Journal: Signal Transduction and Targeted Therapy

Article Title: Subnanomolar MAS-related G protein-coupled receptor-X2/B2 antagonists with efficacy in human mast cells and disease models

doi: 10.1038/s41392-025-02209-8

Figure Lengend Snippet: Biological characterization of PSB-172656. a Selectivity of PSB-172656 (100 nM) for MRGPRX2 over MRGPRX1, MRGPRX3, and MRGPRX4 measured in β -arrestin assays utilizing β -arrestin-CHO cells with recombinant expression of the respective receptor. MRGPRX1 was activated by BAM-22P (8.5 µM), MRGPRX2 was activated by CST-14 (1 µM), MRGPRX3 was activated by PSB-20294 (10 µM), MRGPRX4 was activated by PSB-18061 (2.6 µM). Data represent mean values ± SEM of 3-4 biological replicates, performed in duplicates. Statistical significance was determined by one-way ANOVA (Tukey’s multiple comparisons) test ( ns P > 0.05 and **** P < 0.0001), comparing the effect of each MRGPRX agonist in the presence of PSB-172656 (10 µM) versus their maximal effects (100%) in the absence of the antagonist. b p K i values of PSB-172656 determined using β -arrestin recruitment assays versus structurally diverse MRGPRX2 agonists, versus the peptide agonist SP (EC 80 3 µM); the small-molecule agonist ( R )-ZINC-3573 (EC 80 2 µM); C48/80 (EC 80 18 µM); the tricyclic antidepressant and MRGPRX2 agonist clomipramine (EC 80 13 µM), and CST-14 as a control. Data are means ± SEM of 3 biological replicates, in duplicates. Statistical significance was determined by one-way ANOVA (Tukey’s multiple comparison) test ( ns P > 0.05). For concentration-response curves and structures, see Supplementary Fig. . c TRUPATH G α q dissociation assays in LN229 glioblastoma cells recombinantly expressing MRGPRX2 and G α q RLuc8, Gβ 3 , and Gγ 9 -GFP2, concentration-response curve of the MRGPRX2 peptide agonist CST-14 showing an EC 50 value of 499 ± 207 nM. This effect was completely inhibited after the preincubation of PSB-172656 (100 nM). d TRUPATH G α i1 dissociation assays in LN229 glioblastoma cells recombinantly expressing MRGPRX2 and G α i1 RLuc8, Gβ 3 , and Gγ 9 -GFP2, concentration-response curve of the MRGPRX2 peptide agonist CST-14 showing an EC 50 value of 113 ± 16 nM. CST-14-induced G α i1 dissociation was completely inhibited after preincubation with PSB-172656 (100 nM). Data are means ± SEM of 3 biological replicates, performed in duplicates. e Shift assay to determine inhibition type of PSB-172656 versus CST-14. EC 50 values: without antagonist: 283 ± 45 nM; with 10 nM PSB-172656: 601 ± 89 nM; with 20 nM PSB-172656: 1580 ± 403 nM; with 40 nM PSB-172656: 2510 ± 889 nM; with 100 nM PSB-172656: 4,590 ± 1,580 nM; with 200 nM PSB-172656: 6260 ± 113 nM, faint dashed lines indicate extrapolation. Extrapolated data were not used for analysis but only shown for a comprehensive view. f Schild plot for the effect of PSB-172656 on the concentration-response curves for CST-14. K B value of 6.71 nM (slope,1.08; X-intercept, -8.17 coresponding to -p K B value). g Inhibition mode of PS B -172656 versus ( R )-ZINC-3573. EC 50 values: without antagonist: 2020 ± 333 nM; with 10 nM PSB-172656: 4060 ± 1560 nM; with 20 nM PSB-172656: 6780 ± 572 nM; with 40 nM PSB-172656: 15,900 ± 4020 nM; with 100 nM PSB-172656: 26,300 ± 49,10 nM; with 200 nM PSB-172656: 29,400 ± 4550 nM. h Schild plot for the effect of PSB-172656 on the concentration-response curves for ( R )-ZINC-3573. K B value: 7.59 nM (slope, 0.891; X-intercept, -8.17 corresponding to -p K B value). Data represent means ± SEM of 3-6 biological replicates

Article Snippet: The human mast cell line Laboratory of Allergic Diseases 2 (LAD2) was kindly provided by Dr. A. Kirshenbaum and Dr. D. Metcalfe (Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, MD, USA) and maintained in a complete StemPro-34 medium supplemented with L-glutamine (2 mM), penicillin (100 IU/mL), streptomycin (100 μg/mL), and recombinant human stem cell factor (rhSCF, 100 ng/mL). rhSCF was purchased from PeproTech (Rocky Hill, NJ, USA).

Techniques: Recombinant, Expressing, Control, Comparison, Concentration Assay, Shift Assay, Inhibition