Medicinal chemistry and molecular pharmacology of orphan human Mas-related G protein-coupled receptors X2 and X4 (MRGPRX2 and MRGPRX4)

Abstract

The MRGPRX protein subfamily comprise rhodopsin-like orphan GPCRs, whose endogenous agonists have not been identified yet. They are exclusively expressed in primates, including humans. All Mas-related GPCRs (MRGPRs) were first detected in dorsal root ganglia (DRGs) and trigeminal ganglia (TGs), suggesting a role in pain and itch. The MRGPRX2 subtype and its postulated mouse orthologue MRGPRB2 exhibit unique features being activated by various endogenous and exogenous agonists and due to their predominant expression in mast cells (MCs). Both MRGPRX2 and MRGPRB2 are associated with pseudoallergic reactions, such as asthma, chronic urticaria and skin inflammation. Due to its activation by bile acids and bilirubin, MRGPRX4 has been proposed to the target of high concentrations of these molecules released in cholestasis and being responsible for their provoking of itch. MRGPRX2 and MRGPRX4 represent fundamentally new drug targets. Therefore, the development of potent and selective agonists and antagonists is essential to reveal the (patho)physiological functions of these GPCRs, and validating them as novel drug targets, performing preclinical studies and eventually advance them towards clinical development. In view of the need of new drugs to treat and prevent chronic itch and pain, the design of MRGPRX2, MRGPRB2 and MRGPRX4 ligands represents a crucial step. In this PhD thesis, MRGPRX2, its proposed mouse orthologue MRGPRB2, as well as MRGPRX4 were pharmacologically investigated. Tricyclic benzimidazole derivatives have been identified before as potent MRGPRX2 antagonists, and polar xanthine derivatives as MRGPRX4 agonists with high potency. Structure-activity relationships (SARs) at both GPCRs have been intensively investigated in this study as well as their chemical optimization. This approach resulted in finding the most potent ligands for these GPCRs so far: whereas the MRGPRX2 antagonist CB70 displayed a potency in the nanomolar range (IC50 0.0225 µM in β-arrestin assays), the determined agonistic potency of the MRGPRX4 agonist DM288 was in the high picomolar range (EC50 0.000296 µM in β-arrestin assays). Additionally, tricyclic benzimidazole derivatives were evaluated for their additional antagonism at MRGPRB2 in calcium assays and SAR analysis allowed the identification of the most potent MRGPRB2 antagonist to date (CB70, IC50 0.00169 µM in calcium assays). In order to further characterize CB70, the compound was analyzed for its inhibition mode at MRGPRX2. Several antagonistic hit compounds for MRGPRX4 were evaluated for their potency and selectivity. During the development of calcium and β-arrestin assays for MRGPRX4, a single nucleotide polymorphism (S83L) has been identified in the MRGPRX4 gene that altered the GPCR function and impacted the potency of the investigated MRGPRX4 ligands. Lastly, optimized ligands were tested for their inhibition of mast cell degranulation as well as for their induction of intracellular calcium concentration in stem cell-derived mature human dorsal root ganglia neurons. The successful development of new potent and selective MRGPRX2 and MRGPRX4 ligands will help to further characterize the physiological role of these GPCRs.