Structure Based Inhibition of the Calicivirus RNA-Dependent RNA-Polymerase

Abstract

Caliciviridae are RNA viruses with single stranded positive-oriented genome causing a broad spectrum of diseases such as acute gastroenteritis in humans. The structures of the RNA-dependent RNA-polymerase (RdRp) of several Caliciviruses have been reported. The RdRp is predicted to play a key role in genome replication, as well as in the synthesis and amplification of subgenomic RNA.Starting from crystal structures of human (hNV) and Murine Norwalk virus (MNV) RdRp, we performed an in silico docking search to identify commercially available compounds with predicted high affinity for the enzyme active site. The best candidates were tested in vitro to assay their effective inhibition of MNV and hNV RdRp. The results of such combined computational and experimental screening approach led to the identification of two high-potency inhibitors: EM01 and EM02. The crystal structure of MNV in the presence of the two inhibitors showed a common binding site close to the protein active site where the addition of new nucleotides to the nascent RNA occurs. From such structures we could identify the main residues involved in inhibitor binding. We inserted a point mutation in one of these key residues showing the reduction of inhibitory potency in both MNV and hNV RdRps. Finally, we identified a common moiety present in the two inhibitors likely carrying most of the inhibitory power. Such fragment can be elongated/modified to enhance its druglikeness in order to develop novel anti-viral drugs.