitro</i>. Importantly, we further validated the therapeutic potential of this combination in a mouse model, showing enhanced viral clearance and reduced pathogenesis. These findings suggest that the combination of IMPDH inhibitors with RBV represents a promising therapeutic strategy for RSV and hMPV infection in clinical settings.