Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC 50 = 1.4 μM), suramin sodium salt (IC 50 = 3.6 μM), NF 023 hydrate (IC 50 = 6.2 μM) and tyrphostin AG 538 (IC 50 = 3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.

This chapter describes two types of FRET-based fluorescence assays that can be used to identify and analyze compounds that inhibit the helicase encoded by the hepatitis C virus (HCV). Both assays use a fluorescently labeled DNA or RNA oligonucleotide to monitor helicase-catalyzed strand separation, and they differ from other real-time helicase assays in that they do not require the presence of other nucleic acids to trap the reaction products. The first assay is a molecular beacon-based helicase assay (MBHA) that monitors helicase-catalyzed displacement of a hairpin-forming oligonucleotide with a fluorescent moiety on one end and a quencher on the other. DNA-based MBHAs have been used extensively for high-throughput screening (HTS), but RNA-based MBHAs are typically less useful because of poor signal to background ratios. In the second assay discussed, the fluorophore and quencher are split between two hairpin-forming oligonucleotides annealed in tandem to a third oligonucleotide. This split beacon helicase assay can be used for HTS with either DNA or RNA oligonucleotides. These assays should be useful to the many labs searching for HCV helicase inhibitors in order to develop new HCV therapies that are still desperately needed.

Temporal variability of toxins produced by cyanobacteria in lakes is relatively unknown at time scales relevant to public health (i.e., hourly). In this study, a water quality monitoring buoy was outfitted with an automated water sampler taking preserved samples every 6 h for 68.75 days over a drinking water intake. A total of 251 samples were analyzed by tandem mass spectrometry for 21 cyanotoxin congeners in 5 classes producing 5020 data points. Microcystins (MCs) were the most abundant toxins measured (mean ± sd = 3.9 ± 3.3 μg/L) followed by cyanopeptolins (CPs) (1.1 ± 1.5 μg/L), anabaenopeptins (APs) (1.0 ± 0.6 μg/L), anatoxin-a (AT-A) (0.03 ± 0.06 μg/L), and microginin-690 (MG-690) (0.002 ± 0.01 μg/L). Advanced time series analyses uncovered patterns in cyanotoxin production. The velocity of cyanotoxin concentration varied from −0.7 to 0.9 μg/L/h with a maximum positive velocity just prior to peak toxin concentration during nonbloom periods. A backward-looking moving window of variance analysis detected major increases in cyanotoxin concentration and predicted the two greatest increases in MC. A wavelet analysis identified a significant (p < 0.01) 2.8–4.2 day periodicity in toxin concentration over a ∼25 day period during peak toxin production, which is partially explained by easterly wind velocity (R = −0.2, p < 0.05). Diversity in congener profiles was explored with principle component analysis showing that cyanotoxin dynamics followed a seasonal trajectory where toxin profiles were significantly clustered (ANOSIM R = 0.7, p < 0.05) on a daily basis. Variability in toxin profiles was strongly correlated with time (R = −0.8, p < 0.001) as well as the C:N ratio of the toxin pool (R = 0.17, p < 0.05). The methods employed here should be useful for uncovering patterns in cyanotoxin dynamics in other systems.