A Conserved Inhibitory Mechanism of a Lycorine Derivative against Enterovirus and Hepatitis C Virus

Abstract

Enterovirus 71 (EV71) (Picornaviridae family) and hepatitis C virus (HCV) (Flaviviridae family) are the causative agents of human hand, foot, and mouth disease (HFMD) and hepatitis C, resulting in a severe pandemic involving millions of infections in the Asia-Pacific region and worldwide. The great impact of EV71 and HCV on public health highlights the need to further our understanding of the biology of these two viruses and develop effective therapeutic antivirals. Here, we evaluated a total of 32 lycorine derivatives and demonstrated that 1-acetyllycorine suppressed the proliferation of multiple strains of EV71 in various cells. The results of the drug resistance analysis revealed that 1-acetyllycorine targeted a phenylalanine (F76) in EV71 2A protease (2A(pro)) to stabilize the conformation of a unique zinc finger. Most interestingly, the zinc binding site in EV71 2A(pro) is the exclusive homolog of HCV NS3 among all viruses. Further analysis revealed that 1-acetyllycorine also inhibits HCV with high efficacy, and the mutation on R118 in HCV NS3, which corresponds to F76 in EV71 2A(pro), confers the resistance of HCV to 1-acetyllycorine. These results revealed a conserved mechanism of 1-acetyllycorine against EV71 and HCV through targeting viral proteases. We also documented the significant synergistic anti-EV71 and anti-HCV effects of 1-acetyllycorine with reported inhibitors, supporting potential combination therapy for the treatment of EV71 and HCV infections.

FIG 1

1-Acetyllycorine suppressed EV71 proliferation. (A) Structural conformation of 1-acetyllycorine. (B) Concentration-dependent reduction of EV71 proliferation following treatment with 1-acetyllycorine. RD cells were infected with EV71-GFP at an MOI of 1, and the level of GFP expression was monitored using an epifluorescence microscope at 24 hpi (bottom panel). DAPI was used to visualize the nuclear staining (top panel) in the host cells. (C) Inhibitory effect of 1-acetyllycorine on EV71 RNA synthesis measured through qRT-PCR. The results of each point were presented as the average results from three independent experiments, and the error bars represent the standard errors of the means (n = 3). (D) Dose-dependent reduction of EV71 VP1 expression. The concentration of 1-acetyllycorine in lanes 1 to 5 was 10, 5, 2.5, 1.25, and 0 μM, respectively (top panel). The expression level of GAPDH did not vary (bottom panel).

FIG 2

Quantification of the anti-EV71 effect of 1-acetyllycorine in different cell lines. (A to C) RD, 293T, and Vero cells were treated with 1-acetyllycorine and infected with EV71 (strain Fuyang). EV71 RNA levels were measured using qRT-PCR at 24 hpi. The virus titers in the supernatants were determined at 72 hpi. (D to F) Cytotoxicity in different cell lines. RD, 293T, and Vero cells were incubated with 1-acetyllycorine for 24 h, and the cell viability was measured using a WST-1-based assay. The results of each point are presented as the average results from three independent experiments, and the error bars represent the standard errors of the means (n = 3).

FIG 3

Inhibition by 1-acetyllycorine, NITD008, and ALD of EV71 with different addition times. (A to C) Inhibition of EV71(FY)-Luc pseudotype virus infection of RD cells through addition of 1-acetyllycorine (2 μM) (A), NITD008 (2 μM) (B), and ALD (1 pM) (C) at various times. (D) EV71 subgenomic replicon RNA lacking the P1 region was transfected into RD cells with or without treatment with 1-acetyllycorine (2 μM), NITD008 (2 μM), and ALD (1 pM). The luciferase levels were quantified at 24 hpi. The results of each point are presented as the average results from three independent experiments, and the error bars represent the standard errors of the means (n = 3).

FIG 4

Drug sensitivity of 1-acetyllycorine on EV71 WT and F76L virus. (A) Growth curves of EV71 WT and F76L viruses. (B) EC₅₀s of 1-acetyllycorine against EV71 WT and F76L viruses were measured in parallel using qRT-PCR. (C) EC₅₀s of 1-acetyllycorine, NITD008, and ALD against EV71 WT and F76L subgenomic replicons were measured in parallel by measuring luciferase activity. Each point represents the average results from three independent experiments, and the error bars represent the standard errors of the means (n = 3). (D) Enzymatic activities of EV71 WT 2A^pro with or without 1-acetyllycorine, F76L, and C100A-mutated 2A^pro. Each reaction was performed by using 25 μM protein and 20 μM substrate.

FIG 5

Conserved mechanism of 1-acetyllycorine against EV71 and HCV. (A) Structural comparison of EV71 2A^pro and HCV NS3 proteases. The structures of EV71 2A^pro and HCV NS3 protease were aligned and presented as cartoon diagrams in blue and gold, respectively. (B and C) Unique zinc finger region in EV71 2A^pro and HCV NS3 protease. Key residues were displayed in stick mode with different colors. (D) Inhibitory activity of 1-acetyllycorine on WT or R118L-mutated HCV(JFH-1)-Rluc viruses. (E) Growth curves of HCV WT and R118L-mutated viruses. Each point represents the average results from three independent experiments, and the error bars represent the standard errors of the means (n = 3). (F) Enzymatic activities of HCV WT NS3/4A with or without 1-acetyllycorine and R118L- and D81A-mutated NS3/4A. Each reaction was performed by using 15 μM protein and 10 μM substrate.

FIG 6

Drug-drug interactions of 1-acetyllycorine with other inhibitors of EV71 and HCV proliferation. (A and B) Differential surface plots at the 95% confidence level (CI) were calculated and generated using MacSynergy II for the drug-drug interactions for the combinations of 1-acetyllycorine and NITD008 (A) and 1-acetyllycorine and ALD (B) targeting EV71. The drug-drug interactions for the combinations of 1-acetyllycorine and sofosbuvir (C) and 1-acetyllycorine and RBV (D) on HCV were also evaluated.