Design, synthesis and pharmacological evaluation of novel small molecules as mas-related G protein-coupled receptor X2 (MRGPRX2) antagonist for the treatment of pseudo allergic reactions

Abstract

Pseudo allergic reaction is an immediate systemic fatal hypersensitivity response induced by non-IgE mediated mast cell activation. Mas-related G protein-coupled receptor X2 (MRGPRX2) is a Class A orphan GPCR recently identified as a mast cell receptor responsible for non-IgE-mediated mast cell activation induced by endogenous peptides, exogenous peptides, and several FDA-approved drugs. MRGPRX2 has been recently gained attention as a novel drug target for the treatment of non-IgE-mediated pseudo allergic reactions and several other diseases. Although pseudo allergic reactions are prevalent all over the world, no MRGPRX2 targeted therapeutic drugs are available in the market for the treatment. This thesis aimed to design and develop novel small molecule MRGPRX2 antagonists which can be further developed as a drug to treat pseudo allergic reactions. I first identified natural flavonoid genistein as a lead molecule which inhibited mast cell degranulation, MRGPRX2 activation, Ca2+ influx and pseudo allergic symptoms in compound 48/80 induced pseudo allergic mice model. Second, we have developed a homology model of MRGPRX2 and identified the pharmacophore region of lead molecule genistein interacting with MRGPRX2. A structure aided drug designing, and virtual screening approach was used to design a library of benzo[de]isoquinoline, phenalen-1-one and naphthoate ring-based novel small molecules. After in silico screening for the binding affinity to MRGPRX2, six novel small molecules (GE0117, GE0118, GE0119, GE1109, GE1110, and GE1111) with the highest binding affinity were shortlisted for chemical synthesis. The synthesized small molecules were characterized by LCMS and NMR and had >95% purity. Further, the pharmacological activity of novel small molecules to modulate IgE-independent mast cells activation was investigated via a battery of in vitro experiments (functional and receptor activation assays). In vitro experiments such as human mast cells degranulation assay, β-arrestin based PRESTO Tango assay, secondary messenger Ca2+ flux assay, chemokine, and prostaglandin release assay were used in this thesis. To determine the effect of potent novel small molecules (GE0117, GE1109, and GE1111) on in vivo pseudo allergic and inflammatory reactions, I performed Evans blue extravasation assay in established mouse models and also collected ear tissues for in vivo mast cell degranulation and histological analysis. Moreover, in a systemic anaphylaxis mouse model, the effect of novel small molecule antagonists on compound 48/80 induced body temperature changes and anaphylaxis symptoms was evaluated. The findings demonstrated that the novel small molecule antagonists decreased compound 48/80 induced mast cell degranulation, MRGPRX2 activation, Ca2+ influx, chemokine, and prostaglandin generation. Moreover, antagonist treatment showed significant inhibition of compound 48/80 induced tissue extravasation, in vivo mast cell degranulation and histological changes in surrounding tissue. The antagonist treatment rehabilitated compound 48/80 induced hypothermia and severity of anaphylaxis symptoms in mice. Additionally, the most potent small-molecule antagonist, GE1111, showed its in vitro antagonistic activity against other MRGPRX2 agonists such as cortisatin-14 (CST-14), ciprofloxacin (FDA approved drug), ant venom peptide P17, and its more potent alanine analog (Ala 4 P17). Overall, this thesis presented novel small molecule MRGPRX2 antagonists as a new potential drug candidate to treat MRGPRX2 mediated pseudo allergic reactions.