Initial screen for inhibition of human norovirus Sydney 2012 RdRp activity. The effects of 62 compounds were examined against norovirus RdRp activity using a fluorescent activity assay at 10 µM (A) and 100 µM (B). Five compounds that exhibited more than 25% inhibition at 10 µM and 50% inhibition at 100 µM (red bars) and three soluble compounds that exhibited more than 25% inhibition at both concentrations (green bars) were chosen for further assessment with a counter-screen gel-shift RdRp assay. The positive control (blue bars) is a previously reported norovirus NNI RdRp inhibitor NIC02 (10 µM in (A) and 100 µM in (B)14. The mean values of triplicate datasets with standard deviation are shown. (C) Structure of the eight most potent compounds with 11 and 13 highlighted. (D) A counter-screen gel-shift assay was used to confirm norovirus RdRp inhibitory activity of hit compounds. The eight compounds were examined for inhibition of primed elongation activity. PE44-NoV RNA templates (32 nucleotides) were extended (44 nucleotides) by the RdRp in the absence of any test compounds (0.5% DMSO [vol/vol] negative control) or with test compounds at a fixed concentration of 100 µM. The nucleoside analogue 3′-deoxyguanosine-5′-triphosphate is used as a positive control (10 µM) to demonstrate complete inhibition, and no RdRp was used as a negative control. Full-length gels are presented in Supplementary Fig. S2.