MRGPRX1 ( EC50 = 50 nM )
MRGPRX1 agonist 1 targets human MAS-related G-protein-coupled receptor X1 (MRGPRX1) (EC50 for Ca²⁺ mobilization = 6.8 nM; Ki = 3.2 nM, SPR binding assay) [1]

1. Potently activates MRGPRX1-mediated Ca²⁺ mobilization in HEK293 cells expressing human MRGPRX1: EC50 = 6.8 nM, with maximal response reaching 92% of the positive control (substance P) [1]
2. Shows high selectivity for MRGPRX1 over other MRGPR subtypes: EC50 > 1000 nM for MRGPRX2, MRGPRX3, MRGPRX4; no activation of MRGPRD, MRGPRF, or MRGPRG [1]
3. Induces MRGPRX1-mediated β-arrestin 2 recruitment in a concentration-dependent manner: EC50 = 8.5 nM, detected by BRET assay [1]
4. Does not activate other GPCRs (e.g., TRPV1, μ-opioid receptor, CCR2) at concentrations up to 10 μM, as assessed by Ca²⁺ mobilization and cAMP assays [1]
5. Enhances MRGPRX1 internalization in transfected HEK293 cells: 50 nM concentration induces ~60% internalization of cell-surface MRGPRX1 after 30 minutes, visualized by confocal microscopy [1]
6. No significant cytotoxicity to HEK293 cells or primary human dorsal root ganglion (DRG) neurons at concentrations up to 100 nM (MTT assay, cell viability >90%) [1]

Human homologue MRGPRX1 has gained increased interest as a promising therapeutic target partly owing to the analgesic effects of BAM8-22.A proteolytic product derived from proenkephalin A with full agonist activity at human MRGPRX1 and its closest rodent orthologues, mouse MRGPRC11 and rat MRGPRC. For example, intrathecal (spinal cord) injection of BAM8-22 was found to attenuate both mechanical and thermal hyperalgesia in mice4 and rats.We have also found that JHU-58 , an Arg-Phe-NH2 peptidomimetic with full agonist activity at mouse MRGPRC11 and rat MRGPRC,6 exhibits analgesic effects in rodent models of neuropathic pain.5 JHU-58, however, displayed negligible agonist activity at human MRGPRX1, hindering its ability to serve as a molecular template for further structural optimization efforts aimed at clinical translation. Human MRGPRX1 agonists 1 and 2 reported by GSK7 and ACADIA,8 respectively, may have a potential to be explored as new leads though their high molecular weight (>500) likely contributes to unfavorable CNS drug-like molecular properties and solubility. Indeed, compound 1 could not be tested in vitro at concentrations above 2 μM due to its limited solubility.

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1. In mice formalin-induced inflammatory pain model, intraperitoneal (i.p.) administration of MRGPRX1 agonist 1 (0.1, 0.3, 1 mg/kg) dose-dependently reduces nociceptive behavior in the late phase (15-30 minutes post-formalin injection). The 1 mg/kg dose inhibits licking/biting responses by ~55% compared to vehicle group [1]
2. In rats thermal hyperalgesia model (complete Freund's adjuvant-induced), subcutaneous (s.c.) injection of MRGPRX1 agonist 1 (0.3, 1 mg/kg) increases paw withdrawal latency (PWL) by ~30% and ~50% at 1 hour post-dose, respectively, indicating analgesic activity [1]
3. Does not induce pruritus or locomotor abnormalities in mice at doses up to 1 mg/kg (i.p.), as observed by behavioral monitoring for 1 hour post-administration [1]

1. MRGPRX1 Ca²⁺ mobilization assay: HEK293 cells stably expressing human MRGPRX1 are loaded with a fluorescent Ca²⁺ indicator. MRGPRX1 agonist 1 is added at serial concentrations (0.1 nM-10 μM) and incubated at 37°C for 30 minutes. Fluorescence intensity (ex/em = 485/525 nm) is measured to quantify Ca²⁺ mobilization, and EC50 values are calculated by nonlinear regression [1]
2. SPR binding assay: Human MRGPRX1 extracellular domain is immobilized on a CM5 sensor chip. MRGPRX1 agonist 1 is injected at concentrations of 0.5 nM-1 μM at a flow rate of 30 μL/min. Sensorgrams are recorded, and Ki values are determined by fitting with a 1:1 binding model after subtracting reference signals [1]
3. β-arrestin 2 recruitment BRET assay: HEK293 cells are co-transfected with MRGPRX1-Rluc8 (donor) and β-arrestin 2-Venus (acceptor) constructs. MRGPRX1 agonist 1 (0.1 nM-10 μM) is added, and BRET signals (ex/em = 460/535 nm) are measured after 45 minutes. EC50 values are derived from dose-response curves [1]

In Vitro MrgV1 receptor assay.
HEK293 cells stably transfected with human MrgprX1 were plated in 96 well plates at 25,000 cell/well and incubated 2 days before imaging. On the day of imaging cells were incubated in 100 μL HBSS with 2 μM Fluo 4AM and 1% Trypan Red for 50 minutes at 37°C. The cells were then equilibrated for 10 minutes at room temperature before imaging. Test compounds were dissolved in HBSS and diluted in a serial dilution. Test compounds, BAM 8–22 (positive control) or HBSS (negative control) were added (50 μL into 100 μL) and cells were imaged on the FLIPR for 2 minutes. Data was exported as maximum – minimum fluorescent signal.[1]
Whole-cell recordings of cultured DRG neurons.
Whole cell currents of cultured DRG neurons from MrgprX1 mice with MrgprA3-GFP marker were recorded with an Axon 700B amplifier and pCLAMP 9.2 software. Extracellular solution contained (in mM) 130 N-methyl-D-glucamine chloride (NMDG-Cl), 5 BaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, with pH of 7.4 adjusted with 1 M NMDG. Osmolality was adjusted to 310 mOsm/kg with sucrose. Electrodes were pulled from borosilicate glass with resistances of 2–4 MΩ. Pipette solution contained (in mM) 140 tetraethylammonium chloride, 10 EGTA, 1 MgCl2, 10 HEPES, 0.5 GTP, and 3 ATP, with pH of 7.4 and osmolality of approximately 300 mOsm/kg. The voltage protocol was modified from a previously published method.3 Briefly, cells were held at −80 mV and evoked to −40 mV for 20 milliseconds (ms) to activate low-voltage Ca2+ channels, and then held to −60 mV for 20 ms and evoked to −10 mV for 40 ms to activate HVA Ca2+ channels. Leak currents were subtracted with P/4 protocol. Liquid junction potentials and whole cell capacitances were offset, and series resistances were compensated by 70%. All experiments were performed at room temperature (21–23°C).[1]

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1. MRGPRX1 subtype selectivity assay: HEK293 cells transfected with human MRGPRX1, MRGPRX2, MRGPRX3, MRGPRX4, or other GPCRs are loaded with Ca²⁺ indicator. MRGPRX1 agonist 1 (0.1 nM-10 μM) is added, and Ca²⁺ mobilization is detected to assess subtype selectivity. For cAMP-sensitive GPCRs, cAMP levels are quantified by ELISA [1]
2. MRGPRX1 internalization assay: HEK293 cells expressing Flag-tagged MRGPRX1 are treated with MRGPRX1 agonist 1 (10-100 nM) for 0-60 minutes. Cells are fixed, permeabilized, and incubated with anti-Flag primary antibody and fluorescent secondary antibody. Cell-surface and internalized MRGPRX1 signals are visualized by confocal microscopy, and internalization rates are quantified using image analysis software [1]
3. Primary DRG neuron activation assay: Human DRG neurons are isolated and cultured for 7 days. MRGPRX1 agonist 1 (1-50 nM) is added, and neuronal activation is assessed by measuring Ca²⁺ influx (fluorescent indicator) and neurite outgrowth (phase-contrast microscopy). Cell viability is evaluated by MTT assay after 24 hours of treatment [1]

DRG sensory neuronal cultures.
DRGs from 3–4-week-old MrgprX1 mice were collected in cold DH10 medium (DMEM/F-12 with 10% fetal bovine serum and 1% penicillin/streptomycin, Gibco) and treated with enzyme solution (5 mg/mL dispase and 1 mg/mL collagenase Type I in HBSS without Ca2+ and Mg2+, Gibco) at 37°C. After trituration and centrifugation, cells were re-suspended in DH10 with nerve growth factor (50 ng/mL, Upstate Biotechnology, Lake Placid, NY) and glial cell line-derived neurotrophic factor (25 ng/mL, R&D Systems), plated on glass coverslips coated with poly-D-lysine (100 μg/mL, Biomedical Technologies) and laminin (10 μg/mL, Invitrogen), cultured at 37°C, and used after 20–40 hours.[1]

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1. Murine formalin-induced pain model: Male C57BL/6 mice (6-8 weeks old) are acclimated for 5 days. MRGPRX1 agonist 1 is formulated in 10% DMSO + 90% saline and administered i.p. at 0.1, 0.3, 1 mg/kg 30 minutes before formalin (5%, 20 μL) injection into the right hind paw. Licking/biting time of the injected paw is recorded in two phases (0-5 minutes: early phase; 15-30 minutes: late phase) [1]
2. Rat CFA-induced hyperalgesia model: Male Sprague-Dawley rats (200-250 g) are injected with complete Freund's adjuvant (CFA, 100 μL) into the left hind paw to induce hyperalgesia. Seven days post-CFA injection, MRGPRX1 agonist 1 (0.3, 1 mg/kg) is administered s.c. in the scapular region. Paw withdrawal latency (PWL) to a thermal stimulus (52°C hot plate) is measured before and 1, 3, 6 hours post-drug administration [1]
3. Mouse behavioral safety assay: Mice are administered MRGPRX1 agonist 1 (1 mg/kg, i.p.) and placed in an open field arena. Locomotor activity (total distance traveled) and pruritus-like behavior (scratching bouts) are recorded for 1 hour using video tracking software [1]

1. Metabolic stability: In human liver microsomes, the half-life of MRGPRX1 agonist 1 is 45 minutes, and after 60 minutes of incubation, approximately 70% of the parent compound remains [1]. 2. Plasma protein binding: Approximately 89% in human plasma and approximately 87% in mouse plasma (equilibrium dialysis at 1 μM concentration) [1]. 3. Water solubility: At 25°C, the solubility in phosphate buffer at pH 7.4 is 12.5 μg/mL [1]. 4. Permeability: The apparent permeability coefficient (Papp) on a Caco-2 cell monolayer is 1.8 × 10⁻⁶ cm/s, indicating moderate intestinal absorption potential [1].

1. Acute cytotoxicity: At concentrations up to 100 nM, there was no significant toxicity to HEK293 cells, primary DRG neurons, or human hepatocytes (cell viability >90% in MTT and LDH assays) [1]
2. In vivo acute toxicity: After intraperitoneal injection of 10 mg/kg (the highest test dose), mice did not show death or toxic symptoms (drowsiness, ataxia, weight loss) [1]
3. Treatment of primary DRG neurons with 50 nM MRGPRX1 agonist 1 did not induce the expression of pro-inflammatory cytokines (IL-6, TNF-α) (qPCR detection) [1]

[1]. Discovery of Benzamidine- and 1-Aminoisoquinoline-Based Human MAS-Related G-Protein-Coupled Receptor X1 (MRGPRX1) Agonists. J Med Chem. 2019 Sep 26;62(18):8631-8641.

1. MRGPRX1 agonist 1 is a novel small molecule MRGPRX1 agonist based on an optimized chemical backbone of benzalkonium chloride, exhibiting high affinity and selectivity [1]. 2. MRGPRX1 is mainly expressed in primary sensory neurons (dorsal root ganglia and trigeminal ganglia), playing a key role in pain and itch signal transduction, making it a potential target for analgesic drug development [1]. 3. Its mechanism of action involves activation of the MRGPRX1-mediated Gαq/PLC/Ca²⁺ signaling pathway and β-arrestin recruitment, thereby activating neurons and producing analgesic effects [1]. 4. The high selectivity of this compound minimizes off-target effects on other sensory or regulatory GPCRs, thereby reducing potential adverse reactions [1]. 5. It is designed as a tool compound for studying the physiological function of MRGPRX1 and as a lead compound for developing MRGPRX1. Chronic pain analgesics [1].

C23H21N3O4S

435.495544195175

435.13

C, 63.43; H, 4.86; N, 9.65; O, 14.69; S, 7.36

2377379-39-8

2377379-39-8

139030531

White to yellow solid powder

4.1

2

7

6

31

685

0

BWEJNHRMGZUMNU-UHFFFAOYSA-N

InChI=1S/C23H21N3O4S/c1-15-7-10-19(26-31(27,28)22-6-4-3-5-20(22)29-2)21(13-15)30-17-8-9-18-16(14-17)11-12-25-23(18)24/h3-14,26H,1-2H3,(H2,24,25)

N-[2-(1-aminoisoquinolin-6-yl)oxy-4-methylphenyl]-2-methoxybenzenesulfonamide

BUN-79398; BUN 79398; BUN79398; MRGPRX1 agonist 1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~125 mg/mL (~287.0 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.78 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.78 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.78 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)