Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload

Abstract

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has five genotypes (I, II, III, IV, and V). JEV genotype I circulates widely in some Asian countries. However, current JEV vaccines based on genotype III strains show low neutralizing capacities against genotype I variants. In addition, JE has no specific treatment, except a few supportive treatments. Compound CW-33, an intermediate synthesized derivative of furoquinolines, was investigated for its antiviral activities against JEV in this study. CW-33 exhibited the less cytotoxicity to Syrian baby hamster kidney (BHK-21) and human medulloblastoma (TE761) cells. CW-33 dose-dependently reduced the cytopathic effect and apoptosis of JEV-infected cells. Supernatant virus yield assay pinpointed CW-33 as having potential anti-JEV activity with IC50 values ranging from 12.7 to 38.5 μM. Time-of-addition assay with CW-33 indicated that simultaneous and post-treatment had no plaque reduction activity, but continuous and simultaneous treatments proved to have highly effective antiviral activity, with IC50 values of 32.7 and 48.5 μM, respectively. CW-33 significantly moderated JEV-triggered Ca2+ overload, which correlated with the recovery of mitochondria membrane potential as well as the activation of Akt/mTOR and Jak/STAT1 signals in treated infected cells. Phosphopeptide profiling by LC-MS/MS revealed that CW-33 upregulated proteins from the enzyme modulator category, such as protein phosphatase inhibitor 2 (I-2), Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, and putative 3-phosphoinositide-dependent protein kinase 2. These enzyme modulators identified were associated with the activation of Akt/mTOR and Jak/STAT1 signals. Meanwhile, I-2 treatment substantially inhibited the apoptosis of JEV-infected cells. The results demonstrated that CW-33 exhibited a significant potential in the development of anti-JEV agents.

Structure analysis and antiviral activity of CW-33 analogues against Japanese encephalitis virus

“…CW-33A significantly inhibited the late stage of JEV replication in vitro , including intracellular virus production, viral protein expression and viral genome synthesis (Figs 7 – 10 ). The result indicated antiviral mechanism of CW-33A was consistent with that of CW-33 reported in a prior study that CW-33 declined the late stage of JEV replication via antagonizing the suppression of ERK1/2, Akt/mTOR, and Jak/STAT pathways by JEV 9 . CW-33A might have a clinical application on interfere post-entry stages during JEV replication.…”

“…CW-33 had no effect on the infectivity of virus particles and early entry into cells, but showed a significant inhibition on the late stage of JEV replication cycle 9 . Intracellular infectious virus assay demonstrated that CW-33A at 10 μM has a higher decrease in the intracellular production of JEV infectious particles than CW-33 24 or 48 hpi (Fig.…”

“…CW-33 was previously identified as the lead compound exhibiting the anti-JEV activity in vitro 9 . On the basis of this promising activity, 6 mono substituted analogues with an fluoro, or methoxy group at the C-2, C-3, or C-4 anilino ring of CW-33 were evaluated for their anti-JEV activities included CPE inhibition and Sub-G1 reduction (Figs 2 and 3 ).…”

“…1C ), also called as CW-33, CW-33A to CW-33F. The compounds were refined using high-performance liquid chromatography 9 ; the identity and purity of CW-33 analogues were validated using 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopy. The NMR spectra were listed in Supplemental Figs 1 – 12 and Table 1 .…”

“…The compound CW-33, ethyl 2-(3′,5′-dimethylanilino)-4-oxo-4,5- dihydrofuran-3-carboxylate (Fig. 1A ), exhibits antiviral activity against JEV 9 . The half maximal inhibitory concentration (IC 50 ) of CW-33 to reduce the progeny virus production in vitro is greater than 10 μM, thus the modification of CW-33 structure to improve antiviral activity is essential.…”