Novel small molecule MRGPRX2 antagonists inhibit a murine model of allergic reaction.

BACKGROUND: Activation of Mas-related G protein-coupled receptor X2 (MRGPRX2) is a crucial non-IgE pathway for mast cell activation associated with allergic reactions and inflammation. Only a few peptides and small compounds targeting MRGPRX2 have been reported, with limited information on their pharmacologic activity. OBJECTIVE: We sought to develop novel small molecule MRGPRX2 antagonists to treat MRGPRX2-mediated allergies and inflammation. METHODS: A computational approach was used to design novel small molecules as MRGPRX2 antagonists. The short-listed molecules were synthesized and characterized by liquid chromatography and mass spectrometry as well as nuclear magnetic resonance. Inhibitory activity on MRGPRX2 signaling was assessed in vitro by using functional bioassays (ß-hexosaminidase, calcium flux, and chemokine synthesis) and receptor activation assays (ß-arrestin recruitment and Western blot analysis) in human LAD-2 mast cells and HTLA cells. In vivo effects of the novel MRGPRX2 antagonists were assessed using a mouse model of acute allergy and systemic anaphylaxis. RESULTS: The novel small molecules demonstrated higher binding affinity with MRGPRX2 in the docking study. The half-maximal inhibitory concentration is in the low micromolar range (5-21 µM). The small molecules inhibited not only the early phase of mast cell activation but also the late phase, associated with chemokine and prostaglandin release. Further, Western blot analysis revealed inhibition of downstream phospholipase C-γ, extracellular signal-regulated protein kinase 1/2, and Akt signaling pathway. Moreover, in the mouse models of allergies, small molecule administration effectively blocks acute, systemic allergic reactions and inflammation and prevents systemic anaphylaxis. CONCLUSION: The small molecules might hold a significant therapeutic promise to treat MRGPRX2-mediated allergies and inflammation.

P17 induces chemotaxis and differentiation of monocytes via MRGPRX2-mediated mast cell-line activation.

BACKGROUND: P17, a peptide isolated from Tetramorium bicarinatum ant venom, is known to induce an alternative phenotype of human monocyte-derived macrophages via activation of an unknown G protein-coupled receptor (GPCR). OBJECTIVE: We sought to investigate the mechanism of action and the immunomodulatory effects of P17 mediated through MRGPRX2 (Mas-related G protein-coupled receptor X2). METHODS: To identify the GPCR for P17, we screened 314 GPCRs. Upon identification of MRGPRX2, a battery of in silico, in vitro, ex vivo, and in vivo assays along with the receptor mutation studies were performed. In particular, to investigate the immunomodulatory actions, we used ß-hexosaminidase release assay, cytokine releases, quantification of mRNA expression, cell migration and differentiation assays, immunohistochemical labeling, hematoxylin and eosin, and immunofluorescence staining. RESULTS: P17 activated MRGPRX2 in a dose-dependent manner in ß-arrestin recruitment assay. In LAD2 cells, P17 induced calcium and ß-hexosaminidase release. Quercetin- and short hairpin RNA-mediated knockdown of MRGPRX2 reduced P17-evoked ß-hexosaminidase release. In silico and in vitro mutagenesis studies showed that residue Lys8 of P17 formed a cation-π interaction with the Phe172 of MRGPRX2 and [Ala8]P17 lost its activity partially. P17 activated LAD2 cells to recruit THP-1 and human monocytes in Transwell migration assay, whereas MRGPRX2-impaired LAD2 cells cannot. In addition, P17-treated LAD2 cells stimulated differentiation of THP-1 and human monocytes, as indicated by the enhanced expression of macrophage markers cluster of differentiation 11b and TNF-α by quantitative RT-PCR. Immunohistochemical and immunofluorescent staining suggested monocyte recruitment in mice ears injected with P17. CONCLUSIONS: Our data provide novel structural information regarding the interaction of P17 with MRGPRX2 and intracellular pathways for its immunomodulatory action.

Protective Effect of Genistein against Compound 48/80 Induced Anaphylactoid Shock via Inhibiting MAS Related G Protein-Coupled Receptor X2 (MRGPRX2).

Anaphylactoid shock is a fatal hypersensitivity response caused by non-IgE mediated mast cell activation. These reactions are mediated by a family of G protein-coupled receptors (GPCRs) known as Mas related GPCRX2 (MRGPRX2). Several US FDA approved drugs which are used in day to day life have been reported to cause anaphylactoid shock. Surprisingly, no therapeutic drugs are available which can directly target MRGPRX2 for treatment of anaphylactoid shock. Genistein is a non-steroidal polyphenol known for its diverse physiological and pharmacological activities. In recent studies, Genistein has been reported for its anti-inflammatory activity on mast cells. However, the effects and mechanistic pathways of Genistein on anaphylactoid reaction remain unknown. In the present study, we designed a battery of in-vitro, in-silico and in-vivo experiments to evaluate the anti-anaphylactoid activity of Genistein in order to understand the possible molecular mechanisms of its action. The in-vitro results demonstrated the inhibitory activity of Genistein on MRGPRX2 activation. Further, a mouse model of anaphylactoid shock was used to evaluate the inhibitory activity of Genistein on blood vessel leakage and hind paw edema. Taken together, our findings have demonstrated a therapeutic potential of Genistein as a lead compound in the treatment of anaphylactoid shock via MRGPRX2.

Octadecaneuropeptide, ODN, Promotes Cell Survival against 6-OHDA-Induced Oxidative Stress and Apoptosis by Modulating the Expression of miR-34b, miR-29a, and miR-21in Cultured Astrocytes.

Astrocytes specifically synthesize and release endozepines, a family of regulatory peptides including octadecaneuropeptide (ODN). We have previously reported that ODN rescues neurons and astrocytes from 6-OHDA-induced oxidative stress and cell death. The purpose of this study was to examine the potential implication of miR-34b, miR-29a, and miR-21 in the protective activity of ODN on 6-OHDA-induced oxidative stress and cell death in cultured rat astrocytes. Flow cytometry analysis showed that 6-OHDA increased the number of early apoptotic and apoptotic dead cells while treatment with the subnanomolar dose of ODN significantly reduced the number of apoptotic cells induced by 6-OHDA. 6-OHDA-treated astrocytes exhibited the over-expression of miR-21 (+118%) associated with a knockdown of miR-34b (-61%) and miR-29a (-49%). Co-treatment of astrocytes with ODN blocked the 6-OHDA-stimulated production of ROS and NO and stimulation of Bax and caspase-3 gene transcription. Concomitantly, ODN down-regulated the expression of miR-34b and miR-29a and rescued the 6-OHDA-associated reduced expression of miR21, indicating that ODN regulates their expression during cell death. Transfection with miR-21-3p inhibitor prevented the effect of 6-OHDA against cell death. In conclusion, our study indicated that (i) the expression of miRNAs miR-34b, miR-29a, and miR-21 is modified in astrocytes under 6-OHDA injury and (ii) that ODN prevents this deregulation to induce its neuroprotective action. The present study identified miR-21 as an emerging candidate and as a promising pharmacological target that opens new neuroprotective therapeutic strategies in neurodegenerative diseases, especially in Parkinson's disease.

MRGPRB2/X2 and the analogous effects of its agonist and antagonist in DSS-induced colitis in mice.

The mast cell receptor Mrgprb2, a mouse orthologue of human Mrgprx2, is known as an inflammatory receptor and its elevated expression is associated with various diseases such as ulcerative colitis. We aimed to elucidate the role of Mrgprb2/x2 and the effect of its ligands on a chemically induced murine colitis model. We showed that in Mrgprb2-/- mice, there is a differential regulation of cytokine releases in the blood plasma and severe colonic damages after DSS treatment. Unexpectedly, we demonstrated that known Mrgprb2/x2 agonists (peptide P17, P17 analogues and CST-14) and antagonist (GE1111) similarly increased the survival rate of WT mice subjected to 4% DSS-induced colitis, ameliorated the colonic damages of 2.5% DSS-induced colitis, restored major protein mRNA expression involved in colon integrity, reduced CD68+ and F4/80+ immune cell infiltration and restored cytokine levels. Collectively, our findings highlight the eminent role of Mrpgrb2/x2 in conferring a beneficial effect in the colitis model, and this significance is demonstrated by the heightened severity of colitis with altered cytokine releases and inflammatory immune cell infiltration observed in the Mrgprb2 knockout mice. Elevated expression of Mrgprb2 in WT colitis murine models may represent the organism's adaptive protective mechanism since Mrgprb2 knockout results in severe colitis. On the other hand, both agonist and antagonist of Mrgprb2 analogously mitigated the severity of colitis in DSS-induced colitis model by altering Mrgprb2 expression, immune cell infiltration and inflammatory cytokine releases.

Unlocking the Non-IgE-Mediated Pseudo-Allergic Reaction Puzzle with Mas-Related G-Protein Coupled Receptor Member X2 (MRGPRX2).

Mas-related G-protein coupled receptor member X2 (MRGPRX2) is a class A GPCR expressed on mast cells. Mast cells are granulated tissue-resident cells known for host cell response, allergic response, and vascular homeostasis. Immunoglobulin E receptor (FcεRI)-mediated mast cell activation is a well-studied and recognized mechanism of allergy and hypersensitivity reactions. However, non-IgE-mediated mast cell activation is less explored and is not well recognized. After decades of uncertainty, MRGPRX2 was discovered as the receptor responsible for non-IgE-mediated mast cells activation. The puzzle of non-IgE-mediated pseudo-allergic reaction is unlocked by MRGPRX2, evidenced by a plethora of reported endogenous and exogenous MRGPRX2 agonists. MRGPRX2 is exclusively expressed on mast cells and exhibits varying affinity for many molecules such as antimicrobial host defense peptides, neuropeptides, and even US Food and Drug Administration-approved drugs. The discovery of MRGPRX2 has changed our understanding of mast cell biology and filled the missing link of the underlying mechanism of drug-induced MC degranulation and pseudo-allergic reactions. These non-canonical characteristics render MRGPRX2 an intriguing player in allergic diseases. In the present article, we reviewed the emerging role of MRGPRX2 as a non-IgE-mediated mechanism of mast cell activation in pseudo-allergic reactions. We have presented an overview of mast cells, their receptors, structural insight into MRGPRX2, MRGPRX2 agonists and antagonists, the crucial role of MRGPRX2 in pseudo-allergic reactions, current challenges, and the future research direction.

Role of SCTR/AT1aR heteromer in mediating ANGII-induced aldosterone secretion.

The involvement of secretin (SCT) and its receptor (SCTR) in angiotensin II (ANGII)-mediated osmoregulation by forming SCTR/ angiotensin II type 1 receptor (AT1R) heteromer is well established. In this study, we demonstrated that SCTR/AT1R complex can mediate ANGII-induced aldosterone secretion/release through potentiating calcium mobilization. Through IHC and cAMP studies, we showed the presence of functional SCTR and AT1R in the primary zona glomerulosa (ZG) cells of C57BL/6N (C57), and functional AT1R and non-functional SCTR in SCTR knockout (SCTR-/-) mice. Calcium mobilization studies revealed the important role of SCTR on ANGII-mediated calcium mobilization in adrenal gland. The fluo4-AM loaded primary adrenal ZG cells from the C57 mice displayed a dose-dependent increase in intracellular calcium influx ([Ca2+]i) when exposed to ANGII but not from the SCTR-/- ZG cells. Synthetic SCTR transmembrane (TM) peptides STM-II/-IV were able to alter [Ca2+]i in C57 mice, but not the mice with mutated STM-II/-IV (STM-IIm/IVm) peptides. Through enzyme immunoassay (EIA), we measured the aldosterone release from primary ZG cells of both C57 and SCTR-/- mice by exposing them to ANGII (10nM). SCTR-/- ZG cells showed impaired ANGII-induced aldosterone secretion compared to the C57 mice. TM peptide, STM-II hindered the aldosterone secretion in ZG cells of C57 mice. These findings support the involvement of SCTR/AT1R heterodimer complex in aldosterone secretion/release through [Ca2+]i.