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In silico design of novel probes for the atypical opioid receptor MRGPRX2


The primate-exclusive MRGPRX2 G protein-coupled receptor (GPCR) has been suggested to modulate pain and itch. Despite putative peptide and small-molecule MRGPRX2 agonists, selective nanomolar-potency probes have not yet been reported. To identify a MRGPRX2 probe, we first screened 5,695 small molecules and found that many opioid compounds activated MRGPRX2, including (−)- and (+)-morphine, hydrocodone, sinomenine, dextromethorphan, and the prodynorphin-derived peptides dynorphin A, dynorphin B, and α- and β-neoendorphin. We used these to select for mutagenesis-validated homology models and docked almost 4 million small molecules. From this docking, we predicted ZINC-3573—a potent MRGPRX2-selective agonist, showing little activity against 315 other GPCRs and 97 representative kinases—along with an essentially inactive enantiomer. ZINC-3573 activates endogenous MRGPRX2 in a human mast cell line, inducing degranulation and calcium release. MRGPRX2 is a unique atypical opioid-like receptor important for modulating mast cell degranulation, which can now be specifically modulated with ZINC-3573.

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Figure 1: Validation of MRGPRX2 and MRGPRB2 Agonists.
Figure 2: PRESTO-Tango Screening of MRGPRX2 reveals new agonists.
Figure 3: MRGPRX2 is activated by many opioid scaffolds.
Figure 4: MRGPRX2 is preferentially activated by prodynorphin-derived peptides.
Figure 5: In silico MRGPRX2 homology modeling predicts a selective agonist.
Figure 6: MRGPRX2 mediates intracellular calcium release and degranulation in the LAD2 human mast cell line.

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Support was given by National Institutes of Health (NIH) grants U01104974 (B.L.R., B.K.S. and W.K.K.), the NIH Department of Pharmacology Training Grant (K.L.), a Genentech Foundation Pre-doctoral Fellowship (J.K.), and a PhRMA Foundation Predoctoral Fellowship (K.L.). We thank the National Institute on Drug Abuse Drug Supply Program for supplying the morphine and codeine analogs and the glucuronidated or acetylated metabolites used in this study.

Author information




K.L. performed the in vitro pharmacology and molecular biology and wrote the paper. J.K. designed and developed homology models, carried out docking screens, analyzed results, and wrote the paper. J.L. synthesized the probe enantiomers. X.-P.H. performed GPCRome screening and assisted with in vitro pharmacology experiments. J.D.M. performed binding studies and in vitro pharmacology. W.K.K. assisted in the in vitro small-molecule screening and helped with data and statistical analyses. T.C. performed in vitro pharmacology experiments. H.N. synthesized (+)-TAN-67 and KNT-127. F.I.C. synthesized several compounds and advised structure–activity relationship studies. J.J. supervised chemical synthesis of probe enantiomers. B.L.R. and B.K.S. coordinated and supervised the project, and with the other authors wrote the paper.

Corresponding author

Correspondence to Bryan L Roth.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–12 (PDF 3279 kb)

Supplementary Note

Chemical compound characterization for selective probes (R)-ZINC-3573 and (S)-ZINC-3573 (PDF 348 kb)

Supplementary Data Set 1

PDB file for viewing ZINC-9232 docked in the MRGPRX2 model structure (TXT 207 kb)

Supplementary Data Set 2

PDB file for viewing dextromethorphan docked in the MRGPRX2 model structure (TXT 207 kb)

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Lansu, K., Karpiak, J., Liu, J. et al. In silico design of novel probes for the atypical opioid receptor MRGPRX2. Nat Chem Biol 13, 529–536 (2017).

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