In silico screening for human norovirus antivirals reveals a novel non-nucleoside inhibitor of the viral polymerase

Abstract

Human norovirus causes approximately 219,000 deaths annually, yet there are currently no antivirals available. A virtual screening of commercially available drug-like compounds (~300,000) was performed on the suramin and PPNDS binding-sites of the norovirus RNA-dependent RNA polymerase (RdRp). Selected compounds (n = 62) were examined for inhibition of norovirus RdRp activity using an in vitro transcription assay. Eight candidates demonstrated RdRp inhibition (>25% inhibition at 10 µM), which was confirmed using a gel-shift RdRp assay for two of them. The two molecules were identified as initial hits and selected for structure-activity relationship studies, which resulted in the synthesis of novel compounds that were examined for inhibitory activity. Five compounds inhibited human norovirus RdRp activity (>50% at 10 µM), with the best candidate, 54, demonstrating an IC50 of 5.6 µM against the RdRp and a CC50 of 62.8 µM. Combinational treatment of 54 and the known RdRp site-B inhibitor PPNDS revealed antagonism, indicating that 54 binds in the same binding pocket. Two RdRps with mutations (Q414A and R419A) previously shown to be critical for the binding of site-B compounds had no effect on inhibition, suggesting 54 interacts with distinct site-B residues. This study revealed the novel scaffold 54 for further development as a norovirus antiviral.

The Adenosine Analogue NITD008 has Potent Antiviral Activity against Human and Animal Caliciviruses

“…Compound-induced toxicity of mammalian cells was assessed using CellTitre Blue (CTB) reagent (Promega, Madison, WI, USA), as previously described [19,42,43,44,45]. Briefly, CRFK (3.5 × 10 4 cells/well), RAW264.7 (2 × 10 4 cells/well) or Huh7 (5 × 10 3 cells/well) were seeded into 96-well plates and incubated overnight.…”

“…The effect of NITD008 on FCV and MNV infection was assessed by plaque reduction assays, as previously described [19,42,43,44,45]. Here, CRFK (8 × 10 5 ) or RAW264.7 (2 × 10 6 ) cells were seeded into each well of 6-well plates and incubated overnight for attachment.…”

Norovirus antivirals: Where are we now?

“…To further exploit the broad‐spectrum RdRp binding pocket Site B, a high‐throughput in silico screen of approximately 300 000 commercially available compounds was performed to identify ligands that bind to Site B of the human norovirus and MNV RdRps . Sixty‐two compounds were selected from the screen and assessed for inhibition of the human norovirus RdRp, revealing a hit (compound 11 ) with an IC 50 of 5.0 µM.…”

“…Synthesis of derivatives of this compound produced a novel candidate (compound 54 [2.2.10]) with IC 50 values of 5.6 µM (human norovirus RdRp) and 12.1 µM (MNV RdRp). Further in silico molecular modeling and biological antagonism studies with PPNDS suggested the binding site of compound 54 was indeed within the targeted broad‐spectrum Site B pocket, however, it was relatively ineffective against MNV in cell culture, with an EC 50 of more than 50 µM …”

“…For example, researchers have used in vitro enzyme activity assays, such as viral polymerase assays and protease assays (Figure ) to screen compounds for antiviral activity in a high‐throughput format . In addition, X‐ray crystallography and in silico modeling can be used to examine ligand and viral protein interactions, to further elucidate antiviral mechanisms …”