G protein-coupled receptors (GPCRs) are membrane-embedded proteins, considered one of the most important drug target class, as these receptors accounts for 30-40 % of all marketed drug targets. Unfortunately, it is quite challenging to gain insights into the structure and functioning of GPCRs due to their intrinsic dynamics and high instability when extracted from the cell membrane. Furthermore, GPCRs are difficult to stabilize in the appropriate conformation (e.g. active state) to identify binders with the desired pharmacology. Herein, we describe an approach to mimic the G protein, an endogenous allosteric modulator of GPCRs, via a peptide mimicry method and synthesized rigidified variants of a helical G protein epitope, which interacts with the GPCR.1
A key epitope of the Gs protein, the α5 helix which is responsible for most interactions with the β2 adrenergic receptor (β2AR), was chosen as a template to design the peptidomimetics. Peptide stapling (i.e. covalent linkage of two helical loops) was used as the main strategy to stabilize the helical conformation, as confirmed by circular dichroism. Further structural modifications were introduced to improve the affinity for the receptor and the solubility of the peptide. Simultaneously the peptides were analyzed by radioligand displacement assay, to quantify the stabilization of the receptor in active conformation. This resulted in the identification of peptide mimetics stabilizing the β2AR in an active state, providing a tool comparable to Cb80, a well-known allosteric modulator of the β2AR. The generic character of the Gs peptidomimetics was proven by performing radioligand binding assays on a different Gs-coupled receptor, the dopamine 1 receptor (D1R). During fragment-based screening, these (un)constrained peptidomimetics of the α5 helix in Gs proteins, were able to identify agonist-prone fragments for selected drug targets, and as such they behave as generic tool that could be used in drug discovery programs.2,3