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. 2019 May 30;11(6):496.
doi: 10.3390/v11060496.

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

Daniel Enosi Tuipulotu  1 , Tulio M Fumian  2   3 , Natalie E Netzler  4 , Jason M Mackenzie  5 , Peter A White  6
Affiliations

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

Daniel Enosi Tuipulotu et al. Viruses. .

Abstract

The widespread nature of calicivirus infections globally has a substantial impact on the health and well-being of humans and animals alike. Currently, the only vaccines approved against caliciviruses are for feline and rabbit-specific members of this group, and thus there is a growing effort towards the development of broad-spectrum antivirals for calicivirus infections. In this study, we evaluated the antiviral activity of the adenosine analogue NITD008 in vitro using three calicivirus model systems namely; feline calicivirus (FCV), murine norovirus (MNV), and the human norovirus replicon. We show that the nucleoside analogue (NA), NITD008, has limited toxicity and inhibits calicivirus replication in all three model systems with EC50 values of 0.94 μM, 0.91 µM, and 0.21 µM for MNV, FCV, and the Norwalk replicon, respectively. NITD008 has a similar level of potency to the most well-studied NA 2'-C-methylcytidine in vitro. Significantly, we also show that continual NITD008 treatment effectively cleared the Norwalk replicon from cells and treatment with 5 µM NITD008 was sufficient to completely prevent rebound. Given the potency displayed by NITD008 against several caliciviruses, we propose that this compound should be interrogated further to assess its effectiveness in vivo. In summary, we have added a potent NA to the current suite of antiviral compounds and provide a NA scaffold that could be further modified for therapeutic use against calicivirus infections.

Keywords: antivirals; caliciviruses; norovirus; nucleoside analogue; polymerase inhibitor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
NITD008 is a potent inhibitor of FCV and MNV infection. The antiviral potential of NITD008 was explored against two animal caliciviruses closely related to human norovirus. Panels with black data points/bars are related to MNV and involve 48 h incubations whereas panels with red data points/bars are related to FCV and involve 24 h incubations. (A,B) Infectious virus levels following NITD008 treatment (0.01–10 μM) were quantified by plaque reduction assay. The percentage of maximal viral infectivity is relative to DMSO controls and is plotted as a dose-response curve. The cytotoxic effects of NITD008 (0.2–120 µM) against (C) RAW264.7 and (D) CRFK cell lines, permissive for MNV and FCV respectively, were quantified after treatment using the fluorescent CellTiter-Blue assay. The percentage cell viability is relative to DMSO controls. (E,F) Viral genome levels following NITD008 treatment (1 µM and 5 µM) were quantified by qRT-PCR. The nucleoside analogue 2CMC (10 µM) and DMSO were used as positive and negative controls respectively. Triplicate data from at least two independent experiments are presented for each panel. Error bars represent the mean ± standard deviations.
Figure 2
Figure 2
The interaction between the NITD008 and 2CMC is strongly antagonistic. The combinational effects of 2CMC (0–3 µM) and NITD008 (0–1 µM) against MNV infection were tested over a range of concentrations using a plaque reduction assay. The percentage of inhibition observed across all drug combinations were tabled into a dose-response matrix and analyzed for synergy and antagonism using the Lowe-Additivity model on SynergyFinder. The synergy scores obtained for each drug combination across the entire matrix are presented as a heatmap. Synergistic (positive δ-score) and antagonistic (negative δ-score) interactions are represented in red and green respectively, and no observable effect (δ = 0) is represented as white.
Figure 3
Figure 3
NITD008 is a potent inhibitor of human norovirus replication. (A) Norwalk replicon levels were measured using RT-qPCR following NITD008 treatment (0–20 µM) for 48 h. The norovirus RdRp sequence was amplified for the replicon and β-actin was amplified as the normalizer for the ΔΔCT method (where CT is threshold cycle). (B) The cytotoxic effects of NITD008 (0.2–120 µM) against the hepatoma cell line (Huh7) were assessed after 48 h using the fluorescent CellTitre-Blue assay. The percentage cell viability is relative to DMSO controls. Triplicate data from two independent experiments are presented for each panel. Error bars represent the mean ± standard deviations.
Figure 4
Figure 4
NITD008 effectively clears the Norwalk replicon from replicon-bearing cells. (A) Schematic overview of the clearance-rebound assays performed against the Norwalk replicon. The light green panel represents the clearance phase in which replicon-bearing cells were passaged every 4 days in the presence of DMSO or NITD008 at 1, 5, and 10 µM (~ 5, 25, 50× the EC50 value). The dark green panel represents the rebound phase in which NITD008 is removed from the media and G418 is used to selected for replicon-bearing cells. (B) Clearance of the Norwalk replicon from cells was determined by RT-qPCR. Norwalk replicon levels following NITD008 treatment at every passage are plotted relative to the corresponding DMSO controls. The dotted line represents the qPCR limit of detection. (C) Rebound of the Norwalk replicon was assessed by performing colony formation assays on cells passaged in the presence of NITD008 for either 8 or 12 days. Colonies were fixed and stained with crystal violet before visualization. Triplicate data from at least two independent experiments are presented for each panel. Error bars represent the mean ± standard deviations.
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