Elsevier

Fish & Shellfish Immunology

Volume 81, October 2018, Pages 57-66
Fish & Shellfish Immunology

Full length article
Synthesis and antiviral activity of a new coumarin derivative against spring viraemia of carp virus

https://doi.org/10.1016/j.fsi.2018.07.005Get rights and content

Highlights

  • A coumarin derivative, 7-(4-benzimidazole-butoxy)-coumarin (BBC) was designed and synthesized against SVCV infection.

  • BBC effectively inhibited SVCV replication in host cells and protected cells from morphological damage.

  • BBC leaded to a prolonged survival of zebrafish.

  • BBC can keep an oxidative balance in infected zebrafish and augment interferon response.

Abstract

Coumarin as a lead structure have received a considerable attention in the last three decades for the discovery of antiviral agents. Our previous study indicated that imidazole coumarins possessed antiviral activities against SVCV. Based on the structure-activity relationship in that study, a new imidazole coumarin derivative, 7-(4-benzimidazole-butoxy)-coumarin (BBC), was designed, synthesized and its anti-SVCV activity was evaluated. By comparing inhibitory concentration at half-maximal activity (IC50), we found that BBC (IC50 = 0.56 mg/L) possessed a higher antiviral activity than those imidazole coumarins in our previous study. Besides, BBC can significantly inhibit cell death and reduce cellular morphological damage induced by SVCV. Our further data indicated that intraperitoneal injection of BBC increased the survival rate of zebrafish by 17.5%, decreased viral titer in fish body and inhibited SVCV glycoprotein expression in kidney and spleen. In uninfected zebrafish, the expression levels of ifnγ, ifnφ1, ifnφ2 and rig1 genes were up-regulated after BBC treatment, which indicated that BBC could activate interferon response. In addition, data of the antioxidant enzymes activities and results of the antioxidant enzymes-related genes expressions suggested BBC could reduce SVCV-induced oxidative damage in infected zebrafish. Altogether, BBC is expected to be a therapeutic agent against SVCV infection in the field of aquaculture.

Introduction

As a notable animal disease, spring viraemia of carp virus (SVCV) infection has been an epidemic in Europe, Asia, South America and North America [1,2], and it is regarded as an acute hemorrhagic and contagious disease [3]. Being considered as one of the nine piscine viruses recognized by the International Office of Epizootics, SVCV usually cause high mortality in the cultivation of cyprinid fishes [[3], [4], [5], [6]]. Because it is difficult to eradicate the virus from affected ponds, all aquatic life should be destructed once SVCV infection is established [4]. Regrettably, there is no effective treatment to protect cyprinids from SVCV infection, making it an important public health threat to aquaculture industry. Therefore, it is imperative to develop a safe and effective antiviral strategy to combat highly pathogenic SVCV outbreaks [[6], [7], [8]].

Natural products have been used to treat viral diseases for thousands of years and play an increasingly critical role in drug discovery and development [9,10]. Natural product coumarin, known as benzopyran-2-ones, forms an elite class of natural compounds that possess promising therapeutic perspectives [11]. Based on structural diversity, a variety of coumarin based natural and synthetic derivatives display a wide range of biological activities such as anticancer, antioxidant, anti-inflammatory and antimicrobial efficacies [[11], [12], [13], [14], [15], [16], [17]]. In addition, some investigators have focused on the antiviral activity of coumarins. For instance, it has been found that a series of coumarin-triazine hybrids possess antiviral activities against different strains of Human immunodeficiency viral (HIV) [18]; meanwhile two coumarin-based benzimidazole derivatives were considered as potential anti-HCV (Hepatitis C virus) compounds [19]. Our previous study suggested that imidazole coumarin derivatives with four carbon atoms length of linker were effective to the treatment of SVCV infection in epithelioma papulosum cyprinid (EPC) cells [20].

To obtain a compound with higher anti-SVCV activity, a new benzimidazole coumarin derivative, 7-(4-benzimidazole-butoxy)-coumarin (BBC), was designed, synthetized and identified based on structure-activity relationship in this study. As expected, by real-time quantitative PCR (RT-qPCR), we found that BBC showed higher anti-SVCV activity in EPC cells compared with our previously reported imidazole coumarins [20]. Secondary assays including cytopathic effects (CPE) reduction assays, microtubule structure and nucleus damage test were used to confirm the anti-SVCV activity of BBC in EPC cells. Furthermore, the anti-SVCV activity of BBC in zebrafish was evaluated by RT-qPCR, titer assay and survival rate assay. In order to explore how BBC protected zebrafish from SVCV infection, the effects of BBC on interferon (IFN) response and antioxidant enzymes activities were also investigated.

Section snippets

Cell, virus and zebrafish husbandry

The fish cell line EPC was kindly provided by Prof. Ling-Bing Zeng (Yangtze River Fisheries Research Institute, Wuhan, Hubei, China). Cells were maintained at 25 °C in 5% CO2 atmosphere in Medium 199 (Hyclone, USA) cell culture containing 10% fetal bovine serum (FBS) (ZETA LIFE, USA), streptomycin 100 μg/mL and penicillin 100 U/mL. The SVCV (strain 0504, isolated from common carp in China, kindly provided by Prof. Qiang Li, Key Laboratory of Mariculture, Agriculture Ministry, PRC, Dalian Ocean

Antiviral activity of BBC against SVCV infection in EPC cells

By analyzing the data of 1H NMR (500 MHz, MeOD) and 13C NMR (126 MHz, MeOD), compound 3 was identified as 7-(4-benzimidazole-butoxy)-coumarin (BBC) (Fig. 1B). CCK-8 data on cell viability determined that BBC had little cytotoxic effect on EPC cells under the examined doses (0.04–10 mg/L) (Fig. S1). To determine the antiviral activity of BBC on SVCV replication in EPC cells, the expression of SVCV glycoprotein (G), phosphoprotein (P) and nucleoprotein (N) was analyzed by RT-qPCR in a seven-point

Discussion

Among a wide range of biological activities, coumarin compounds have shown a large potential to be used as a novel member of antiviral drugs [33,34]. Toddacoumaquinone, a coumarin-naphthoquinone dimer, was explored with antiviral activity against HSV-1 and HSV-2 [35]. Additionally, Lee et al. found that two sesquiterpene coumarins possessed antiviral activity against H1N1 [36]. In our previous study, two imidazole coumarins (7-(3-benzoimidazol)-coumarin) and 7-(4-(4-methyl-imidazole)-coumarin)

Acknowledgement

This work was supported by the Shaanxi Science and Technology Co-ordination Innovation Engineering Project (2015KTTSNY01-02).

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