Pharmacomodulation of a ligand targeting the HBV capsid hydrophobic pocket

Abstract

Hepatitis B virus (HBV) is a small enveloped retrotranscribing DNA virus and an important human pathogen. Its capsid-forming core protein (Cp) features a hydrophobic pocket proposed to be central notably in capsid envelopment. Indeed, mutations in and around this pocket can profoundly modulate, and even abolish, secretion of enveloped virions. We have recently shown that Triton X-100, a detergent used during Cp purification, binds to the hydrophobic pocket with micromolar affinity. We here performed pharmacomodulation of pocket binders through systematic modifications of the three distinct chemical moieties composing the Triton X-100 molecule. Using NMR and ITC, we found that the flat aromatic moiety is essential for binding, while the number of atoms of the aliphatic chain modulates binding affinity. The hydrophilic tail, in contrast, is highly tolerant to changes in both length and type. Our data provide essential information for designing a new class of HBV antivirals targeting capsid-envelope interactions.

Fig. 1. Workflow to assess binding to the hydrophobic pocket. (A) Organization of the five helices of Cp149 dimer (PDB 1QGT) coloured on one monomer. (B) Structure of the HBV capsid, assembled from 120 copies of Cp149 dimer. (C) Structure of the Cp149 dimer in surface representation. The residues of the hydrophobic pocket impacted by the interaction of TX100 are highlighted in blue. The eight residues whose NMR signals were used to quantify CSPs are highlighted in red. The pocket entrance is indicated by an arrow. Naturally occurring mutations are plotted in green. (D) Sequence of the full-length core protein (Cp183) with mutations highlighted. (E) Structure of the TX100 molecule (compound 1a), with the hydrophilic tail in red, the aromatic moiety in blue, and the alkyl part in para-position in orange. (F) Solution-state NMR spectra of the ²H¹³C¹⁵N-Cp149 apo dimer in grey, and with four equivalents TX100 in red ([Cp149_monomer] = 80 μM and [TX100] 320 μM). Peaks used to assess CSPs are circled and labelled with their amino-acid type and position in the sequence. The assigned apo spectrum is shown in Fig. S2, and the full set of CSPs between the two spectra are shown in Fig. S3. (G) ITC raw data and binding isotherms displaying the titration of 4-hexylphenol (compound 2a) against Cp149 reassembled capsids at pH 7.5 and 298 K (K_D = 13 ± 3 μM). For an overview of ITC runs see Fig. S4.

Fig. 2. Effects of modulations of the TX100 aromatic ring and hydrophilic tail. (A) Chemical structures of compounds tested from categories 1 and 2. (B) NMR CSPs induced by the compounds for the eight selected capsid resonances (for full spectra see Fig. S8 and S9†). CSP averages are indicated by dotted lines. Residue numbers of the representative peaks are given below the CSPs; all CSPs throughout the manuscript are shown at the same scale.

Fig. 3. Effects of modulations of the TX100 hydrophobic moiety. (A) Compounds tested from categories 3 and 4. (B) NMR chemical-shift perturbations induced by compounds on the eight selected capsid resonances (for full spectra see Fig. S10 and S11†). CSPs averages are indicated by dotted lines.

Fig. 4. Effect of modulations of the TX100 aromatic ring, and concurrently on the hydrophilic and hydrophobic groups. (A) Compounds tested from categories 5 and 6. (B) NMR CSPs induced by the compounds on the eight selected capsid resonances (for full spectra see Fig. S12 and S13†). CSPs averages are indicated by dotted lines.

Fig. 5. Computed models of OP and TX100 bound in the hydrophobic pocket of the Cp149 dimer. Docked model of (A) OP (compound 1c) and (B) TX100 (1a) in the hydrophobic pocket of Cp149 dimer. Residues located in the direct proximity of ligands (<5 Å) are represented in blue sticks. (C and D) Comparison of bound TX100 in the cryo-EM structure (in magenta, protein in wheat, PDB 7PZK) with the docked models (in green, protein in grey) for (C) TX100 and (D) OP. Neighbouring residues V13, E64, M93, K96 and F97 are shown as sticks. In both cryo-EM structure and docked models, TX100 contains n = 3 ethylene oxide units in the PEG tail.

Fig. 6. Solid-state NMR can probe the impact of the ligand's hydrophilic tail on the hydrophobic pocket. (A) Comparison of 2D extracts from ¹³C–¹³C-DARR solid-state NMR spectra of Cp149 capsids reassembled without compounds (empty pocket, grey), and in presence of TX100 (bound pocket, red), OP (blue), or 4HF (orange). Full aliphatic regions of DARR spectra are shown in Fig. S14. (B) Comparison of ¹³C-CSPs induced by the three compounds. Three groups could be identified based on their behaviour when bound to 4HF: P5 at the entrance of the pocket showing a mixture of free/bound pocket; the residues showing intermediate CSPs; and the residues showing no CSPs with 4HF. (C) Structure of Cp183-TX100 (PDB 7PZK) with residues involved in the three CSP classes shown in orange. The right panel is rotated by 180° to display the bottom of the hydrophobic pocket and the proximity with F97 sidechain.

Fig. 7. Summary of interacting molecules in the hydrophobic pocket of the HBV core protein. Overview of the different moieties attached to either side of the central aromatic ring, classified as function of their CSPs. Only compounds with CSPs above 0.038 ppm are shown.