Figure 2: Identification of Barbadin (compound #42) as an inhibitor of the interaction between β2-adaptin and β-arrestin.

(a–c) Concentration-response curves of compounds #1, #33 and #42 selected from the BRET-based screen (Fig. 1) using the same β2-adaptin/β-arrestin1 interaction assay. Dotted line represents the level of AVP-promoted BRET upon pre-incubation with DMSO. Data are the mean±s.e.m. of three independent experiments. (d) Chemical structure of Barbadin (IUPAC name: 3-amino-5-(4-benzylphenyl)-3H,4H-thieno[2,3-d]pyrimidin-4-one). (e,f) Docking pose of Barbadin (green sticks) within the groove of β2-adaptin platform subdomain (grey ribbon (e), or surface (f)) that is the site of interaction with β-arrestin (orange ribbon and sticks (f)). β2-adaptin residues known to interact with β-arrestin are labelled and shown as grey stick. Hydrogen-bonds interactions between Barbadin and both Tyr-888 and Glu-902 are depicted as magenta dotted lines (e). Superimposition of Barbadin with the β-arrestin1 C-terminus peptide as in the co-crystal structure (PDB entry 2IV8), where Phe-388, Phe-391 and Arg-395 are the three key residues for β-arrestin binding interaction (shown from top to bottom as orange sticks) (f). (g) Thermal denaturation of β2-adaptin (residues 700–937) and concentration-dependent stabilization effect of Barbadin. The maximum of the first derivatives of fluorescence (dF/dT) data from differential scanning fluorimetry corresponds to the melting temperatures (Tm, indicated with a dotted line) of β2-adaptin in the presence of DMSO, Barbadin or β-arrestin1 C-tail peptide (positive control) at the indicated concentrations.