Gs protein peptidomimetics as allosteric modulators of the β2-adrenergic receptor.

Lotte-Emilie Boyhus,Jacob Hartvig Løper,Micha Ben Achim Kunze,Mia Danielsen,Phuong Thu Tran,Nina Smidt Bengtson,Daniel Sejer Pedersen,Jesper Mosolff Mathiesen,Kresten Lindorff-Larsen,Xavier Kubiak,Tjerk J Sminia,Søren G F Rasmussen

doi:10.1039/c7ra11713b

Lotte-Emilie Boyhus, Jacob Hartvig Løper + Show 10 more

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https://doi.org/10.1039/c7ra11713b

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Journal: RSC advances	Publication Date: Jan 1, 2018
Citations: 9	License type: CC BY 3.0

Affiliation: University of Copenhagen

Abstract

A series of Gs 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 Gs protein (PDB 3SN6). We hypothesised that such peptidomimetics may function as allosteric modulators that target the intracellular Gs protein binding site of the β2AR. Peptidomimetics were designed to mimic the 15 residue C-terminal α-helix of the Gs 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 Gs protein for the intracellular binding site to block ISO-induced cAMP formation, but are unable to stabilise an active-like receptor conformation.

Highlights

The importance of G protein-coupled receptors (GPCRs) within drug discovery is undisputed
Hamm and co-workers previously reported that a peptide comprised of the last 12 amino acid residues from the G protein a subunits (Ga) Cterminus (GasCT12) was capable of inhibiting Gs protein coupling to the b2adrenergic receptor (b2AR) and increased agonist affinity for the receptor.[15]
In our hands the corresponding proteinogenic 15-mer peptide (GasCT15) did not block agonist induced cAMP formation in b2AR cell membranes, whereas nanobody 80 (Nb80) signi cantly inhibited the maximal efficacy of ISO (Fig. 1c)

Summary

Introduction

The importance of G protein-coupled receptors (GPCRs) within drug discovery is undisputed. GPCR ligands generally bind to the extracellular side of the receptor and target the orthosteric or allosteric binding sites, or both as bivalent ligands.[3] On the other hand, the intracellular surface of GPCRs has largely been ignored in the development of allosteric modulators Such allosteric modulators could conceivably be designed to target the receptor surface responsible for recruiting intracellular transducers such as the G proteins and arrestins and be useful pharmacological tool compounds for studying GPCR signal transduction and possibly provide a new avenue for drug discovery. The structure (PDB 5X7D) clearly shows the ligand occupying the G protein binding pocket.[5]

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Conclusion