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

Abstract

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.

Figure 1. Validation of MRGPRX2 and MRGPRB2 Agonists

A–D) Average concentration response curves in the FLIPR intracellular calcium release assay (n=3 in triplicate) in MRGPRX2-inducible cells, designated +X2 or –X2 for +/−tetracycline-induced receptor expression, for previously published MRGPRX2 agonists. Y axis is fold change calcium release over basal. E) Concentration response curves in FLIPR intraceullar calcium release assay for stable cell lines expressing either the proposed orthologues human MRGPRX2 (solid lines) or mouse MRGPRB2 (dotted lines) with X2-activating peptides Cortistatin-14 and Substance P and small molecule agonist TAN-67. Y axis is fold change calcium release over basal (n=3 in quadruplicate) F) Concentration response curves for Cortistatin-14, Substance P, and small molecule agonist TAN-67 with MRGPRX2-Tango (solid lines) and MRGPRB2-Tango (dotted lines) in the PRESTO-Tango arrestin recruitment assay (n=3 in triplicate). Y axis is fold change response over basal luminescent signal. All error bars represent SEM.

Figure 2. PRESTO-Tango Screening of MRGPRX2 reveals new agonists

A) Venn diagram depicting pooled PRESTO-Tango screening actives for MRGPRX1, MRGPRX2, and MRGPRX4 for all hits with greater than 2-fold activation over basal (known false-positives and duplicates excluded). B-C) Average concentration response curves (n=3 in triplicate wells for all, except ADL5859 n=1) for the five compounds from the screening show low micromolar activation of MRGPRX2. D) Concentration response curve for previously published MRGPRX2 peptide agonists at the MRGPRX2-Tango construct. Y axes shown as % of TAN-67 activity. All error bars represent SEM.

Figure 3. MRGPRX2 is activated by many opioid scaffolds

A–D) Average concentration response curves (n=3 in triplicate) for structurally related compounds (morphinans, benzomorphans, morphine and analogues, and codeine and analogues, respectively) in intracellular calcium release assay where Y axis is fold change calcium release over baseline. Error bars represent SEM. E) Summary of major findings in structure activity relationships for opioid scaffolds at MRGPRX2, including size of N-substituent, stereochemistry of chiral centers, and bulk on the 3-position of the morphinan.

Figure 4. MRGPRX2 is preferentially activated by prodynorphin-derived peptides

A–B) Averaged concentration response curves (n=2 or 3 in triplicate, see Supplementary Table 2) for pro-dynorphin-derived peptides in MRGPRX2 Tet-On cells where Y axis is fold change calcium release over baseline. Error bars represent SEM. C, D) Average concentration response curves (n=2 or 3 in triplicate, see Supplementary Table 2) depicting non-prodynorphin-derived peptides with minimal activity compared to dynorphins in A and B. Error bars represent SEM.

Figure 5. In silico MRGPRX2 homology modeling predicts a selective agonist

Workflow depicting MRGPRX2 homology model construction (top left) followed by identification of a putative binding site (top middle) that was confirmed by testing the mutations E164Q and D184N (top right, average dose response, n=3 in triplicate, shown with dextromethorphan (DXM). Then, ~3.7 million molecules were docked to predict the agonist ZINC-9232 (bottom left). Further iteration and testing revealed the tool and selective compounds (S)- and (R)-ZINC-5373 (bottom middle and right). Docking pose of selective compound is supported with mutation experiments in PRESTO-Tango (bottom right, average dose response curve, average of n=4 in triplicate). Error bars on graphs shown is representative of SEM.

Figure 6. MRGPRX2 mediates intracellular calcium release and degranulation in the LAD2 human mast cell line

A) Average concentration response for MRGPRX2-selective agonists ZINC-6932 and ZINC-3573 in the calcium mobilization assay (n=3 in triplicate). B) Average concentration response (n=3 in triplicate) for MRGPRX2 probes ZINC-3573 (R) and ZINC-3573 (S) in the β-hexosaminidase degranulation assay. C). Bar graph depicting fold change %degranulation (baseline = average DMSO of all plates) induced by EC80 concentration of drug following 25 nM MRGPRX2 siRNA transfection; NT = Non Targeting pool, siRNA 2+3 = MRGPRX2 siRNA pool. Statistics = Two-way ANOVA with a Sidak post-hoc test (p <0.05 = *, p=0.031, 0.031, 0.033, and 0.017 for TAN, Dyn, Morphine, and ZINC-3573, respectively). n=3 for all except ZINC-3573, n=2. All replicates in triplicate wells. D) Concentration response curves for MRGPRX2-activating opioid ligands and the structurally related, κ-opioid receptor ligand (−)-cyclazocine in the calcium mobilization assay (n=3 in triplicate). E) Average concentration response in intracellular calcium release with MRGPRX2-activating (+/−)-TAN-67 and canonical opioid receptor ligands DADLE, DAMGO, Salvinorin A, and BW373U86 (n=3 in triplicate). F) Bar graph depicting baseline normalized percent degranulation induced for each 10 μM (+/−)-TAN-67, DMSO, and calcium ionophore ionomycin in the presence of absence of biotin-labeled IgE antibodies. (Streptavidin in all wells, average of n=2 in triplicate.) G). Average concentration response curves for MRGPRX2-activating opiates (n=3 in triplicate.) in the β-hexosaminidase degranulation assay. H) Degranulation concentration response with canonical opioid receptor agonists and MRGPRX2-activating (+/−)-TAN-67, (n=3 in triplicate). All error bars demonstrate SEM.