Salinomycin inhibits orthopoxvirus infection in vitro and in vivo by blocking endosomal acidification.

The recent global spread of monkeypox virus (MPXV) highlights the urgent need for effective antiviral therapies. Through high-throughput screening of an FDA-approved small molecule drug library, we identified salinomycin as a potent inhibitor of orthopoxvirus infection. Salinomycin showed nanomolar anti-MPXV activity in human keratinocytes (HaCaT) and lung epithelial cells (A549), with selectivity indices >100. Importantly, its efficacy was validated in primary human fibroblasts, reducing infectious viral titers by ∼2.6 log units (440-fold) versus controls. Mechanistic studies revealed salinomycin acts at the viral post-entry stage, blocking membrane fusion via disrupting endosomal acidification, without affecting clathrin-mediated endocytosis. This host-directed mechanism was validated by DiO fusion assays, acridine orange staining, and low-pH pulse rescue experiments. In a lethal intranasal vaccinia virus (VACV-WR) murine challenge model, oral salinomycin (1 mg/kg/day) significantly reduced lung viral loads, mitigated histopathology, and conferred full mortality protection. Furthermore, salinomycin synergized with tecovirimat, an approved viral egress inhibitor. In fixed-ratio combinations, salinomycin's EC50 dropped ∼4.0-fold (30.12 nM to 7.50 nM), and tecovirimat's ∼3.2-fold (11.96 nM to 3.73 nM). Bliss independence analysis confirmed strong synergy, with a maximum ΔBliss value of 16.97. Notably, the combination achieved 100% survival in a high-dose VACV-WR model, an effect unmatched by either monotherapy. Collectively, these preliminary in vitro and in vivo data identify salinomycin as a potential host-targeted anti-orthopoxvirus inhibitor, especially in combination with tecovirimat. Further comprehensive preclinical evaluations, including full toxicological profiling and validation in clinically relevant models, are needed to define its translational potential for MPXV and other orthopoxvirus infections.