The Mechanism of Action of Hepatitis B Virus Capsid Assembly Modulators Can Be Predicted from Binding to Early Assembly Intermediates

Abstract

Interfering with the self-assembly of virus nucleocapsids is a promising approach for the development of novel antiviral agents. Applied to hepatitis B virus (HBV), this approach has led to several classes of capsid assembly modulators (CAMs) that target the virus by either accelerating nucleocapsid assembly or misdirecting it into noncapsid-like particles, thereby inhibiting the HBV replication cycle. Here, we have assessed the structures of early nucleocapsid assembly intermediates, bound with and without CAMs, using molecular dynamics simulations. We find that distinct conformations of the intermediates are induced depending on whether the bound CAM accelerates or misdirects assembly. Specifically, the assembly intermediates with bound misdirecting CAMs appear to be flattened relative to those with bound accelerators. Finally, the potency of CAMs within the same class was studied. We find that an increased number of contacts with the capsid protein and favorable binding energies inferred from free energy perturbation calculations are indicative of increased potency.

Figure 1:

HBV capsid structure and assembly. (A) Structure of Cp149 dimer taken from the capsid structure (PDB code 3J2V). The names of helices α1–5 are indicated; yellow and green helices form the assembly interface, while the dimerization interface helices, α3-α4, are colored blue and purple respectively. (B) Overlap of GLS4 and AT130 binding sites after alignment of the bound protein structures (PDB codes 5E0I and 4G93, respectively). Both compounds occupy a similar space in the HAP pocket and interact with Trp102. (C) HBV capsid assembly process based on experimental data and mathematical modeling.^,–

Figure 2:

Comparison of standard deviation ellipses (SDEs) for spike and base angle distributions for selected simulations. SDEs are centered on the average values of a distribution. Their width and height are based on the standard deviation of the corresponding variables, while their rotation is based on the correlation between two variables. The ellipses shown here are scaled to envelop 90% of the sampled distributions. The locations of the starting structures in the graphs are shown in Figure S9. With the exception of Hexa SDE, all other SDEs are for tetramer structures. (A) SDEs of base and spike angles for the simulated apo structures and for PPA compounds, which accelerate assembly. (B) SDEs of base and spike angles for selected bound misdirecting CAMs. (C) SDEs of base and spike angles for SBAs, which do not alter the empty capsid assembly, and GLPs, which cause formation of misformed capsids. In (B) and (C) the results for the apo tetramer and hexamer are added for comparison. (D) Definitions of spike and base angles. See Methods and Supporting Information for details.

Figure 3:

Standard deviation ellipses for hexamers with one bound GLS4, GLP-26, or AT130. Apo hexamer is added for comparison.

Figure 4:

Selected snapshots of tetramers from our simulations illustrate the effect of spike and base angles on the curvature of the assembly intermediates. See Figure 2D for base and spike definitions. θ_b and θ_s correspond to the values of base and spike angles for each structure, respectively. Increases in either spike or base angle induce more curvature in the tetramer structures, with the most curved structures having large base and spike angles.

Figure 5:

Comparison of hydrogen bonding with HBV capsid protein between GLP/SBA derivatives, and HAP compounds. Hydrogens are omitted for clarity. The CAM is colored in green and protein residues are colored in cyan. (A) Hydrogen bonding with GLS4. (B) Hydrogen bonding with GLP-26.

Figure 6:

Setup and results of our FEP calculations. The structural differences from HAP12 are shown in red. All differences in binding free energies are shown in kcal/mol. Black values were calculated from experimental EC₅₀s from Table 4 using Eq. 1, while blue values were obtained from FEP calculations.