Antiviral efficacy of bromo-anilino substituents of 4,5-dihydrofuran-3-carboxylate compound CW-33 against Japanese encephalitis virus.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, occasionally causes severe central nervous system disorders in the risk zone where more than 3 billion people reside. Our prior studies demonstrated antiviral potential of 4,5-dihydrofuran-3-carboxylate compound CW-33 (ethyl 2-(3',5'-dimethylanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate) and its derivative CW-33A ((ethyl 2-(2-fluoroanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate) against JEV infection ((Int. J. Mol. Sci. 2016, 17: E1386; Sci. Rep. 2018, 8: 16595). This study synthesized six new CW-33 derivatives containing chloro, or bromo groups at the C-2, C-3, or C-4 of anilino ring of CW-33, and assessed the antiviral activity and mechanisms of these chloro- and bromo-anilino substitutedderivatives. CW-33K, CW-33L and CW-33M had the bromo-substituents at the C-2, C-3, or C-4 of anilino ring of CW-33, respectively, showing the higher anti-JEV activity than CW-33 and other derivatives. CW-33K (ethyl 2-(2-bromoanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate) exhibited the highest antiviral efficacy and therapeutic index. The IC50 value of CW-33K was less than 5 µM for reducing JEV-induced cytopathic effect, virus infectivity and virus yield. CW-33K significantly inhibited the JEV replication at the early and late stages, suppressing viral RNA synthesis and intracellular JEV particle production. The study demonstrated that the CW-33 derivative with a bromosubstitutionat the C-2 anilino ring improved the antiviral activity JEV, providing the structure-antiviral activity relationship for the development of anti-JEV agents.

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

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 Ca(2+) 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.

Robust Design of Effective Allosteric Activators for Rsp5 E3 Ligase Using the Machine Learning Tool ProteinMPNN.

We used the deep learning tool ProteinMPNN to redesign ubiquitin (Ub) as a specific and functionally stimulating/enhancing binder of the Rsp5 E3 ligase. We generated 20 extensively mutated─up to 37 of 76 residues─recombinant Ub variants (UbVs), named R1 to R20, displaying well-folded structures and high thermal stabilities. These UbVs can also form stable complexes with Rsp5, as predicted using AlphaFold2. Three of the UbVs bound to Rsp5 with low micromolar affinity, with R4 and R12 effectively enhancing the Rsp5 activity six folds. AlphaFold2 predicts that R4 and R12 bind to Rsp5's exosite in an identical manner to the Rsp5-Ub template, thereby allosterically activating Rsp5-Ub thioester formation. Thus, we present a virtual solution for rapidly and cost-effectively designing UbVs as functional modulators of Ub-related enzymes.

Molecular interaction of the antiviral compound CW­33 and its analogues with the NS2B­NS3 protease of the Japanese encephalitis virus.

In a previous study from our group, a novel compound, namely CW­33 (ethyl 2­(3',5'­dimethylanilino)­â€‹4­oxo­4,5­dihydrofuran­3­carboxylate) was identified that exhibited antiviral activity for Japanese encephalitis virus (JEV). The viral NS2B­NS3 serine protease serves an important role in cytoplasmic cleavage events that occur during viral polyprotein maturation. The inhibition of viral RNA and protein syntheses was responsible for the antiviral activities of the novel furanonaphthoquinone derivatives that were discovered for the prevention of JEV infection. Consequently, the present study examined the molecular docking simulation of JEV protease with compound CW­33 and its analogues, and developed quantitative structure­activity relationship (QSAR) models to assess the potential antiviral activities of these compounds with regard to JEV. Molecular docking simulation indicated the potential ligand­protein interactions associated with the antiviral activities of these compounds. According to the results of the QSAR models, the secondary amine group was an important moiety required for compound bioactivity, which enabled the formation of hydrogen bonding with the residue Glu155. Furthermore, the aromatic ring mapping of the phenyl moiety of each compound was predicted to form a π­cation interaction with residue Arg76, whereas the hydrophobic feature represented by the ethyl moiety exhibited hydrophobic contacts with residue Glu74. Finally, the hydrophobic substituents in the meta­position of the phenyl ring further contributed to the efficacy of the antiviral activity. These results unravel the structural characteristics that are required for binding of CW­33 to the JEV protease and can be used for potential therapeutic and drug development purposes for JEV.

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

Japanese encephalitis virus (JEV) is a member of neurotropic flaviviruses transmitted by mosquito bites, causing severe central nervous system disorders. Current JEV genotype III vaccines have a low protection against genotype I isolates in the risk zone. The lead compound CW-33, ethyl 2-(3',5'-dimethylanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate, demonstrates the antiviral activity against JEV with an IC50 values of 38.5 µM for virus yield reduction (Int J Mol Sci 2016,17: E1386). This study synthesized fourteen CW-33 analogues containing a fluoro atom or one methoxy group at the C-2, C-3, or C-4 of anilino ring, and then evaluated for their antiviral activity and mechanism. Among 6 amalogues, CW-33A (ethyl 2-(2-fluoroanilino)-4-oxo- 4,5-dihydrofuran-3-carboxylate), and CW-33D (ethyl 2-(3-methoxyanilino)-4-oxo- 4,5-dihydrofuran-3-carboxylate exhibited antiviral potentials in viral cytopathic effect (CPE) inhibition. CW-33A significantly suppressed the viral protein expression, genome synthesis and intracellular JEV particle production, showing a higher inhibitory effect on JEV yield than CW-33 and CW-33D. The study demonstrated that a mono-fluoro substitution on at the C-2 anilino ring of CW-33 improved the antiviral activity JEV, revealing the structure-activity relationship for developing novel agents against JEV infection.

The axial misalignment between ocular lens and cornea observed by MRI (I)--at fixed accommodative state.

Lens tilt and decentration relative to the corneal axis are two of the factors which cause aberration in human eyes. Although many techniques have been developed to quantitatively measure these factors, however, they are mostly confined in 2D observations. To extend the view from 2D to 3D, MR imaging technique becomes a good candidate due to its capability of 3D-image reconstruction and with fairly good spatial resolution for that purpose. In this study a total of six eyes of six young students at this Institute were examined by using a 1.5T MRI machine incorporating with a commercial 3-in. surface coil at Taipei Veterans General Hospital. From a 45 degrees flipped reflective mirror, the subject could focus to a target at a distance of 60 cm from the eye for MR imaging in monocular vision. Quadric surface models were used to fit cornea and lens surfaces in the post image processes. Tilts of the two lens surfaces and the decentration of lens centre with respect to corneal axis were determined through coordinate transformations. One month later, retest was carried out on five of the six subjects. The results show that the average tilts of anterior and posterior lens surfaces, and lens decentration are 3.7+/-2.5 degrees, 3.3+/-1.4 degrees and 0.11+/-0.07 mm (H), -0.06+/-0.38 mm (V), respectively, for the test; 2.1+/-2.4 degrees, 1.9+/-1.8 degrees and -0.02+/-0.28 mm (H), -0.45+/-0.28 mm (V) for the retest. No statistically significant difference (by Wilcoxon Signed Ranks test) is revealed for the tilts of both lens surfaces (p(phia)=0.375, p(phip)=0.225) and for decentration (p(H)=0.343, p(V)=0.345) between test and retest in this longitudinal observation. The surface shapes of the ocular anterior components do not seem to be unified by only one type but ellipsoid, elliptic paraboloid, hyperboloid and paraboloid are all possible. Changes in shape type in the ocular anterior components were also found when measured in a period of one month.