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Towards the development of norovirus antiviral drugs: zebrafish larvae as an innovative model system to study the replication of human norovirus

Boek - Dissertatie

Human noroviruses (HuNoVs) are single stranded, positive sense RNA viruses belonging to the Caliciviridae family. HuNoV infections result in vomiting and watery non-bloody diarrhea. Infections occur worldwide and are an important health issue that affects all age groups, resulting annually in 200,000 deaths. Infections occur via the fecal-oral route, by consumption of contaminated food or water and by person-to-person contact. HuNoV is very contagious and robust in the environment, therefore large outbreaks occur frequently mostly in health institutions, long-term care facilities and other semi-closed environments. The current therapy is merely supportive as there are still no antivirals to treat and/or prevent HuNoV infections. An anti-norovirus drug that could be used as a prophylactic or to treat HuNoV infections, is highly needed. Different steps in the viral replication cycle have been explored as antiviral targets (Chapter 1).Given the diversity of human viral pathogens that can cause an acute gastroenteritis, we explored the possibility for a syndrome-based treatment by studying the in vitro antiviral effect of 2'‑C‑nucleosides on rota-, noro- and sapovirus (Chapter 3). The most potent nucleoside analogue, 7-deaza-2'-C-methyladenosine (7DMA), was able to inhibit the replication of these viruses with an EC50 <5 µM. Mechanistically, we demonstrate that the 2'‑C‑methyl nucleoside analogues act by inhibiting the transcription of the rotavirus genome. In this chapter, we provide the first evidence that a single viral diarrhea-targeted treatment can be developed through a viral polymerase targeting small molecule.One of the main reasons for the lack of antiviral therapies against viral gastroenteritis was the lack of suitable cell culture systems and/or animal models. The HuNoV was not easily cultivated in vitro for a long time; therefore, most antiviral research is being performed using the mouse norovirus (MNV) or the HuNoV GI.1 replicon. Only recently, it was reported that HuNoV can replicate in a human B-cell line (BJAB) and in stem-cell-derived enteroids. We established a new small animal model to study HuNoV replication, using zebrafish larvae (Chapter 4). Both HuNoV GI and GII replicate to high titers in zebrafish larvae, when inoculated in the yolk at three days post fertilization. HuNoV replication peaks at day two post infection and is detectable for at least six days. HuNoV GII.4 could be passaged from larva to larva two consecutive times. By immunohistochemistry we could detect HuNoV antigens in cells of the hematopoietic lineage and the intestine. Moreover, the HuNoV replication could be inhibited by >2 log10 by simply adding a known norovirus inhibitor 2CMC to the swimming water of the HuNoV-infected zebrafish larvae. Overall, we were able to establish a simple and robust in vivo replication model that will be a major asset in the anti-norovirus drug discovery campaigns.The viral non-structural proteins constitute an attractive target for antiviral drug design.The first non-structural protein we explored was the norovirus protease (NS6). The protease is essential to cleave the polyprotein precursor into the individual mature non-structural proteins. Together with the team of Prof. Hong Liu, a class of peptidomimetic aldehydes was designed based on the chemical structure of rupintrivir, a known norovirus protease inhibitor (Chapter 5). We initially selected compound 4 (EC50 MNV: 0.71±0.24 μM, EC50 HuNoV GI.1: 1.21±0.58 μM). Compound 4 was able to efficiently clear the HuNoV GI.1 replicon from the host cells after two passages. After three months of selective pressure, a compound 4res GI.1 replicon variant was generated, resulting in a ~10-fold increase in EC50 for both compound 4 and rupintrivir against the compound 4res GI.1 replicon variant. The resistant variant carries a mutation (I109V) in a highly conserved region of the viral protease. By using a cell-based FRET-sensor approach, we confirmed that compound 4 inhibits not only the proteolytic activity of the GI.1 protease, but also of the GII and GV proteases. After a new round of derivatives were synthetized, compound 10d was found to be ~100-fold more potent. Finally, both compound 4 and compound 10d showed to be active in vivo by inhibiting the replication of HuNoV GII.4 in infected zebrafish larvae when the compounds were injected as a single dose in the pericardial cavity. Overall, we identified a new class of norovirus inhibitors with in vitro and in vivo activity and a very high barrier to resistance.The second non-structural protein we explored was the norovirus RNA-dependent RNA polymerase (RdRp), an essential protein for viral RNA replication. Together with the team of Prof. Romano Silvestri, ~1000 drug-like small molecules were designed and screened for antiviral activity against MNV (Chapter 6). The molecule 3-(3,5-dimethylphenyl)sulfonyl-5-chloroindole N‑(phenylmethanol-4-yl) carboxamide (RS5105) was identified as an interesting hit (EC50 MNV: 0.5±0.1 µM). A series of analogues was synthesized of which RS5111 had an improved potency/selectivity (EC50 MNV: 0.2±0.1 µM and EC50 HuNoV GI replicon: 1.2±0.6 µM). Time-of-drug-addition (TOA) studies revealed that RS5111 acts at a time point that coincides with the onset of viral RNA replication, although RS5111 did not directly inhibit the activity of the MNV RdRp. After six months of selective pressure, two MNV RS5111res variants were independently selected and both were found to harbor one mutation in VPg and three mutations in the RdRp. After reverse engineering S131T and Y154F as single mutations into the MNV backbone, we did not find a markedly RS5111res phenotype. Insertion of the other single mutations or combination of mutations is ongoing. The antiviral effect of RS5111 was assessed in HuNoV-infected zebrafish larvae. By simply adding RS5111 to the swimming water 0.5-1 log10 fewer HuNoV GII.6 RNA copies were detected. We here present a class of novel norovirus inhibitors with a high barrier to resistance and antiviral activity in vitro and in vivo against the most relevant HuNoV genogroups.In conclusion, we here provided evidence that a syndrome related broad-spectrum antiviral drug may be feasible. Moreover, we were able to establish zebrafish larvae as a robust in vivo model to study HuNoV replication. Finally, in collaboration with medicinal chemists we were able to identify and characterize novel anti-norovirus molecules, targeting the highly conserved viral protease or polymerase.
Jaar van publicatie:2020