Structure-based inhibition of Norovirus RNA-dependent RNA polymerases.

Caliciviridae are RNA viruses with a single-stranded, positively oriented polyadenylated genome, responsible for a broad spectrum of diseases such as acute gastroenteritis in humans. Recently, analyses on the structures and functionalities of the RNA-dependent RNA polymerase (RdRp) from several Caliciviruses have been reported. The RdRp is predicted to play a key role in genome replication, as well as in synthesis and amplification of additional subgenomic RNA. Starting from the crystal structures of human Norovirus (hNV) RdRp, we performed an in silico docking search to identify synthetic compounds with predicted high affinity for the enzyme active site. The best-ranked candidates were tested in vitro on murine Norovirus (MNV) and hNV RdRps to assay their inhibition of RNA polymerization. The results of such combined computational and experimental screening approach led to the identification of two high-potency inhibitors: Suramin and NF023, both symmetric divalent molecules hosting two naphthalene-trisulfonic acid heads. We report here the crystal structure of MNV RdRp alone and in the presence of the two identified inhibitors. Both inhibitory molecules occupy the same RdRp site, between the fingers and thumb domains, with one inhibitor head close to residue 42 and to the protein active site. To further validate the structural results, we mutated Trp42 to Ala in MNV RdRp and the corresponding residue (i.e., Tyr41 to Ala) in hNV RdRp. Both NF023 and Suramin displayed reduced inhibitory potency versus the mutated hNV RdRp, thus hinting at a conserved inhibitor binding mode in the two polymerases.