Inhibition of Mast Cell Degranulation by Novel Small Molecule MRGPRX2 Antagonists

MRGPRX2, expressed on mast cells (MCs), is associated with non–immunoglobulin E (IgE)–mediated MC activation. The authors intended to identify potent and selective MRGPRX2 antagonists for potential treatment of MC-mediated diseases.Experiments used Chinese hamster ovary (CHO) cells overexpressing human MRGPRX2, human embryonic kidney (HEK) cells expressing MRGPRB2 (the murine ortholog of MRGPRX2), MRGPRB2 knockout (KO) mice, MRGPRX2 knock-in (KI) mice, and Laboratory of Allergic Diseases (LAD2) MCs. They measured β-arrestin recruitment and inositol monophosphate (IP-1) accumulation. The former regulates G-protein (including MRGPRX2) signaling leading to IP-1 that releases Ca+ from endoplasmic reticulum. IV Evans blue was used to assess vascular permeability in the KO and KI mice before intradermal (ID) treatment with placebo, MRGPRX2, or goat anti-mouse IgE. Ex vivo human skin from operative samples had ID injection with MRGPRX2 antagonists followed by ID MRGPRX2 agonists and subsequent serial histamine release quantification.When exposed to various endogenous and exogenous MRGPRX2 ligands, the CHO and HEK cells produced β-arrestin and IP-1. Similarly treated LAD2 cells degranulated and produced a different MC product, β-hexosaminidase, but MRGPRX2 KO LAD2 cells did not. ID injection of MRGPRX2 agonists increased vascular permeability in wild-type mice, but not in the MRGPRB2 KO mice. These in vitro and in vivo studies indicated that the observed MC degranulation was fully mediated by MRGPRX2. Using iterative screening and modifications, the team optimized 2 small molecules (EP262 and EP9907) as specific MRGPRX2 antagonists. Both completely inhibited MRGPRX2 activation in the CHO cells, but did not in other MRGPRX molecules, and were also potent inhibitors of MC degranulation in multiple ex vivo human MCs and in LAD2 cells. KI mice injected with MRGPRX2 agonists triggered MC degranulation as evidenced by increased Evans blue vascular permeability. When EP262 and EP9907 were given orally, the KI mice had a dose-dependent reduction of MC degranulation and vascular permeability in response to all the MRGPRX2 agonists tested.MRGPRX2 antagonists potently inhibit MC degranulation in vitro and in vivo, providing a possible future therapeutic option for disorders of MC activation.For the generalist, the design of this study is less critical than the clinical importance of MRGPRX2 activation and the potential therapeutic implications of its antagonism. The phenotypic result of MC degranulation is independent of the receptor triggering it. Engagement of MRGPRX2 by numerous endogenous (eg, some eosinophil products, neuropeptides, endorphins, antimicrobial proteins) and exogenous (eg, narcotics, vancomycin, neuromuscular blocking agents, quinolones, icatibant, radiocontrast media) ligands is independent of IgE and thus independent of prior exposure and sensitization. MRGPRX2 is not only responsible for some forms of non–IgE-mediated MC activation (including anaphylaxis), but some studies show it to be upregulated in atopic and other MC disorders, such as chronic spontaneous urticaria. Thus, there is potential for additive or synergistic effects when MRGPRX2 and the high-affinity IgE receptor (FcɛR1) or other MC receptors are activated simultaneously. Monoclonal antibodies blocking IgE have no effect on MRGPRX2. The possible future addition of safe, selective, and effective MRGPRX2 antagonists has great potential for treatment of mast cell disorders that may help our pediatric patients in the future.