MRGPRX2 activation as a rapid, high-throughput mechanistic-based approach for detecting peptide-mediated human mast cell degranulation liabilities

Lafleur, Michel; Werner, Jonathan; Fort, Madeline M.; Lobenhofer, Edward K.; Balázs, Mercedesz; Goyos, Ana

doi:10.1080/1547691x.2020.1757793

Cited by 29 publications

(14 citation statements)

References 89 publications

(103 reference statements)

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“…Among these hits, human Tango-MRGPRX2 was found to be dose-dependently activated by P17 with an EC 50 value of 4.13 mM (95% CI, 1.85-9.18 mM), whereas cortistatin-14 (CST-14), a positive control, was able to activate MRGPRX2 with an EC 50 of 0.49 mM (95% CI, 0.27-0.88 mM 31 ) (Fig 1 , Aii). Because MRGPRX2-mediated calcium release has been evidently reported before, 41,42 we have therefore evaluated the effect of P17 on Ca 21 Locating the pharmacophore region of peptide P17 using in silico 3-dimensional homology modeling and in vitro validation of peptide-receptor interaction site…”

Section: Identification Of Mrgprx2 As a Receptor For P17 And 3-dimensional Homology Modeling To Determine Specific Aa Residues Involved Imentioning

confidence: 99%

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

Duraisamy

Singh

Kumar

et al. 2022

Journal of Allergy and Clinical Immunology

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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 b-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 b-arrestin recruitment assay. In LAD2 cells, P17 induced calcium and b-hexosaminidase release. Quercetin-and short hairpin RNA-mediated knockdown of MRGPRX2 reduced P17evoked b-hexosaminidase release. In silico and in vitro mutagenesis studies showed that residue Lys 8 of P17 formed a cation-p interaction with the Phe 172 of MRGPRX2 and [Ala 8 ] 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

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Section: Identification Of Mrgprx2 As a Receptor For P17 And 3-dimensional Homology Modeling To Determine Specific Aa Residues Involved Imentioning

confidence: 99%

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

Duraisamy

Singh

Kumar

et al. 2022

Journal of Allergy and Clinical Immunology

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“…The plethora of recent literature reports further substantiating the role of MRGPRX2 in drug-induced allergenic responses, i.e., drugs having off-target activity on MRGPRX2, suggests that the probability of encountering this preclinical safety signal is not uncommon (Porebski et al 2018;Grimes et al 2019;Jiang et al 2019;Zheng et al 2019;Hu et al 2020;Mencarelli et al 2020;Ogasawara et al 2020;Sahid et al 2020). As an example, LaFleur et al (2020) similarly grappled with injection site reactions in preclinical animal studies and used human CD34 þ stem cell-derived mature human mast cells expressing MRGPRX2 to validate the translational relevance of using in vitro screens against MRGPRX2 and provide a comparison between two (Ca 2þ based and b-arrestin) commerciallyavailable assays for this purpose. Lansu et al (2017) used in silico modeling tools to assess the structure-activity nature of drugs interacting with MRGPRX2.…”

Section: Discussionmentioning

confidence: 99%

In vitro prediction of in vivo pseudo-allergenic response via MRGPRX2

John

Dalsgaard

Jeppesen

et al. 2021

Journal of Immunotoxicology

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In development of peptide therapeutics, rodents are commonly-used preclinical models when screening compounds for efficacy endpoints in the early stages of discovery projects. During the screening process, some peptides administered subcutaneously to rodents caused injection site reactions manifesting as localized swelling. Screening by postmortem evaluations of injection site swelling as a marker for local subcutaneous histamine release, were conducted in rats to select drug candidates without this adverse effect. Histological analysis of skin samples revealed that the injection site reactions were concurrent with mast cell degranulation, resulting in histamine release. Mast cell activation can be mediated by MRGPRX2, a GPCR that induces a pseudo-allergenic immune response. The present study demonstrates that a commercially-available cell-based MRGPRX2 assay reliably identifies compounds that induce histamine release or localized edema in ex vivo human and rodent skin samples. In vitro screening was subsequently implemented using the MRGPRX2 assay as a substitute for postmortem injection site evaluation, thus achieving a significant reduction in animal use. Thus, in cases where injection site reactions are encountered during in vivo screening, to enable faster screening during the early drug discovery process, an MRGPRX2 in vitro assay can be used as an efficient, more ethical tool with human translational value for the development of safer pharmacotherapies for patients.

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“…A good correlation was observed between the histamine release assay and the MRGPRX2 assay with dual endpoints. Sensitivity of 100% was seen with both the β‐arrestin and Ca 2+ endpoints, while corresponding specificities were 93% and 83%, respectively 137 . β‐arrestin and Ca 2+ endpoints were similar, that is, no ligand bias was observed, but note that some ligands activate one (eg, G‐protein‐biased icatibant) or other, or both, pathways (eg, compound 48/80) 47,138 .…”

Section: Distinguishing and Diagnosing Mrgprx2‐ And Ige/fcεri‐mediate...mentioning

confidence: 97%

MRGPRX2, drug pseudoallergies, inflammatory diseases, mechanisms and distinguishing MRGPRX2‐ and IgE/FcεRI‐mediated events

Baldo

2023

Brit J Clinical Pharma

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MRGPRX2, a novel Gaq‐coupled human mast cell receptor, mediates non‐immune adverse reactions without the involvement of antibody priming. Constitutively expressed by human skin mast cells, MRGPRX2 modulates cell degranulation producing pseudoallergies manifesting as itch, inflammation and pain. The term pseudoallergy is defined in relation to adverse drug reactions in general and immune/non‐immune‐mediated reactions in particular. A list of drugs with MRGPRX2 activity is presented, including a detailed examination of three important and widely used approved therapies: neuromuscular blockers, quinolones and opioids. For the clinician, the significance of MRGPRX2 is considered as an aid in distinguishing and ultimately identifying specific immune and non‐immune inflammatory reactions. Anaphylactoid/anaphylactic reactions, neurogenic inflammation and inflammatory diseases with a clear or strongly suspected association with MRGPRX2 activation are examined. Inflammatory diseases include chronic urticaria, rosacea, atopic dermatitis, allergic contact dermatitis, mastocytosis, allergic asthma, ulcerative colitis and rheumatoid arthritis. MRGPRX2‐ and allergic IgE/FcεRI‐mediated reactions may be clinically similar. Importantly, the usual testing procedures do not distinguish the two mechanisms. Currently, identification of MRGPRX2 activation and diagnosis of pseudoallergic reactions is generally viewed as a process of exclusion once other non‐immune and immune processes, particularly IgE/FcεRI‐mediated degranulation of mast cells, are ruled out. This does not take into account that MRGPRX2 signals via β‐arrestin, which can be utilized to detect MRGPRX2 activation by employing MRGPRX2 transfected cells to assess MRGPRX2 activation via two pathways, the G‐protein‐independent β‐arrestin pathway and the G‐protein‐dependent Ca2+ pathway. Testing procedures, interpretations for distinguishing mechanisms, patient diagnosis, agonist identification and drug safety evaluations are addressed.

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MRGPRX2 activation as a rapid, high-throughput mechanistic-based approach for detecting peptide-mediated human mast cell degranulation liabilities

Abstract: Goyos (2020) MRGPRX2 activation as a rapid, high-throughput mechanistic-based approach for detecting peptide-mediated human mast cell degranulation liabilities,

Cited by 29 publications

References 89 publications

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

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

In vitro prediction of in vivo pseudo-allergenic response via MRGPRX2

MRGPRX2, drug pseudoallergies, inflammatory diseases, mechanisms and distinguishing MRGPRX2‐ and IgE/FcεRI‐mediated events

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