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Hepatitis C virus nonstructural protein 3 (HCV NS3) helicase is believed to be essential for viral replication and has become an attractive target for the development of antiviral drugs. A fluorescence resonant energy transfer helicase assay was established for fast screening of putative inhibitors selected from virtual screening using the program DOCK. Soluble blue HT (1) was first identified as a novel HCV helicase inhibitor. Crystal structure of the NS3 helicase in complex with soluble blue HT shows that the inhibitor bears a significantly higher binding affinity mainly through a 4-sulfonatophenylaminophenyl group, and this is consistent with the activity assay. Subsequently, fragment-based searches were utilized to identify triphenylmethane derivatives for more potent inhibitors. Lead optimization resulted in a 3-bromo-4-hydroxyl substituted derivative 12 with an EC(50) value of 2.72 microM to Ava.5/Huh-7 cells and a lower cytotoxicity to parental Huh-7 cells (CC(50) = 10.5 microM), and it indeed suppressed HCV replication in the HCV replicon cells. Therefore, these inhibitors with structural novelty may serve as a useful scaffold for the discovery of new HCV NS3 helicase inhibitors.

Previously reported findings by our group showed that non-structural protein 3 (NS3) of Japanese encephalitis virus (JEV) was localized mainly in the JEV-induced convoluted membrane (CM), which has been proposed to originate from rough endoplasmic reticulum (rER), Golgi apparatus or the trans-Golgi network (TGN), and serves as a reservoir for viral proteins during virus assembly. Earlier findings indicated that NS3 of Kunjin virus interacts with microtubules. In addition, one of the Golgi-associated proteins, tumour susceptibility protein 101 (TSG101), associates with microtubules and is required for budding of retroviral particles. To clarify the association of NS3 with microtubules or with TSG101 during JEV assembly, we applied immunofluorescence, co-immunoprecipitation and immunoelectron microscopic methods. Virus infection, as well as transfection with an NS2B-NS3 expression plasmid, induced microtubule rearrangement. When cells were treated with colchicine, which interferes with microtubule polymerization, NS3 still associated with tubulin and TSG101. Furthermore, tubulin and TSG101 were co-localized with NS3 in the CM by immunogold labelling. Our observations indicate that microtubules and TSG101 associate with NS3, which is incorporated into the JEV-induced structure during JEV replication.

Six new polyisoprenyl benzophenonoids, (±)-garcinialiptone A (1, 2), garcinialiptone B (3), (−)-cycloxanthochymol (4), garcinialiptone C (5), and garcinialiptone D (6), along with three known compounds, xanthochymol (7), isoxanthochymol (8), and cycloxanthochymol (9), were isolated from the fruits of Garcinia subelliptica. The structures of 1-6 were elucidated by spectroscopic analysis. Biological evaluation showed that all compounds 1-9 exhibited cytotoxic activity against a small panel of human tumor cell lines (A549, DU145, KB, vincristine-resistant KB).Garcinia subelliptica Merr. (Guttiferae) is a tree that serves as a dye source in tropical and subtropical Asia. Several xanthones and phloroglucinol derivatives have been isolated from this plant, 1-14 and found to exhibit various biological activities, including inhibitory activity against DNA topoisomerases I and II,13 and antioxidant,2 anti-inflammatory,9 , 10 and cytotoxic 12 effects. Polyisoprenylated benzophenones isolated from the Guttiferae family feature a bicyclo[3.3.1]-nonane-2,4,9-trione skeleton substituted with a benzoyl group and prenyl or geranyl groups. These polyprenylated benzoylphloroglucinol derivatives are classified into three types according to the relative position of the benzoyl group. 16 Type A, with the benzoyl group linked at C-1, is exemplified by nemorosone16 and garcinielliptone FB;12 type B, with the benzoyl group at C-3, is represented by xanthochymol, the main component of G. subelliptica; and type C, with the benzoyl group at C-5, is typified by garcinielliptone K. 11 Many polycyclic polyprenylated acylphloroglucinol derivatives, also isolated from plants of the family Guttiferae, undergo secondary cyclizations involving the β-diketone and olefinic groups to afford admantanes, homoadamantanes, dihydrofuro-fused structures, and related structures. 17 The occurrence of cyclized secondary metabolites is also documented for polyprenylated benzoylphloroglucinol derivatives.In a continuing search for novel plant-derived antitumor agents, it was found that an acetone extract of the fruits of G. subelliptica showed moderate cytotoxicity against Hela and WiDr cells. Bioassay-directed fractionation of G. subelliptica resulted in the isolation of six new cytotoxic polyisoprenyl benzophenonoids, (±)-garcinialiptone A (1, 2, Results and DiscussionExtraction of the fruits of G. subelliptica with acetone, followed by chromatography, led to the isolation of nine compounds, including the known compounds 7-9, as well as six new polyprenylated benzoylphloroglucinol compounds (1-6).Compounds 1 and 2 [the (+) and (−) isomers of garcinialiptone A], obtained as yellow amorphous solids, had almost identical HRESIMS, UV, IR, and NMR spectra. Compound 1 was assigned the molecular formula C 38 H 48 O 6 (corresponding to 15 degrees of unsaturation) on the basis of positive-ion HRESIMS of the peak at m/z 623.3377 [M + Na] + . Signals for hydroxy (3403 cm −1 ), carbonyl (1742, 1700 cm −1 ), and aromatic (1599, 1549, 1440 cm −1 ) groups were fou...

Infection with a mutant Japanese encephalitis virus (JEV) strain RP-2ms showed reduced neurovirulence than wild type or RP-9 strains after inoculation in BALB/c mice. However, higher intracellular viral titer was detected in Rp-2ms infected cultured cells. Localizations of non-structural 3 (NS3) and envelope (E) proteins were demonstrated by immunocytochemistry. NS3 protein was primarily found in the pyramidal neurons in cerebrum, in the molecular and granular layers of cerebellum. Neither E nor NS3 protein was detected in Purkinje cells of the cerebellum. Immunoelectron microscopic observations showed that E and NS3 proteins were positive in JEV-induced membranous systems, mainly hypertrophic rough endoplasmic reticulum (rER) and membrane vesicle structure (MVS) but not smooth membrane structure. Virus particles were seen in the Golgi apparatus, rER, nuclear envelope, MVS and cytoplasmic vacuoles. Different mechanisms of intracellular trapping in vivo provide a possible basis for attenuation of RP-2ms strains of JEV.

Astragalus membranaceus polysaccharide (APS) components are main ingredients of TCM and have proven efficacy to activate T cells and B cells, enhancing immunity in humans. In this study, elevated cytokine and anti-PD-1 antibody titers were found in mice after immunization with APS. Therefore, phage-display technology was utilized to isolate specific anti-programmed death-1 (PD-1) antibodies from mice stimulated by APS and to confirm whether the isolated anti-PD-1 antibody could inhibit the interaction of PD-1 with the programmed death-ligand 1 (PD-L1), resulting in tumor growth inhibition. The isolated single-chain fragment variable (scFv) S12 exhibited the highest binding affinity of 20 nM to PD-1, completed the interaction between PD-1 and PD-L1, and blocked the effect of PD-L1-induced T cell exhaustion in peripheral blood mononuclear cells in vitro. In the animal model, the tumor growth inhibition effect after scFv S12 treatment was approximately 48%. However, meaningful synergistic effects were not observed when scFv S12 was used as a cotreatment with ixabepilone. Moreover, this treatment caused a reduction in the number of tumor-associated macrophages in the tumor tissue. These experimental results indirectly indicate the ability of APS to induce specific antibodies associated with the immune checkpoint system and the potential benefits for improving immunity in humans.

Six new meroterpenoids, diplomeroterpenoids A-F (1-6), two new chalcone-lignoids, diplochalcolins A and B (7, 8), and 13 known compounds were isolated from the root extract of Mimosa diplotricha. Diplomeroterpenoids A-F consist of a 4H-chromen-4-one and a diterpenoid unit, and their absolute configurations were determined by X-ray crystallographic analysis. Compounds 1-3 and 5 showed potent inhibitory activity on protein farnesyl transferase, with IC values from 5.0 to 8.5 μM. Compound 1 showed antiproliferative activity against human hepatoblastoma HepG2 cells with a GI value of approximately 8.6 μM.

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