Biophysics-Guided Lead Discovery of HBV Capsid Assembly Modifiers

Hepatitis B virus (HBV) is the leading cause of chronic liver pathologies worldwide. HBV nucleocapsid, a key structural component, is formed through the self-assembly of the capsid protein units. Therefore, interfering with the self-assembly process is a promising approach for the development of novel antiviral agents. Applied to HBV, this approach has led to several classes of capsid assembly modulators (CAMs). Here, we report structurally novel CAMs with moderate activity and low toxicity, discovered through a biophysics-guided approach combining docking, molecular dynamics simulations, and a series of assays with a particular emphasis on biophysical experiments. Several of the identified compounds induce the formation of aberrant capsids and inhibit HBV DNA replication in vitro, suggesting that they possess modest capsid assembly modulation effects. The synergistic computational and experimental approaches provided key insights that facilitated the identification of compounds with promising activities. The discovery of preclinical CAMs presents opportunities for subsequent optimization efforts, thereby opening new avenues for HBV inhibition.


FigureFigure S2 :
Figure S1: 2D plot of the two largest principal components in our PCA analysis of apo tetramer.The structures that were selected for docking are marked in red.

Figure S6 :
FigureS6: BioAnalyzer data of the purified Cp149 protein indicating that the sample is clean.The Cp149 protein band migrates lower than its anticipated monomer MW (16.8 kDa) in the Bioanalyzer plot, but this is likely due to it having a theoretical pI of 4.9 at physiological pH.

Figure S9 :
Figure S9: TEM images of assembled products without compound (A,B), and with GT-32 (C,D), with one image from each replica.

Figure S10 :
Figure S10: TEM images of assembled products with GT-39 (A, B), and GT-46 (C, D), with one image from each replica.

Figure S11 :
Figure S11: Illustration of the differences between the three structures used for docking.A) Tetra 1 structure (blue) with the red rectangle pointing to the most distinct region in the binding pocket between the 3 structures.Differences in Tetra 2 and Tetra 3 structures after backbone alignment are shown in red and green, respectively.B) Structural comparison of the distinct region.

Figure S12 :
Figure S12: Illustration of docked structures for the most promising compounds in comparison to GLS4.Top Left: GT-32, top right: GT-39, bottom right: GT-46, bottom left comparison of GLS4 (purple) and GT-32 binding.The compounds are shown in the VdW representation.Carbon, oxygen and nitrogen of the compounds are shown as orange, red and blue, respectively.Three residues known to form interactions with CAMs are also displayed in cyan: W102, T128 and L140.Most hydrogens are omitted for clarity.

Figure S13 :
Figure S13: Example of a simulated system, the periodic box of the starting state of dimer of dimers with bound GT-32 it shown in top view (left), and side view (right).The AB dimer and the CD dimer are shown in blue and red, respectively, using cartoon representation.Water molecules are shown as red dots.GT-32 is colored gray and is shown in vdW representation.Finally ions are shown as spheres, with yellow and cyan corresponding to sodium and chloride ions, respectively.

Figure S14 :
Figure S14: Illustration of an image processed using ImageJ.A) The original, unprocessed image.B) Image highlighting particles identified by ImageJ, ranging in size from 800 to 2000 pixels.

Table S1 :
Summarized results of HBV DNA inhibition and toxicity testing for the first round of selected compounds.N/A stands for not applied.

Table S2 :
Summarized results of HBV DNA inhibition and toxicity testing for the second round of selected compounds.

Table S5 :
The range of sampled base and spike angles in deg for all simulated systems.The values are based on the calculated SDEs.