Evaluation of Potential Anti-Hepatitis A Virus 3C Protease Inhibitors Using Molecular Docking

Abstract

Hepatitis A virus (HAV) infection is a major cause of acute hepatitis worldwide and occasionally causes acute liver failure and can lead to death in the absence of liver transplantation. Although HAV vaccination is available, the prevalence of HAV vaccination is not adequate in some countries. Additionally, the improvements in public health reduced our immunity to HAV infection. These situations motivated us to develop potentially new anti-HAV therapeutic options. We carried out the in silico screening of anti-HAV compounds targeting the 3C protease enzyme using the Schrodinger Modeling software from the antiviral library of 25,000 compounds to evaluate anti-HAV 3C protease inhibitors. Additionally, in vitro studies were introduced to examine the inhibitory effects of HAV subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication in hepatoma cell lines using luciferase assays and real-time RT-PCR. In silico studies enabled us to identify five lead candidates with optimal binding interactions in the active site of the target HAV 3C protease using the Schrodinger Glide program. In vitro studies substantiated our hypothesis from in silico findings. One of our lead compounds, Z10325150, showed 47% inhibitory effects on HAV genotype IB subgenomic replicon replication and 36% inhibitory effects on HAV genotype IIIA HA11-1299 replication in human hepatoma cell lines, with no cytotoxic effects at concentrations of 100 μg/mL. The effects of the combination therapy of Z10325150 and RNA-dependent RNA polymerase inhibitor, favipiravir on HAV genotype IB HM175 subgenomic replicon replication and HAV genotype IIIA HA11-1299 replication showed 64% and 48% inhibitory effects of HAV subgenomic replicon and HAV replication, respectively. We identified the HAV 3C protease inhibitor Z10325150 through in silico screening and confirmed the HAV replication inhibitory activity in human hepatocytes. Z10325150 may offer the potential for a useful HAV inhibitor in severe hepatitis A.

Keywords: 3C protease; HAV; in silico screening; molecular docking; protease inhibitors.

Figure 1

Molecular docking study and the structure of ligands for hepatitis A virus (HAV) 3C protease in the present study: (A) docking of Z10325150 in the active site of HAV 3C protease; (B) electrostatic potential of HAV 3C protease in complex with Z10325150; (C,D) 3D and 2D interactions between Z10325150 and HAV 3C protease; (E) structure of ligands for HAV 3C protein.

Figure 1

Molecular docking study and the structure of ligands for hepatitis A virus (HAV) 3C protease in the present study: (A) docking of Z10325150 in the active site of HAV 3C protease; (B) electrostatic potential of HAV 3C protease in complex with Z10325150; (C,D) 3D and 2D interactions between Z10325150 and HAV 3C protease; (E) structure of ligands for HAV 3C protein.

Figure 2

Effects of Z2351109846, Z10325150, Z1452073950, Z287374370, and Z1208291016 on cell viabilities of HuhT7 cells. Cell viabilities of HuhT7 cells treated with Z2351109846 (A), Z10325150 (B), Z1452073950 (C), Z287374370 (D), and Z1208291016 (E). HuhT7 cells were treated with each drug for 48 h. Cell viabilities were determined via dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium (MTS) assay. Data are expressed as means and standard deviations of triplicate determinations from three independent experiments. Statistical significance was analyzed using the two-tailed Student’s t-test.

Figure 3

Z2351109846, Z10325150, Z1452073950, and Z287374370 inhibited HAV genotype IB subgenomic replicon replication. HuhT7 cells transfected with the HAV genotype IB subgenomic replicon. After 24 h of transfection, cells were treated with Z2351109846 (A), Z10325150 (B), Z1452073950 (C), Z287374370 (D), and Z1208291016 (E) for 48 h, and luciferase activity was determined after 72 h of transfection. Data are presented as the means and standard deviations of triplicate determinations from at least three independent experiments. Statistical significance was analyzed using the two-tailed Student’s t-test: * p < 0.05.

Figure 4

Effects of Z2351109846, Z10325150, Z1452073950, Z287374370, and Z1208291016 on cell viabilities of Huh7 cells. Cell viabilities of Huh7 cells treated with Z2351109846 (A), Z10325150 (B), Z1452073950 (C), Z287374370 (D), and Z1208291016 (E). Huh7 cells were treated with each drug for 72h. Cell viabilities were determined by dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium (MTS) assay. Data are expressed as means and standard deviations of triplicate determinations from three independent experiments. Statistical significance was analyzed using the two-tailed Student’s t-test: * p < 0.05.

Figure 5

Z10325150 could inhibit HAV genotype IIIA HA11-1299 replication. Huh7 cells infected with HAV genotype IIIA HA11-1299 were treated with Z2351109846 (A), Z10325150 (B), Z1452073950 (C), Z287374370 (D), and Z1208291016 (E) for 72 h. HAV RNA levels were measured using real-time RT-PCR. Actin mRNA was used as an internal control. Data are presented as the means and standard deviations of triplicate determinations from at least three independent experiments. Statistical significance was analyzed using the two-tailed Student’s t-test: * p < 0.05.