Induction of Hepatitis B Core Protein Aggregation Targeting an Unconventional Binding Site

Reviewer #1 (Public Review):

Summary:

In this paper, the authors present an interesting strategy to interfere with the HBV life cycle: the preparation of geranyl and peptides' dimers that could impede the correct assembly of hepatitis B core protein HBc into viable capsids. These dimers are of different nature, depending on the HBc site the authors plan to target. A preliminary study with geranyl dimers (targeting a hydrophobic site of HBc) was first investigated. The second series deals with peptide-PEG linker-peptide dimers, targeting the tips of HBc dimer spikes.

Strengths:

This work is very well conducted, combining ITC experiments (for determination of dimers' KD), cellular effects (thanks to the grafting of previously developed dimers with polyarginine-based cell penetrating peptide) HBV infected HEK293 cells and Cryo-EM studies.

The findings of these research teams unambiguously demonstrated the interest of such dimeric structures in impeding the correct HBV life cycle and thus, could bring solutions in the control of its development. Ultimately, a new class of HBV Capside Assembly Modulators could arise from this study.

There is no doubt that this work could bring very interesting information for people working on VHB.

Weaknesses:

Some minor corrections must be made, especially for a more precise description of the strategy and the chemical structure of the designed new VHB capsid assembly modulators.

Reviewer #2 (Public Review):

Summary:

Vladimir Khayenko et al. discovered two novel binding pockets on HBc with in vitro binding and electron microscopy experiments. While the geranyl dimer targeting a central hydrophobic pocket displayed a micromolar affinity, the P1-dimer binding to the spike tip of HBc has a nanomolar affinity. In the turbidity assay and at the cellular level, an HBc aggregation from peptide crosslinking was demonstrated.

Strengths:

The study identifies two previously unexplored binding pockets on HBc capsids and develops novel binders targeting these sites with promising affinities.

Weaknesses:

While the in vitro and cellular HBc aggregation effects are demonstrated, the antiviral potential against HBV infection is not directly evaluated in this study.

Reviewer #3 (Public Review):

Summary:

HBV is a continuing public health problem and new therapeutics would be of great value. Khayenko et al examine two sites in the HBc dimer as possible targets for new therapeutics. Older drugs that target HBc bind at a pocket between two HBc dimers. In this study Khayenko et al examine sites located in the four helix bundle at the dimer interface.

The first site is a pocket first identified as a triton100 binding site. The authors suggest it might bind terpenes and use geraniol as an example. They also test a decyl maltose detergent and a geraniol dimer intended for bivalent binding. The KDs were all in the 100µM range. Cryo-EM shows that geraniol binds the targeted site.

The second site is at the tip of the spike. Peptides based on a 1995 study (reference 43) were investigated. The authors test a core peptide, two longer peptides, and a dimer of the longest peptide. A deep scan of the longest monomer sequence shows the importance of a core amino acid sequence. The dimeric peptide (P1-dimer) binds almost 100 fold better than the monomer parent (P1). Cryo-EM structures confirm the binding site. The dimeric peptide caused HBc capsid aggregation When HBc expressing cells were treated with active peptide attached to a cell penetrating peptide, the peptide caused aggregation of HBc antigen mirroring experiments with purified proteins.

Strengths:

The two sites have not been well investigated. This paper marks a start. The small collection of substrates investigated led to discovery of a dimeric peptide that leads to capsid aggregation, presumably by non-covalent crosslinking. The structures determined could be very useful for future investigations.

Weaknesses:

In this draft, the rational for targets for the triton x100 site is not well laid out. The target molecules bind with KDs weaker that 50µM. The way the structural results are displayed, one cannot be sure of the important features of binding site with respect to the the substrate. The peptide site and substrates are better developed, but structural and mechanistic details need to be described in greater detail.