Target-based design, synthesis and activity analyses of hepatitis B virus core protein allosteric modulators

[Objective] Globally, approximately 254 million people suffer from chronic hepatitis B virus(HBV)infection, and this virus causes approximately 1.079 million deaths annually due to complications such as liver cirrhosis and hepatocellular carcinoma.This study aims to develop novel anti-hepatitis B drugs with high efficacy, low toxicity, and optimal pharmacological properties using a precise, structure-based drug design strategy.Specifically, the approach combines multiple-site binding in the accommodating region, structural diversity modification of the solvent-exposed region, and bio-electronic substitution to overcome the limitations of existing antiviral drugs, which fail to eliminate the covalently closed circular DNA(cccDNA)of HBV and don't achieve a functional cure for chronic hepatitis B.[Methods] Using NVR3-778 as the lead compound, the structural analysis based on its binding mode with the HBV core protein revealed two key modification sites.One is to introduce hydrophobic groups in the available region at the 2nd position of the central benzene ring, and the other is to introduce hydrophobic groups in the solvent-exposed region around the sulfonylamine group for modification.Accordingly, benzene-sulfonamine compounds(Series Ⅰ)and 2-bromo-substituted benzamide compounds(Series Ⅱ)were designed and synthesized.The inhibitory activity of the target compounds against HBV DNA replication and the live cell content in HepAD38 cells were quantitatively detected by quantitative real-time polymerase chain reaction(qRT-PCR)and CCK-8 method, respectively.The anti-HBV activity and cytotoxicity of the target compounds were evaluated.The molecular docking and 100 ns molecular dynamics simulations of the target compounds were conducted using Schrödinger software to further verify the experimental results, elucidate binding modes, stability, and interactions, and supplement the results of in vitro biological evaluation.[Results] Benzene-sulfonamide compounds(Series Ⅰ)and 2-bromo-substituted benzamide compounds(Series Ⅱ)showed varying degrees of anti-HBV activity and cytotoxicity in the HepAD38 cell line, confirming the rationality and effectiveness of the initial design strategy of this study.Among these compounds, the 2-bromo-substituted thiourea benzamide compound 18 maintained a high antiviral activity(EC50=0.37 μmol/L)while showing lower cytotoxicity(CC50>100 μmol/L)and a higher selectivity index(SI>267.38).Molecular docking analysis and dynamic simulation results indicated that for compound 18, the amide nitrogen atom and amide oxygen atom on the compound formed hydrogen bonds with the key amino acids Thr128 and Trp102 of the HBV core protein, which was the key to maintaining the antiviral activity of the compound.Furthermore, upon introducing the thiourea group into the solvent-exposed region, the two nitrogen atoms of this group formed new and stable hydrogen bonds with the important amino acid residues Leu140 on the B chain, further enhancing its binding stability, thereby elucidating the structural basis for its excellent anti-HBV activity and validating the rationality of the experimental design.[Conclusion] Based on the analysis of the binding mode of the lead compound NVR3-778 with the HBV core protein, and through the precise drug design strategy based on multiple-site binding in the accommodating region, structural diversity modification of the solvent-exposed region, and bio-electronic substitution, benzene-sulfonamide compounds(Series Ⅰ)and 2-bromo-substituted benzamide compounds(Series Ⅱ)were successfully developed.From the experimental results, it was found that introducing a bromine atom at the 2-position of the central benzene ring could enhance the antiviral activity and reduce cytotoxicity.Introducing thiourea groups into the solvent-exposed region was more effective than using sulfonamide or urea groups in enhancing activity and reducing toxicity.Among them, the derivatives with electron-withdrawing groups on the benzene ring exhibited better activity.This novel 2-bromo-substituted thiourea benzamide compound 18 exhibits strong anti-HBV activity with low toxicity.This finding provides important guidance for subsequent drug design and further optimization, and offers a new candidate compound for the treatment of chronic hepatitis B.Its combination with other drugs targeting cccDNA may provide new ideas for achieving a functional cure.