The human 7-transmembrane orphan receptor family MRGPRX: native expression and identification, development and pharmacological characterization of agonists and antagonists

Abstract

The human MRGPRX receptors belong to the orphan 7-transmembrane receptors (7TMRs) within the Rhodopsin family of 7TMRs. The MRGPRX receptor subfamily is exclusively expressed in primates, which represents a hurdle for elucidating the (patho)physiological roles of these receptors. In this PhD thesis the four human MRGPRX receptors were pharmacologically investigated. The primary assay employed in this study was the β-arrestin assay using β-galactosidase complementation due to its high specificity for the expressed receptor. Additional assays including calcium mobilization and cAMP accumulation were also employed.<br /> MRGPRX1 is the best investigated member of the MRGPRX subfamily of 7TMRs. Our efforts were focused on screening several compound libraries in order to identify new antagonists for this receptor subtype. Only one compound, MIRA-1, was found to have an antagonistic activity at MRGPRX1 in the performed β-arrestin assays. However, the chemical structure (instable ester) and the steep concentration-response curve made us refrain from further investigation of this hit compound. Screening of further compound libraries should be performed in the future to identify ligands that are more suitable for drug development. <br /> MRGPRX2 was paired to the peptide agonist CST-14, but several other agonists have been described in the literature. However, no antagonists have been described so far. In our screening approach for antagonists using the β-arrestin assay several hits were identified, e.g. vitamin K3 (menadione) from a compound library. One hit from another compound library, designated CB8, with a tricyclic benzimidazole scaffold showed an IC50 value of 2.42 µM. It was decided to develop this hit further. Our efforts led to an establishment of structure-activity relationships (SARs) and to successive optimization of the parent compound. The best antagonist so far (CB63) exhibited an IC50 value of 6.38 nM. The mode of action of these antagonists was determined to be competitive versus CST-14. In addition, initial pharmacokinetic data of the best three antagonists were determined. <br /> Our results provide valuable tools for investigating the MRGPRX2 receptor. <br /> The MRGPRX3 receptor is an orphan receptor with no reported pharmacological tools for now. This receptor was cloned, and a β-arrestin cell line with high expression was established. Screening of all available compound libraries during the time of this thesis resulted in no hits. The MRGPRX3 receptor remains an enigmatic receptor. <br /> MRGPRX4 is an orphan receptor. The drug nateglinide was reported at the final stage of this thesis to act as a weak, non-selective agonist. Several screening and deorphanization approaches in the course of this thesis resulted in no progress until an artificial agonist was discovered. Related compounds were subsequently tested and initial SARs were established. Our efforts led to the optimization of both, efficacy and potency of the agonists. The best agonist with increased metabolic stability, showed EC50 values in the nanomolar range in both, β-arrestin and calcium mobilization assays. Using these agonists we demonstrated only Gq coupling for this 7TMR. Further synthesis resulted in an agonist which demonstrated increased efficacy with the best signal-to-noise ratio in the β-arrestin assay so far. For a deeper understanding of this receptor a search for a native cell line led us to stem cells differentiated to DRG-like neurons, and to the detection of MRGPRX4 in several lymphocyte subpopulations. The obtained information proved the expression and consequently a potential physiological role of MRGPRX4 in the immune system. Moreover, calcium mobilization assays showed a high signal with efficient Gq coupling. Screening for antagonists has also been carried out and several hits with different scaffolds have been identified. This opens up the door for the development of more potent and selective antagonists. <br /> All in all, the results of this thesis enrich our understanding of the so far not-well characterized MRGPRX receptor subfamily and provide important tools for future investigations.