Original article
Novel triacsin C analogs as potential antivirals against rotavirus infections

https://doi.org/10.1016/j.ejmech.2012.02.010Get rights and content

Abstract

Recently our group has demonstrated that cellular triglyceride (TG) levels play an important role in rotavirus replication. In this study, we further examined the roles of the key enzymes for TG synthesis (lipogenesis) in the replication of rotaviruses by using inhibitors of fatty acid synthase, long chain fatty acid acyl-CoA synthetase (ACSL), and diacylglycerol acyltransferase and acyl-CoA:cholesterol acyltransferase in association with lipid droplets of which TG is a major component. Triacsin C, a natural ACSL inhibitor from Streptomyces aureofaciens, was found to be highly effective against rotavirus replication. Thus, novel triacsin C analogs were synthesized and evaluated for their efficacies against the replication of rotaviruses in cells. Many of the analogs significantly reduced rotavirus replication, and one analog (1e) was highly effective at a nanomolar concentration range (ED50 0.1 μM) with a high therapeutic index in cell culture. Our results suggest a crucial role of lipid metabolism in rotavirus replication, and triacsin C and/or its analogs as potential therapeutic options for rotavirus infections.

Graphical abstract

Novel triacsin C analogs were synthesized and found to be highly effective against rotavirus replication at low micromolar or nanomolar concentration ranges with high therapeutic indices in cell culture.

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Highlights

► Rotaviral diarrheal remained as one of the most important causes for mortality worldwide. ► There is no specific antiviral drug for rotavirus infection. ► Triacsin C, a fungal metabolite, was found to be highly effective against rotavirus replication. ► One triacsin C analog, TC20, was highly effective in a nanomolar concentration range with a high therapeutic index. ► Triacsin C and/or its analogs are potential therapeutic options for rotavirus infections.

Introduction

Rotaviruses are non-enveloped, icosahedral viruses with an 11-segment double-stranded RNA genome [1]. The capsid of rotavirus is composed of the outer capsid proteins, VP4 and VP7, and the major inner capsid protein, VP6. Rotaviruses are divided into 7 (A to G) antigenically distinct serogroups based on VP6. Among them, group A rotaviruses are the leading cause of severe gastroenteritis in infants and children worldwide, associated with over 500,000 deaths in children younger than 5 years of age each year, although attenuated live vaccines are available [1], [2], [3]. Majority of rotavirus infection-associated mortality occurs in the developing countries. Nonetheless, nearly 1 in 80 children is hospitalized with rotavirus gastroenteritis by 5 years of age in the US [1], [2], [3]. Since there are no specific antiviral agents for rotavirus infection, the treatment options for rotavirus infection are limited to providing oral rehydration solution to restore and maintain hydration until the infection resolves [4]. However, the development of rotavirus antivirals to reduce the severity of diseases and duration of rotavirus-related hospitalization has been impeded by limited information on the therapeutic targets for rotavirus infections.

Previously it was shown that disruption of lipid rafts and/or lipid droplets decrease infectious rotaviruses by inhibition of rotavirus morphogenesis [5], [6]. Lipid droplets are cellular organelles for storage of neutral fats such as TG and cholesterol ester, and play a crucial role in regulating cellular lipid levels. Lipid rafts are microdomains in cell membrane enriched in cholesterol, glycosphingolipids, and proteins. These structures are found important for infectious virus particle formation of human hepatitis C virus (HCV) [7], [8] and dengue virus [9], suggesting the importance of lipid homeostasis in virus replication. Recently our group has demonstrated that rotavirus replication induced an increase in the TG levels in cells, and suppression of increase in TG levels by the farnesoid X receptor (FXR) agonists significantly inhibited rotavirus replication [10].

Lipogenesis is the process of producing fats from acetyl-CoA, which is then stored as an energy source. During lipogenesis, fatty acid (FA) is synthesized (de novo synthesis) from acetyl-CoA, and subsequently esterified with glycerol to form TG. Numerous enzymes participate in lipogenesis, which include fatty acid synthase (FASN), long chain fatty acid acyl-CoA synthetase (ACSL) 1–6, and diacylglycerol acyltransferase (DGAT) 1/2 and Acyl-CoA:cholesterol acyltransferase (ACAT)1/2 [11], [12], [13], [14], [15]. Lipolysis is the reverse pathway of lipogenesis to generate acetyl-CoA and energy from TG in which process multiple enzymes including lipases are involved. In this study, we found that the commercially available inhibitors for FASN, ACSL, DGAT and ACAT significantly reduced the replication of rotaviruses in vitro. Since triacsin C, a fungal metabolite from Streptomyces aureofaciens, is an ACSL inhibitor and showed the most potent inhibition on rotavirus replication, we synthesized its analogs and examined their antiviral effects against rotavirus. The synthetic sequence is straightforward and efficient (Scheme 1, Scheme 2), which would provide flexibility for further optimization. Among the triacsin C and its analogs, 1e is the most potent against rotavirus replication with the effective dose that reduce 50% of the virus replication (ED50) of 0.1 μM. Our results suggest that lipogenic enzymes may represent potential therapeutic targets for rotavirus infection, and triacsin C analogs may be useful as therapeutic options for rotavirus infection.

Section snippets

Biochemical studies

The effects of commercially available inhibitors for lipogenesis as well as newly synthesized triacsin C analogs (described below) were examined against in rotavirus replication using SA11 rotavirus strain in MA104 cells. Various chemical inhibitors including FASN inhibitors (cerulenin and C75), ACSL inhibitors (triacsin C and troglitazone), DGAT inhibitors (A922500 and betulinic acid), and ACAT inhibitors (CI-976, hexadecylamino-p-amino benzoic acid [PHB]) (Fig. 1) were obtained from

Chemistry

Various triascin C analogs were synthesized from (E,E,E)-2,4,7-undecatrienal (1), which was prepared by a modification of the reported procedure [16], [17] (Scheme 1). Hence, bromination of (E)-2-hexen-1-ol (2) with phosphorus tribromide in dichloromethane followed by a displacement reaction with 3-(tetrahydropyranyloxy)propynyl magnesium bromide and a catalytic amount of copper cyanide afforded enyne 3. Removal of the THP protecting group of 3 with oxalic acid in refluxing methanol followed by

Results and discussion

All inhibitors, cerulenin, C75, triacsin C, troglitazone, A922500, betulinic acid, CI-976 and PHB, significantly reduced the replication of SA11 rotavirus in cells with ED50 values of 0.2–28.5 μM (Table 1). The ED50 values of cerulenin, C75, A922500, betulinic acid and PHB were similar at 11.3–28.5 μM, while troglitazone and CI-976 had lower ED50 values of 5.8 μM and 4.3 μM, respectively (Table 1). The TD50 of the inhibitors in MA104 cells varied at 8.5–85.4 μM (Table 1). Among the tested commercial

General

NMR spectra were obtained from a 400-MHz spectrometer (Varian Inc.), in CDCl3, unless otherwise indicated, and reported in ppm. Mass spectra were taken from an API 2000-triple quadrupole ESI-MS/MS mass spectrometer (from Applied Biosystems). Chemicals and solvents including chloromethanesulfonyl chloride, 3-chloropropane-1-sulfonyl chloride, and propane-2-sulfonyl chloride were purchased from Fisher Scientific and Aldrich Chemical Co. A modification of the reported procedure [16], [17] was used

Cells, antisera and reagents

The MA104 cell line was maintained in minimum essential medium (MEM) containing 5% fetal bovine serum and antibiotics (penicillin and streptomycin). Antibodies specific to rotavirus VP6 or β-actin were obtained from Santa Cruz Biotechnology Inc. Rotavirus strains including Wa (human G1) and SA11 (primate G3) were obtained from American Type Culture Collection (ATCC, Manassas, VA).

Nonspecific cytotoxic effects

Confluent MA104 grown in 24-well plates were treated with various concentrations of each compound for 24 h. Cell

Acknowledgments

This work was supported by NIH U01AI081891. The authors thank Samira Najm for technical assistance. SH would like to thank the International Cooperation Program for Excellent Lectures of Shandong Provincial Education Department, P. R. China.

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