Guanine nucleotide exchange factor DOCK11-binding peptide fused with a single chain antibody inhibits hepatitis B virus infection and replication.

Hepatitis B virus (HBV) infection is a major global health problem with no established cure. Dedicator of cytokinesis 11 (DOCK11), known as a guanine nucleotide exchange factor (GEF) for Cdc42, is reported to be essential for the maintenance of HBV. However, potential therapeutic strategies targeting DOCK11 have not yet been explored. We have previously developed an in vitro virus method as a more efficient tool for the analysis of proteomics and evolutionary protein engineering. In this study, using the in vitro virus method, we screened and identified a novel antiasialoglycoprotein receptor (ASGR) antibody, ASGR3-10M, and a DOCK11-binding peptide, DCS8-42A, for potential use in HBV infection. We further constructed a fusion protein (10M-D42AN) consisting of ASGR3-10M, DCS8-42A, a fusogenic peptide, and a nuclear localization signal to deliver the peptide inside hepatocytes. We show using immunofluorescence staining that 10M-D42AN was endocytosed into early endosomes and released into the cytoplasm and nucleus. Since DCS8-42A shares homology with activated cdc42-associated kinase 1 (Ack1), which promotes EGFR endocytosis required for HBV infection, we also found that 10M-D42AN inhibited endocytosis of EGFR and Ack1. Furthermore, we show 10M-D42AN suppressed the function of DOCK11 in the host DNA repair system required for covalently closed circular DNA synthesis and suppressed HBV proliferation in mice. In conclusion, this study realizes a novel hepatocyte-specific drug delivery system using an anti-ASGR antibody, a fusogenic peptide, and DOCK11-binding peptide to provide a novel treatment for HBV.

Hydrogel-Stiffening and Non-Cell Adhesive Properties of Amphiphilic Peptides with Central Alkylene Chains.

Invited for the cover of this issue is the group of Takahiro Muraoka at Tokyo University of Agriculture and Technology and collaborators. The image depicts nanofiber formation of an amphiphilic peptide with a central alkylene chain that shows non-cell adhesive properties. Read the full text of the article at 10.1002/chem.202100739.

Hydrogel-Stiffening and Non-Cell Adhesive Properties of Amphiphilic Peptides with Central Alkylene Chains.

Amphiphilic peptides bearing terminal alkyl tails form supramolecular nanofibers that are increasingly used as biomaterials with multiple functionalities. Insertion of alkylene chains in peptides can be designed as another type of amphiphilic peptide, yet the influence of the internal alkylene chains on self-assembly and biological properties remains poorly defined. Unlike the terminal alkyl tails, the internal alkylene chains can affect not only the hydrophobicity but also the flexibility and packing of the peptides. Herein, we demonstrate the supramolecular and biological effects of the central alkylene chain length inserted in a peptide. Insertion of the alkylene chain at the center of the peptide allowed for strengthened ß-sheet hydrogen bonds and modulation of the packing order, and consequently the amphiphilic peptide bearing C2 alkylene chain formed a hydrogel with the highest stiffness. Interestingly, the amphiphilic peptides bearing internal alkylene chains longer than C2 showed a diminished cell-adhesive property. This study offers a novel molecular design to tune mechanical and biological properties of peptide materials.

Glycine Substitution Effects on the Supramolecular Morphology and Rigidity of Cell-Adhesive Amphiphilic Peptides.

Self-assembling peptides that are capable of adopting ß-sheet structures can generate nanofibers that lead to hydrogel formation. Herein, to tune the supramolecular morphologies, mechanical properties, and stimuli responses of the hydrogels, we investigated glycine substitution in a ß-sheet-forming amphiphilic peptide. Glycine substitution generally enhances conformational flexibility. Indeed, glycine substitution in an amphiphilic peptide weakened the hydrogels or even inhibited the gelation. However, unexpectedly, glycine substitution at the center of the peptide molecule significantly enhanced the hydrogel stiffness. The central glycine substitution affected the molecular packing and led to twisted ß-sheet structures and to nanofiber bundling, which likely led to the stiffened hydrogel. Importantly, the supramolecular structures were accurately predicted by molecular dynamics simulations, demonstrating the helpfulness of these techniques for the identification of self-assembling peptides. The hydrogel formed by the amphiphilic peptide with the central glycine substitution had cell adhesive function, and showed a reversible thermal gel-to-sol transition. Thus, glycine substitution is effective in modulating self-assembling structures, rheological properties, and dynamics of biofunctional self-assembling peptides.