Optimization of the antiviral spectrum of cyclophilin inhibitors targeting respiratory viruses.

We evaluated the antiviral activity of a library of 36 urea-based small-molecule cyclophilin inhibitors (SMCypIs) against human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV). Structure-spectrum analysis revealed virus-specific structural requirements for antiviral efficacy. By combining the key determinants identified for each virus, we rationally designed optimized dual-acting derivatives. Among these, the optimized compound F832/33 emerged as a potent, broad-spectrum antiviral agent with nanomolar activity against both hRSV and hMPV, submicromolar inhibition of cyclophilin (Cyp) peptidyl-prolyl isomerase (PPIase) activity, and no detectable cytotoxicity. Interestingly, no direct correlation was observed between Cyp PPIase inhibition and antiviral potency, suggesting that Cyp engagement rather than enzymatic inhibition per se is required for antiviral activity. Competition assays with alisporivir confirmed that the antiviral effects of SMCypIs depend on their specific interaction with Cyp. Beyond its dual efficacy, F832/33 displayed strong antiviral activity against additional clinically relevant respiratory pathogens, including parainfluenza virus type 3, enterovirus D68, and both ancestral and Omicron severe acute respiratory syndrome coronavirus 2 strains, in immortalized cell lines and fully differentiated human nasal epithelial cells, a physiologically relevant model of respiratory infection. Collectively, our results demonstrate that structure-activity relationship-guided optimization can be successfully applied to expand the antiviral spectrum of SMCypIs, positioning F832/33 as a promising host-targeted broad-spectrum antiviral candidate. Further in vivo studies are warranted to evaluate its therapeutic potential and clarify its precise mechanism of action.