Screening of a library of FDA-approved drugs identifies several enterovirus replicaton inhibitors that target viral protein 2C

Enteroviruses (EV) represent many important pathogens of humans. Unfortunately, no antiviral compounds currently exist to treat infections with these viruses. We screened the Prestwick Chemical Library®, a library of approved drugs, for inhibitors of coxsackievirus B3 and identified pirlindole as potent novel inhibitor and confirmed the inhibitory action of dibucaine, zuclopenthixol, fluoxetine and formoterol. Upon testing viruses of several EV species, we found that dibucaine and pirlindole inhibited EV-B and EV-D, and that dibucaine also inhibited EV-A, but none of them inhibited EV-C or rhinoviruses (RV). In contrast, formoterol inhibited all enteroviruses and rhinoviruses tested. All compounds acted through inhibition of genome replication. Mutations in the coding sequence of the CVB3 2C protein conferred resistance to dibucaine, pirlindole and zuclopenthixol - but not formoterol - suggesting that 2C is the target for this set of compounds. Importantly, dibucaine bound to CVB3 protein 2C in vitro whereas binding to a 2C protein carrying the resistance mutations was reduced, providing an explanation of how resistance is acquired.