Al Hamwi, GhazlGhazlAl HamwiAlnouri, Mohamad WessamMohamad WessamAlnouriVerdonck, SvenSvenVerdonckLeonczak, PiotrPiotrLeonczakChaki, ShaswatiShaswatiChakiFrischbutter, StefanStefanFrischbutterKolkhir, PavelPavelKolkhirMatthey, MichaelaMichaelaMattheyKopp, ConstantinConstantinKoppBednarski, MarekMarekBednarskiRiedel, Yvonne K.Yvonne K.RiedelMarx, DanielDanielMarxClemens, SophieSophieClemensNamasivayam, VigneshwaranVigneshwaranNamasivayamGattner, SusanneSusanneGattnerThimm, DominikDominikThimmSylvester, KatharinaKatharinaSylvesterWolf, KatharinaKatharinaWolfKremer, Andreas EmanuelAndreas EmanuelKremerJonghe, Steven deSteven deJongheWenzel, DanielaDanielaWenzelKotańska, MagdalenaMagdalenaKotańskaAli, HydarHydarAliHerdewijn, Piet A.M.M.Piet A.M.M.HerdewijnMüller, Christa ElisabethChrista ElisabethMüller2025-06-102025-06-102025https://publica.fraunhofer.de/handle/publica/48848010.1038/s41392-025-02209-82-s2.0-10500315333740254631The MAS-related G protein-coupled receptor-X2 (MRGPRX2), an orphan receptor expressed on mast cells (MCs), is upregulated upon inflammation and induces hypersensitivity and inflammatory diseases. In contrast to the large number of MRGPRX2 agonists, only a few antagonists have been described, and no optimization has been reported to improve potency, selectivity, and drug-like properties. Antagonists with ancillary inhibition of the putative mouse ortholog MRGPRB2 have not been described. Here, we present a multi-disciplinary approach involving chemistry, biology, and computational science, resulting in the development of a small-molecule MRGPRX2 antagonist (PSB-172656, 3-ethyl-7,8-difluoro-2-isopropylbenzo[4,5]imidazo [1,2-a] pyrimidin-4(1H)-one) based on a fragment screening hit. The compound exhibits metabolic stability, low cytotoxicity, and competitive blockade of MRGPRX2 activation induced by a diverse range of agonists. It displays subnanomolar potency in Ca²⁺ mobilization assays (K<inf>i</inf> value 0.142 nM) and was found to block MRGPRX2-mediated Gα<inf>q</inf> and Gα<inf>i1</inf> dissociation, in addition to β-arrestin-2 recruitment. PSB-172656 is selective for MRGPRX2 versus all other MRGPRX subtypes. Its effect on MCs was confirmed in cell lines, including rat basophilic leukemia cells (RBL-2H3) recombinantly expressing human MRGPRX2, human Laboratory of Allergic Diseases 2 (LAD2) MCs, and native human skin MCs. PSB-172656 was found to additionally block the putative mouse ortholog of MRGPRX2, MRGPRB2, as determined in Ca²⁺ mobilization assays (K<inf>i</inf> 0.302 nM), and to prevent mouse tracheal contractions, local allergic reactions, and systemic anaphylactic symptoms. PSB-172656 constitutes a unique pharmacological tool and has the potential to be developed as a drug for mast cell-mediated hypersensitivity reactions and chronic inflammatory diseases, addressing a huge unmet medical need.enfalsejournal article