Abstract by Ziyang Chen

Functional diversity of γ-aminobutyric acid (GABA) type B receptors (GBRs) results from the distinct subcellular localizations of the two receptor subtypes, GB1a/2R and GB1b/2R. The latter predominantly localizes in dendritic spines generating slow inhibitory postsynaptic currents. The N-terminal of GB1a/2R contains two sushi domains (SD 1 and 2) that directly interact with amyloid precursor protein (APP) and thereby facilitate trafficking of GB1a/2R to glutamatergic axon terminals regulating glutamate release. Moreover, adherence junction-associated protein 1 (AJAP-1) and PILRα-associated neural protein (PIANP) also bind directly to SD1 while their function remains elusive. We envisioned that GB1a-SD-involved APP, AJAP-1 and PIANP interactions provide a promising approach to selective modulate the function of presynaptic GB1a/2R.

In this study, we mapped the GB1a-SD binding epitopes within APP, AJAP-1 and PIANP employing the SPOT technology. Subsequent systematic truncation and elongation scans redefined the binding epitopes to 14-mer peptides. We further performed systematic structure-activity relationship (SAR) studies of the redefined SD-binding peptides including Ala, D-amino acids, N-methylation, deep mutational and in silico non-canonical amino acid (ncAA)-based positional scans. The SAR-obtained data guided the design of low nanomolar affinity inhibitors of the SD-specific interactions. Further characterization revealed that the designed potent peptides are highly stable in human plasma and non-toxic in a cell-based assay. Conjugation of developed peptides to cell-penetrating peptides (CPPs) resulted in excellent cell membrane permeability, however, causing significant decrease in affinity. Truncation of the poly-Arg CPP-tag restored the high target affinity while maintaining excellent permeability. Furthermore, pull-down experiments validated the developed peptides can compete with native SDs-specific interactions and are able enriched GB1a subunit from mice brain lysates. Finally, cellular functional assay confirmed that designed peptides selectively modulate the function of GB1a/2Rs.

In summary, the designed first-in-class peptide-based probes targeting the GB1a-SD involved interactions exhibit potentially regioselective modulation of presynaptically localized GB1a/2Rs. These peptides represent promising tool compounds to reveal the therapeutic potential of SD-specific protein-protein interactions (PPIs).