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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Protein Data Bank
Allen, J.A. & Roth, B.L. Strategies to discover unexpected targets for drugs active at G protein-coupled receptors. Annu. Rev. Pharmacol. Toxicol. 51, 117–144 (2011).
Overington, J.P., Al-Lazikani, B. & Hopkins, A.L. How many drug targets are there? Nat. Rev. Drug Discov. 5, 993–996 (2006).
Huang, X.P. et al. Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65. Nature 527, 477–483 (2015).
Rask-Andersen, M., Masuram, S. & Schiöth, H.B. The druggable genome: evaluation of drug targets in clinical trials suggests major shifts in molecular class and indication. Annu. Rev. Pharmacol. Toxicol. 54, 9–26 (2014).
Fredriksson, R. & Schiöth, H.B. The repertoire of G-protein-coupled receptors in fully sequenced genomes. Mol. Pharmacol. 67, 1414–1425 (2005).
Kroeze, W.K. et al. PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nat. Struct. Mol. Biol. 22, 362–369 (2015).
Ngo, T. et al. Identifying ligands at orphan GPCRs: current status using structure-based approaches. Br. J. Pharmacol. 173, 2934–2951 (2016).
Isberg, V. et al. Computer-aided discovery of aromatic l-α-amino acids as agonists of the orphan G protein-coupled receptor GPR139. J. Chem. Inf. Model. 54, 1553–1557 (2014).
Mason, J.S., Bortolato, A., Congreve, M. & Marshall, F.H. New insights from structural biology into the druggability of G-protein-coupled receptors. Trends Pharmacol. Sci. 33, 249–260 (2012).
Zylka, M.J., Dong, X., Southwell, A.L. & Anderson, D.J. Atypical expansion in mice of the sensory neuron-specific Mrg G-protein-coupled receptor family. Proc. Natl. Acad. Sci. USA 100, 10043–10048 (2003).
Dong, X., Han, S., Zylka, M.J., Simon, M.I. & Anderson, D.J. A diverse family of GPCRs expressed in specific subsets of nociceptive sensory neurons. Cell 106, 619–632 (2001).
Lembo, P.M. et al. Proenkephalin A gene products activate a new family of sensory neuron-specific GPCRs. Nat. Neurosci. 5, 201–209 (2002).
Tatemoto, K. et al. Immunoglobulin E-independent activation of mast cell is mediated by Mrg receptors. Biochem. Biophys. Res. Commun. 349, 1322–1328 (2006).
Kamohara, M. et al. Identification of MrgX2 as a human G-protein-coupled receptor for proadrenomedullin N-terminal peptides. Biochem. Biophys. Res. Commun. 330, 1146–1152 (2005).
Subramanian, H. et al. β-Defensins activate human mast cells via Mas-related gene X2. J. Immunol. 191, 345–352 (2013).
Robas, N., Mead, E. & Fidock, M. MrgX2 is a high potency cortistatin receptor expressed in dorsal root ganglion. J. Biol. Chem. 278, 44400–44404 (2003).
Malik, L. et al. Discovery of non-peptidergic MrgX1 and MrgX2 receptor agonists and exploration of an initial SAR using solid-phase synthesis. Bioorg. Med. Chem. Lett. 19, 1729–1732 (2009).
Johnson, T. & Siegel, D. Complanadine A, a selective agonist for the Mas-related G protein-coupled receptor X2. Bioorg. Med. Chem. Lett. 24, 3512–3515 (2014).
McNeil, B.D. et al. Identification of a mast-cell-specific receptor crucial for pseudo-allergic drug reactions. Nature 519, 237–241 (2015).
Southern, C. et al. Screening β-arrestin recruitment for the identification of natural ligands for orphan G-protein-coupled receptors. J. Biomol. Screen. 18, 599–609 (2013).
Sromek, A.W. et al. Preliminary pharmacological evaluation of enantiomeric morphinans. ACS Chem. Neurosci. 5, 93–99 (2014).
Wang, M.H. et al. Activation of opioid mu-receptor by sinomenine in cell and mice. Neurosci. Lett. 443, 209–212 (2008).
Nagase, H. et al. The pharmacological profile of delta opioid receptor ligands, (+) and (−) TAN-67 on pain modulation. Life Sci. 68, 2227–2231 (2001).
White, K.L. et al. Identification of novel functionally selective κ-opioid receptor scaffolds. Mol. Pharmacol. 85, 83–90 (2014).
Horn, F. et al. GPCRDB: an information system for G-protein-coupled receptors. Nucleic Acids Res. 26, 275–279 (1998).
Lin, H., Sassano, M.F., Roth, B.L. & Shoichet, B.K. A pharmacological organization of G protein-coupled receptors. Nat. Methods 10, 140–146 (2013).
Irwin, J.J. & Shoichet, B.K. ZINC--a free database of commercially available compounds for virtual screening. J. Chem. Inf. Model. 45, 177–182 (2005).
Eswar, N. et al. Comparative protein structure modeling using MODELLER. Curr. Protoc. Protein Sci. 50, 2.9.1–2.9.31 (2007).
Yang, Q. & Sharp, K.A. Building alternate protein structures using the elastic network model. Proteins 74, 682–700 (2009).
Mysinger, M.M. & Shoichet, B.K. Rapid context-dependent ligand desolvation in molecular docking. J. Chem. Inf. Model. 50, 1561–1573 (2010).
Mysinger, M.M. et al. Structure-based ligand discovery for the protein–protein interface of chemokine receptor CXCR4. Proc. Natl. Acad. Sci. USA 109, 5517–5522 (2012).
Carlsson, J. et al. Ligand discovery from a dopamine D3 receptor homology model and crystal structure. Nat. Chem. Biol. 7, 769–778 (2011).
Jacobson, M.P., Friesner, R.A., Xiang, Z. & Honig, B. On the role of the crystal environment in determining protein side chain conformations. J. Mol. Biol. 320, 597–608 (2002).
Ballesteros, J.A. & Weinstein, H. Integrated methods for the construction of three-dimensional models and computational probing of structure–function relations in G-protein-coupled receptors. Methods Neurosci. 25, 366–428 (1995).
O'Connor, C. et al. NMR structure and dynamics of the agonist dynorphin peptide bound to the human kappa opioid receptor. Proc. Natl. Acad. Sci. USA 112, 11852–11857 (2015).
Irwin, J.J. & Shoichet, B.K. Docking screens for novel ligands conferring new biology. J. Med. Chem. 59, 4103–4120 (2016).
Jacquet, Y.F., Klee, W.A., Rice, K.C., Iijima, I. & Minamikawa, J. Stereospecific and nonstereospecific effects of (+)- and (−)-morphine: evidence for a new class of receptors? Science 198, 842–845 (1977).
Baldo, B.A. & Pham, N.H. Histamine-releasing and allergenic properties of opioid analgesic drugs: resolving the two. Anaesth. Intensive Care 40, 216–235 (2012).
Rosow, C.E., Moss, J., Philbin, D.M. & Savarese, J.J. Histamine release during morphine and fentanyl anesthesia. Anesthesiology 56, 93–96 (1982).
Kumar, K. & Singh, S.I. Neuraxial opioid-induced pruritus: an update. J. Anaesthesiol. Clin. Pharmacol. 29, 303–307 (2013).
Hutchinson, M.R. et al. Exploring the neuroimmunopharmacology of opioids: an integrative review of mechanisms of central immune signaling and their implications for opioid analgesia. Pharmacol. Rev. 63, 772–810 (2011).
Yamasaki, H. Pharmacology of sinomenine, an anti-rheumatic alkaloid from Sinomenium acutum. Acta Med. Okayama 30, 1–20 (1976).
Zajac, M. et al. [Recreational usage of dextromethorphan—analysis based on internet users experiences]. Przegl. Lek. 70, 525–527 (2013).
Scimemi, A. & Beato, M. Determining the neurotransmitter concentration profile at active synapses. Mol. Neurobiol. 40, 289–306 (2009).
Podvin, S., Yaksh, T. & Hook, V. The emerging role of spinal dynorphin in chronic pain: a therapeutic perspective. Annu. Rev. Pharmacol. Toxicol. 56, 511–533 (2016).
Sweetnam, P.M., Neale, J.H., Barker, J.L. & Goldstein, A. Localization of immunoreactive dynorphin in neurons cultured from spinal cord and dorsal root ganglia. Proc. Natl. Acad. Sci. USA 79, 6742–6746 (1982).
Rojewska, E., Makuch, W., Przewlocka, B. & Mika, J. Minocycline prevents dynorphin-induced neurotoxicity during neuropathic pain in rats. Neuropharmacology 86, 301–310 (2014).
Bienenstock, J. et al. Mast cell/nerve interactions in vitro and in vivo. Am. Rev. Respir. Dis. 143, S55–S58 (1991).
Barelier, S., Sterling, T., O'Meara, M.J. & Shoichet, B.K. The recognition of identical ligands by unrelated proteins. ACS Chem. Biol. 10, 2772–2784 (2015).
Akuzawa, N., Obinata, H., Izumi, T. & Takeda, S. Morphine is an exogenous ligand for MrgX2, a G-protein-coupled receptor for cortistatin. J. Cell Animal Biol. 2, 004–009 (2007).
Wu, H.E., Schwasinger, E.T., Terashvili, M. & Tseng, L.F. dextro-Morphine attenuates the morphine-produced conditioned place preference via the sigma(1) receptor activation in the rat. Eur. J. Pharmacol. 562, 221–226 (2007).
Kirshenbaum, A.S. et al. Characterization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 established from a patient with mast cell sarcoma/leukemia; activation following aggregation of FcepsilonRI or FcgammaRI. Leuk. Res. 27, 677–682 (2003).
Jordan, M., Schallhorn, A. & Wurm, F.M. Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 24, 596–601 (1996).
Staats, H.F. et al. A mast cell degranulation screening assay for the identification of novel mast cell activating agents. MedChemComm 4, 88–94 (2013).
Pei, J., Kim, B.H. & Grishin, N.V. PROMALS3D: a tool for multiple protein sequence and structure alignments. Nucleic Acids Res. 36, 2295–2300 (2008).
Jacobson, M.P. Epilepsy in aging populations. Curr. Treat. Options Neurol. 4, 19–30 (2002).
Sharp, K.A. Polyelectrolyte electrostatics: salt dependence, entropic, and enthalpic contributions to free energy in the nonlinear Poisson–Boltzmann model. Biopolymers 36, 227–243 (1995).
Meng, E.C., Shoichet, B.K. & Kuntz, I.D. Automated docking with grid-based energy evaluation. J. Comput. Chem. 13, 505–524 (1992).
Li, J., Zhu, T., Cramer, C.J. & Truhlar, D.G. New class IV charge model for extracting accurate partial charges from wave functions. J. Phys. Chem. A 102, 1820–1831 (1998).
Chambers, C.C., Hawkins, G.D., Cramer, C.J. & Truhlar, D.G. Model for aqueous solvation based on class IV atomic charges and first solvation shell effects. J. Phys. Chem. 100, 16385–16398 (1996).
Paruch, K. et al. Discovery of Dinaciclib (SCH 727965): a potent and selective inhibitor of cyclin-dependent kinases. ACS Med. Chem. Lett. 1, 204–208 (2010).
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.
The authors declare no competing financial interests.
Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–12 (PDF 3279 kb)
Chemical compound characterization for selective probes (R)-ZINC-3573 and (S)-ZINC-3573 (PDF 348 kb)
PDB file for viewing ZINC-9232 docked in the MRGPRX2 model structure (TXT 207 kb)
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). https://doi.org/10.1038/nchembio.2334
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