The 8-bromobaicalein inhibited the replication of dengue, and Zika viruses and targeted the dengue polymerase.

Dengue and Zika viruses are mosquito-borne flaviviruses burdening millions every year with hemorrhagic fever and neurological symptoms. Baicalein was previously reported as a potential anti-flaviviral candidate and halogenation of flavones and flavanones potentiated their antiviral efficacies. Here, we reported that a chemically modified 8-bromobaicalein effectively inhibited all dengue serotypes and Zika viruses at 0.66-0.88 micromolar in cell-based system. The compound bound to dengue serotype 2 conserved pocket and inhibited the dengue RdRp activity with 6.93 fold more than the original baicalein. Moreover, the compound was mildly toxic against infant and adult C57BL/6 mice despite administering continuously for 7 days. Therefore, the 8-bromobaicalein should be investigated further in pharmacokinetics and efficacy in an animal model.

Alkyne-Tagged Apigenin, a Chemical Tool to Navigate Potential Targets of Flavonoid Anti-Dengue Leads.

A flavonoid is a versatile core structure with various cellular, immunological, and pharmacological effects. Recently, flavones have shown anti-dengue activities by interfering with viral translation and replication. However, the molecular target is still elusive. Here we chemically modified apigenin by adding an alkyne moiety into the B-ring hydroxyl group. The alkyne serves as a chemical tag for the alkyne-azide cycloaddition reaction for subcellular visualization. The compound located at the perinuclear region at 1 and 6 h after infection. Interestingly, the compound signal started shifting to vesicle-like structures at 6 h and accumulated at 24 and 48 h after infection. Moreover, the compound treatment in dengue-infected cells showed that the compound restricted the viral protein inside the vesicles, especially at 48 h. As a result, the dengue envelope proteins spread throughout the cells. The alkyne-tagged apigenin showed a more potent efficacy at the EC50 of 2.36 ± 0.22, and 10.55 ± 3.37 µM, respectively, while the cytotoxicities were similar to the original apigenin at the CC50 of 70.34 ± 11.79, and 82.82 ± 11.68 µM, respectively. Molecular docking confirmed the apigenin binding to the previously reported target, ribosomal protein S9, at two binding sites. The network analysis, homopharma, and molecular docking revealed that the estrogen receptor 1 and viral NS1 were potential targets at the late infection stage. The interactions could attenuate dengue productivity by interfering with viral translation and suppressing the viral proteins from trafficking to the cell surface.

Dibromopinocembrin and Dibromopinostrobin Are Potential Anti-Dengue Leads with Mild Animal Toxicity.

Dengue infection is one of the most deleterious public health concerns for two-billion world population being at risk. Plasma leakage, hemorrhage, and shock in severe cases were caused by immunological derangement from secondary heterotypic infection. Flavanone, commonly found in medicinal plants, previously showed potential as anti-dengue inhibitors for its direct antiviral effects and suppressing the pro-inflammatory cytokine from dengue immunopathogenesis. Here, we chemically modified flavanones, pinocembrin and pinostrobin, by halogenation and characterized them as potential dengue 2 inhibitors and performed toxicity tests in human-derived cells and in vivo animal model. Dibromopinocembrin and dibromopinostrobin inhibited dengue serotype 2 at the EC50s of 2.0640 ± 0.7537 and 5.8567 ± 0.5074 µM with at the CC50s of 67.2082 ± 0.9731 and >100 µM, respectively. Both of the compounds also showed minimal toxicity against adult C57BL/6 mice assessed by ALT and Cr levels in day one, three, and eight post-intravenous administration. Computational studies suggested the potential target be likely the NS5 methyltransferase at SAM-binding pocket. Taken together, these two brominated flavanones are potential leads for further drug discovery investigation.

Cardol triene inhibits dengue infectivity by targeting kl loops and preventing envelope fusion.

Dengue virus causes a global burden that specific chemotherapy has not been established. A previous report suggested that anacardic acid inhibited hepatitis C virus infection. Here, we explored structure activity relationship of anacardic acid, cardanol, and cardol homologues with anti-DENV cellular infectivities. Cardol triene showed the highest therapeutic index at 29.07 with the CC50 and EC50 of 207.30 ± 5.24 and 7.13 ± 0.72 µM, respectively. Moreover, we observed that the more unsaturated the hydrocarbon tail, the higher the CC50s in all head groups. High CC50s were also found in HepG-2, THP-1, and HEK-293 cell lines where cardol triene CC50s were 140.27 ± 8.44, 129.77 ± 12.08, and 92.80 ± 3.93 µM, respectively. Cardol triene expressed pan-dengue inhibition with the EC50s of 5.35 to 8.89 µM and kl loops of dengue envelope proteins were major targets. The strong binding energy at T48, E49, A50, P53, K128, V130, L135, M196, L198, Q200, W206, L207, I270, and L277 prevented cellular pH-dependent fusion. Zika virus kl loops were aligned in the closed position preventing cardol triene to bind and inhibit fusion and infectivity. This study showed for the first time that cardol triene had a potential for further development as anti-dengue inhibitors.

Simplified dengue virus microwell plaque assay using an automated quantification program.

The plaque assay is essential for virion quantitation but the classic protocol requires considerable efforts. A simplified dengue 96-well plaque assay with automated quantitation program is an alternative to access the level of infectious virus. Dengue plaque assay was simplified using LLC/MK2 cells and virus mixing simultaneously before semisolid addition. Results were obtained using a flatbed scanner and analysis by the self-written program optimized to manual reads. The newly developed microwell system was accurate to the standard assay because 19 independent titrations from all subtypes obtained from both systems differed less than a log10 p.f.u./ml with no significance (p>0.05) with good correlation (R2=0.9058). Coefficient of variations within and between assays, indicating assay reliability and repeatability, were 19.29%, and 12.50%, respectively. This method serves various experimental designs in drug discovery that requires viral titers assessment. Effective concentrations (EC90) results showed no significant difference between 24- and 96-well assays (p>0.05). Compound screening for potential antivirals and clinical isolate titrations were successfully arranged. The method contains distinguished features including protocol simplicity, less reagent consumption in microwell format, convenient and affordable data acquisition and analysis system.