[1,2,4]Triazolo[1,5-a]pyrimidine derivative (Mol-5) is a new NS5-RdRp inhibitor of DENV2 proliferation and DENV2-induced inflammation.

Dengue fever is an acute infectious disease caused by dengue virus (DENV) and transmitted by Aedes mosquitoes. There is no effective vaccine or antiviral drug available to date to prevent or treat dengue disease. Recently, RNA-dependent RNA polymerase (RdRp), a class of polymerases involved in the synthesis of complementary RNA strands using single-stranded RNA, has been proposed as a promising drug target. Hence, we screened new molecules against DENV RdRp using our previously constructed virtual screening method. Mol-5, [1,2,4]triazolo[1,5-a]pyrimidine derivative, was screened out from an antiviral compound library (~8000 molecules). Using biophysical methods, we confirmed the direct interactions between mol-5 and purified DENV RdRp protein. In luciferase assay, mol-5 inhibited NS5-RdRp activity with an IC50 value of 1.28 ± 0.2 µM. In the cell-based cytopathic effect (CPE) assay, mol-5 inhibited DENV2 infectivity with an EC50 value of 4.5 ± 0.08 µM. Mol-5 also potently inhibited DENV2 RNA replication as observed in immunofluorescence assay and qRT-PCR. Both the viral structural (E) and non-structural (NS1) proteins of DENV2 were dose-dependently decreased by treatment with mol-5 (2.5-10 µM). Mol-5 treatment suppressed DENV2-induced inflammation in host cells, but had no direct effect on host defense (JAK/STAT-signaling pathway). These results demonstrate that mol-5 could be a novel RdRp inhibitor amenable for further research and development.

Q63, a novel DENV2 RdRp non-nucleoside inhibitor, inhibited DENV2 replication and infection.

Dengue virus (DENV) annually infects 400 million people worldwide. Unfortunately, there is lack of widely protective vaccine or drugs against DENV. The viral RNA-dependent RNA polymerase (RdRp) of NS5 protein is highly conserved among different DENV subtypes, thus presenting itself as an attractive target for drug design. In the current research, SPRi was performed to screen compounds against DENV2 RdRp and 5(1H)-Quinazolinone,2-(4-bromophenyl)-2,3,4,6,7,8-hexahydro-7,7-dimethyl-1,3-diphenyl (Q63) was successfully screened out with a KD of 0.9 µM. Then, ITC and molecular docking assay was performed to access the binding mechanism between Q63 and DENV2 RdRp. Meanwhile, Q63 also decreased the intermediate dsRNA production, which was the product of RdRp. Further the antiviral effects of Q63 were evaluated on mosquito C6/36 cells and mammalian BHK-21 cells. Q63 reduced CPE and cell toxicity effect after DENV2 infection on C6/36 and BHK-21 cells, with an EC50 of 2.08 µM. Time of addition assay revealed that Q63 affected the early genome RNA replication stage, including genome RNA replication. In addition, Q63 down-regulated STAT1 phosphorylation, ISG15 and ISG54 after DENV2 infection. In summary, Q63 was found to be a novel RdRp non-nucleoside inhibitor and a potential lead compound for coping with DENV infectious disease in the future.

Tatanan A from the Acorus calamus L. root inhibited dengue virus proliferation and infections.

BACKGROUND: Acorus calamus l. (Acoraceae) is a well-known traditional Chinese medicinal plant, whose root are historically mainly used to treat neurodegenerative diseases, and for cholera treatment. This datum strongly indicates the antimicrobial activity of A. calamus. PURPOSE: Our goal is to find the active constituents of A. calamus to treat dengue virus (DENV) infections, and to study the effects and mechanisms of these active substances. METHODS: The root of A. calamus was extracted by ethanol. Mosquito larva C6/36 cells were used for DENV2 replication and transfection host. Mouse kidney fibroblast cells (BHK-21) were used as a host cell to study the infection ability of the virus. DENV2-induced cytopathic effect (CPE) and plaque assay were used to evaluate the inhibitory effect of A. calamus extracts on DENV2 infectivity inhibition. The levels of E and NS1 protein expression were measured by real-time PCR and western blot assays. RESULTS: 12 compounds were isolated from ethanol extract of A. calamus root, tatanan A showed the best anti-DENV ability among these 12 compounds, which significantly alleviated DENV2-induced CPE and cytotoxicity effects, with an EC50 of 3.9 µM. In addition, RNA replication assay further confirmed the antivirus ability of tatanan A. Time-addition assay showed that tatanan A affected the early stage of viral RNA replication, which in turn inhibited mRNA and protein levels of DENV2. CONCLUSIONS: These results demonstrated the anti-DENV2 effect of tatanan A, in inhibiting DENV2 RNA replication and infections. In summary, tatanan A was found to be a novel natural DENV inhibitor and a potential candidate for the treatment of DENV infectious disease.

Inhibition of dengue viral infection by diasarone-I is associated with 2'O methyltransferase of NS5.

Dengue virus (DENV) is the most prevalent mosquito borne viral pathogen worldwide. However, antiviral drugs against this infection are not available. To identify novel anti-DENV compound from traditional Chinese medicine, we discovered the ethanol extract of Acorus tatarinowii Schott containing potent anti-DENV activity and diasarone-I was isolated from this extract. Diasarone-I has antiviral effect with half maximal effective concentration (EC50) of 4.5µM and half maximal cytotoxicity concentration (CC50) of >80µM. Time of drug addition assay suggested that this compound inhibited at RNA replication step in the DENV life cycle. Further, in silico analysis indicated that diasarone-I might act as an inhibitor of 2'O Methyltransferase of NS5. Diasarone-I has also decreased the DENV2-induced STAT1 phosphorylation and ISGs. In summary, we suggest that diasarone-I may be a 2'O Methyltransferase inhibitor and might serve as a potential candidate for the treatment of DENV2 infections.