New fluoxetine analogues as anti-enterovirus agents targeting 2C protein

There are currently no antiviral drugs available to treat or prevent life-threatening human non-poliovirus enterovirus infections, such as those caused by CV-B3, EV-A71 or EV-D68. Our aim is to develop novel inhibitors that target the non-structural ATPase/Helicase 2C protein, which is involved in the RNA replication process that is essential for enterovirus replication, among other functions. In this study, we describe the optimization of (S)-fluoxetine, a promising hit identified through drug repurposing that binds to an allosteric site on the CV-B3 2C ATPase domain. Our optimization process was guided by rational design, X-ray crystallographic structures, computational docking, and validation by enzyme and cell-based assays, leading to several new inhibitors, among which compound 53 (CV-B3 EC₅₀ = 0.5 μM and EV-D68 EC₅₀ = 0.4 μM), a novel anti-enterovirus with higher selectivity indexes than (S)-fluoxetine.