The development of a bioassay expressing the human MRGPRX2 receptor for ex vivo drug discovery

Successful discovery of potent and selective ligands for therapeutically relevant receptors is indispensable for future clinical applications to treat a wide array of diseases. The mas-related G protein-coupled receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, governs to a significant extent the human immune response. A number of ligands for MRGPRX2 have been identified including human cathelicidin (LL-37) and antimicrobial peptides from plants (PAMPS). Future drug discovery to identify MRGPRX2 specific ligands undoubtedly will benefit from a continued screening of small molecule and peptide libraries. Mast cells (MCs) are generally difficult and expensive to maintain in vitro and, therefore, are not economical to use for screening large numbers of molecules. Herein, we present the development of a bioassay expressing the human MRGPRX2 receptor, linked to inwardly rectifying potassium (GIRK1/2) channels that are opened upon binding of β-γ subunits of a Gi/o-type protein following activation of MRGPRX2. This communication presents a method-focused study demonstrating a simple, yet robust and inexpensive electrophysiological approach (voltage clamp) using oocytes from Xenopus laevis to perform high-throughput screening of libraries. Unexpectedly, this bioassay also supports the claim that the MRGPRX2 receptor can couple efficiently to Gi/o-type proteins, a pathway not yet described. Furthermore, the approach allows structure-function research using mutated MRGPRX2 genes and can study reciprocal cross talks between MRGPRX2 and other co-expressed membrane-bound proteins, such as voltage- or ligand-gated ion channels, mimicking in vivo conditions.