Synthesis and antiviral evaluation of cytisine derivatives against dengue virus types 1 and 2

Glycyrrhizic Acid Derivatives Bearing Amino Acid Residues in the Carbohydrate Part as Dengue Virus E Protein Inhibitors: Synthesis and Antiviral Activity

“…DENV-1 isolate CMUH 2018-4 and DENV-2 strain 16681 used in this study were propagated in monkey kidney epithelial Vero E6 cells (Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan). The cultured media of infected Vero E6 cells were harvested 5 days post-infection, and virus titer of the stock was determined using the mean (50%) tissue culture infectious dose (TCID50) per mL [ 45 , 59 , 60 ]. Vero E6 cells and human lung epithelial A549 cells (BCRC, Hsinchu, Taiwan) that were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin, were used to explore antiviral potential and the mechanisms of GL conjugates with methyl or ethyl esters of L- and D-amino acids (Compounds 2 – 21 ) against DENV1 and DENV2.…”

“…In the antiviral screening assay, each tested GL conjugate at 10 μM was used to survey the anti-DENV activity in Vero E6 cells using cytopathic effect (CPE) and infectivity reduction assays as described in our prior report [ 45 , 59 , 60 ]. In CPE and infectivity reduction assays, Vero E6 cells were infected by DENV2 at an MOI of 0.005 and simultaneously treated 10 μM of indicated GL conjugate for 96 h, and then photographed by microscope for examining the levels of DENV2-induced CPE in infected cells treated with or without GL conjugate.…”

“…Meanwhile, the cells were further performed using immunofluorescence assay (IFA) with anti-DENV NS4B antibodies (GeneTex, Inc., Hsinchu, Taiwan) and anti-rabbit IgG antibodies conjugated with AF555 (ThermoFisher, Waltham, MA, USA) plus DAPI (4′,6-diamidino-2-phenylindole) nuclear staining. Finally, the DENV-2 infectivity was measured according to the ratio of DENV NS4B-positive cells to DAPI-stained cells in the well of mock-treated or treated cells, as described in [ 45 , 59 , 60 ]. In the antiviral potency assay, Vero E6 and A549 cells were infected with the fixed amount of DENV (DENV1 at an MOI of 0.1, or DENV2 (at MOIs of 0.05 and 0.005), and simultaneously treated with 0.1, 1, and 10 μM of active GL conjugates (Compounds 3 , 6 , 11 , and 21 ), respectively.…”

“…The time-of-addition assays consisted of three modes including attachment treatment/removal, entry-treatment/removal, and post-entry treatment/removal modes [ 59 , 60 ]. In the attachment-treatment/removal mode, Vero E6 cells monolayer in 6-well plate was placed at 4 °C for 2 h and incubated with the fresh-prepared mixture of DENV-2 (MOI = 0.005) with the active compound at indicated concentrations for 2 h at 4 °C, and then washed with phosphate-buffered saline (PBS).…”

“…After active compound removal (washout), the cells were incubated at 37 °C for 96 h, and then were analyzed by immunofluorescent and DAPI staining assay for determining the residual DENV-2 infectivity, as described above. The inhibitory activity of the transient treatment by active compounds on attachment, entry, and post-entry stages of DENV replication in vitro was determined based on the residual infectivity [ 59 , 60 ].…”