Flavonoids of Andrographis paniculata regulate hepatitis B virus replication and hepatocellular carcinoma progression: evidence from computational and experimental studies.

BACKGROUND: The HBx protein of hepatitis B virus (HBV) plays a crucial role in HBV pathogenesis, yet current treatments like HIV reverse transcriptase (RT) inhibitors, which target HBV RT due to similar active sites, have severe side effects, risk of drug resistance, and high costs. The present study investigates the anti-hepatitis B virus (HBV) properties of Andrographis paniculata (AP) and Thespesia populnea (TP) on HBV expressing HepG2.2.15 cells and by computational analysis. METHODS: In vitro cytotoxicity, reverse transcriptase inhibitory, DNA and pgRNA quantification by qRT-PCR, time course analysis of HBsAg and HBeAg, and HBX-HBXIP interaction inhibition studies were conducted. The interaction of HBX with HBXIP, and phytocompounds' interaction with HBx was analyzed through molecular docking and dynamics studies. RESULTS: AP exhibits lower cytotoxicity (CC(50) = 832.915 µg/mL) than TP (CC(50) = 593.122 µg/mL) after 24 h, with Tenofovir disoproxil fumarate (TDF) showing minimal cytotoxicity (CC(50) > 500 µM). Both AP and TP significantly decreased intracellular HBV DNA with a > 2(5) fold reduction at higher concentrations (125-500 µg/mL) but had no significant effect on pgRNA level. AP and TP 500 µg/mL effectively inhibited HBsAg secretion (95% and 80% inhibition, respectively), over 120 h. AP also showed inhibition of HBeAg secretion (75-82%), while TP exhibited a higher inhibition of 90% at 24 h. TDF showed consistent but lower inhibitory effects on HBsAg and HBeAg. The HBx-HBXIP interaction inhibition assay showed AP's greater inhibitory capacity (IC(50) < 62.5 µg/mL) compared to TP (IC(50) = 806.69 µg/mL). Computational studies further validated these findings, showing stable binding interactions of AP compounds (flavonoids) with HBx protein (with Arg138 and His139, Lys140, and Trp141 residues participating in the interaction with HBXIP), corroborating their potential in disrupting HBV replication. Molecular dynamics simulations confirmed the stability of these interactions over 100ns. CONCLUSIONS: AP exhibits potent anti-HBV activities, making it a promising candidate for further therapeutic development.