MRGPRX2 receptor activation as a rapid, high-throughput mechanistic-based approach for detecting peptide-mediated human mast cell degranulation liabilities.

Abstract

Abstract Mast cells play key roles in allergy, anaphylaxis and pathogen/toxin defense. They contain cytoplasmic granules with a wide spectrum of pleotropic mediators (e.g. histamine, tryptase, serotonin) that are released upon activation. Mast cell degranulation (MCD) occurs upon clustering of the IgE receptor in presence of antigen or via non-IgE mediated mechanisms, one of which is by means of the Mas-related G protein coupled receptor (MRGPRX2). MRGPRX2 is a non-selective, low affinity binding receptor that responds to many basic biogenic amines and peptides. Consequently, MCD is an important potential safety liability for peptide therapeutics. To improve screening of peptides for this liability, we assessed a MRGPRX2-CHO cell-based receptor activation assay using positive (n=30) and negative (n=29) control peptides, which were previously characterized for MCD liability in CD34+ bone marrow-derived human mast cell cultures. MRGPRX2-CHO cells were engineered to selectively activate beta-arrestin upon receptor stimulation. In addition to beta-arrestin, Ca2+ was also measured to determine the potential of ligand bias (selective engagement of G-protein dependent or independent pathways). The MRGPRX2-CHO receptor assay had a sensitivity of 97% (false negative 3%) and specificity of 93% (false positive 7%) against the outcome in human mast cell cultures. No clear evidence of ligand bias was observed. Because MRGPRX2 is overexpressed in CHO cells, the assay may slightly over predict, and does not inform on other mechanisms of MCD not mediated by MRGPRX2. Assessing MRGPRX2 receptor activation provides a rapid, high-throughput and economical mechanism-based approach for detecting peptide-mediated human MCD liabilities.