Inhibition of human Norovirus replication by a novel class of oindolylarylsulfones in vitro and in zebrafish larvae

Human noroviruses are the leading cause of viral gastroenteritis outbreaks worldwide. Potent and safe antiviral therapy is urgently needed to reduce the burden of norovirus disease in vulnerable populations and as prophylaxis, i.e. to reduce transmission during an outbreak. After screening ~1000 compounds from the Drug Design and Synthesis Center at Sapienza University against the GV mouse norovirus (MNV) in infected RAW 264.7 cells, the molecule 3-(3,5-dimethylphenyl)sulfonyl-5-chloroindole N-(pheylmethanol-4-yl)carboxamide (RS5105) had anti-norovirus activity (EC50 0.53±0.07 mM). After structural modifications, RS5111 was the most potent derivative with EC50 values of 0.16 ± 0.06 mM against MNV and 1.2 ± 0.6 mM against the human norovirus (HuNoV) GI replicon. Time-of-drug-addition studies revealed that RS5111 acted at the onset of viral replication, like the nucleosides 2’-C-methylcytidine and favipiravir. Although RS5111 did not directly inhibit the activity of the MNV RNA-dependent RNA polymerase (RdRp), it rendered the catalytic site less efficient, thus could act as an allosteric inhibitor of the RdRp. After six months of selective pressure, two RS5111-resistant variants were independently selected and both harbor one mutation in VPg (which acts as a primer at the 5’-end of the viral genome) and three mutations in the RdRp. Susceptibility of the reverse engineered mutants to RS5111-treatment is being evaluated in CD300lf-expressing Huh-7-Lunet cells. Finally, RS5111 efficiently reduced HuNoV GII viral RNA levels by 0.5-1 log10 in HuNoV-infected zebrafish larvae water. Overall, we here present a class of novel norovirus inhibitors, active in vitro and in vivo against the most relevant HuNoV genogroups, with a high barrier to resistance.