G_s protein peptidomimetics as allosteric modulators of the β₂-adrenergic receptor

A series of G_s protein peptidomimetics were designed and synthesised based on the published X-ray crystal structure of the active state β₂-adrenergic receptor (β₂AR) in complex with the G_s protein (PDB 3SN6). We hypothesised that such peptidomimetics may function as allosteric modulators that target the intracellular G_s protein binding site of the β₂AR. Peptidomimetics were designed to mimic the 15 residue C-terminal α-helix of the G_s protein and were pre-organised in a helical conformation by (i, i + 4)-stapling using copper catalysed azide alkyne cycloaddition. Linear and stapled peptidomimetics were analysed by circular dichroism (CD) and characterised in a membrane-based cAMP accumulation assay and in a bimane fluorescence assay on purified β₂AR. Several peptidomimetics inhibited agonist isoproterenol (ISO) induced cAMP formation by lowering the ISO maximal efficacy up to 61%. Moreover, some peptidomimetics were found to significantly decrease the potency of ISO up to 39-fold. In the bimane fluorescence assay none of the tested peptidomimetics could stabilise an active-like conformation of β₂AR. Overall, the obtained pharmacological data suggest that some of the peptidomimetics may be able to compete with the native G_s protein for the intracellular binding site to block ISO-induced cAMP formation, but are unable to stabilise an active-like receptor conformation.