Synthesis and structure-activity relationship study of saponin-based membrane fusion inhibitors against SARS-CoV-2.

We previously discovered that triterpenoid saponin platycodin D inhibits the SARS-CoV-2 entry to the host cell. Herein, we synthesized various saponin derivatives and established a structure-activity relationship of saponin-based antiviral agents against SARS-CoV-2. We discovered that the C3-glucose, the C28-oligosaccharide moiety that consist of (→3)-ß-d-Xyl-(1 â†’ 4)-α-l-Rham-(1 â†’ 2)-ß-d-Ara-(1 â†’ ) as the last three sugar units, and the C16-hydroxyl group were critical components of saponin-based coronavirus cell entry inhibitors. These findings enabled us to develop minimal saponin-based antiviral agents that are equipotent to the originally discovered platycodin D. We found that our saponin-based antiviral agents inhibited both the endosomal and transmembrane protease serine 2-mediated cell surface viral entries. Cell fusion assay experiment revealed that our newly developed compounds inhibit the SARS-CoV-2 entry by blocking the fusion between the viral and host cell membranes. The effectiveness of the newly developed antiviral agents over various SARS-CoV-2 variants hints at the broad-spectrum antiviral efficacy of saponin-based therapeutics against future coronavirus variants.

Inhibitors of synaptic vesicle exocytosis reduce surface expression of postsynaptic glutamate receptors.

Bafilomycin A1, a vacuolar H+-ATPase inhibitor, and botulinum toxin B and tetanus toxin, both vesicle fusion inhibitors, are widely known exocytosis blockers that have been used to inhibit the presynaptic release of neurotransmitters. However, protein trafficking mechanisms, such as the insertion of postsynaptic receptors and astrocytic glutamate-releasing channels into the plasma membrane, also require exocytosis. In our previous study, exocytosis inhibitors reduced the surface expression of astrocytic glutamate-releasing channels. Here, we further investigated whether exocytosis inhibitors influence the surface expression of postsynaptic receptors. Using pH-sensitive superecliptic pHluorin (SEP)-tagged postsynaptic glutamate receptors, including GluA1, GluA2, GluN1, and GluN2A, we found that bafilomycin A1, botulinum toxin B, and/or tetanus toxin reduce the SEP fluorescence of SEP-GluA1, SEP-GluA2, SEP-GluN1, and SEP-GluN2A. These findings indicate that presynaptic vesicle exocytosis inhibitors also affect the postsynaptic trafficking machinery for surface expression. Finally, this study provides profound insights assembling presynaptic, postsynaptic and astrocytic viewpoints into the interpretation of the data obtained using these synaptic vesicle exocytosis inhibitors.

Direct interaction with 14-3-3γ promotes surface expression of Best1 channel in astrocyte.

BACKGROUND: Bestrophin-1 (Best1) is a calcium-activated anion channel (CAAC) that is expressed broadly in mammalian tissues including the brain. We have previously reported that Best1 is expressed in hippocampal astrocytes at the distal peri-synaptic regions, called microdomains, right next to synaptic junctions, and that it disappears from the microdomains in Alzheimer's disease mouse model. Although Best1 appears to be dynamically regulated, the mechanism of its regulation and modulation is poorly understood. It has been reported that a regulatory protein, 14-3-3 affects the surface expression of numerous membrane proteins in mammalian cells. METHODS: The protein-protein interaction between Best1 and 14-3-3γ was confirmed by yeast-two hybrid assay and BiFC method. The effect of 14-3-3γ on Best1-mediated current was measured by whole-cell patch clamp technique. RESULTS: We identified 14-3-3γ as novel binding partner of Best1 in astrocytes: among 7 isoforms of 14-3-3 protein, only 14-3-3γ was found to bind specifically. We determined a binding domain on the C-terminus of Best1 which is critical for an interaction with 14-3-3γ. We also revealed that interaction between Best1 and 14-3-3γ was mediated by phosphorylation of S358 in the C-terminus of Best1. We confirmed that surface expression of Best1 and Best1-mediated whole-cell current were significantly decreased after a gene-silencingof 14-3-3γ without a significant change in total Best1 expression in cultured astrocytes. Furthermore, we discovered that 14-3-3γ-shRNA reduced Best1-mediated glutamate release from hippocampal astrocyte by recording a PAR1 receptor-induced NMDA receptor-mediated current from CA1 pyramidal neurons in hippocampal slices injected with adenovirus carrying 14-3-3γ-shRNA. Finally, through a structural modeling, we found critical amino acid residues containing S358 of Best1 exhibiting binding affinities to 14-3-3γ. CONCLUSIONS: 14-3-3γ promotes surface expression of Best1 channel in astrocytes through direct interaction.