MRGPRX2/MRGPRB2 EXPRESSING CELL BASED ASSAY TO DETECT PSEUDO-ALLERGIC DRUG REACTIONS AND TO IDENTIFY BLOCKERS TO PREVENT THE ADVERSE REACTIONS

Abstract

The present invention relates to cells and methods for detecting compounds that induce a pseudo-allergic-type reaction and methods for reducing the severity of a pseudo-allergic-type reaction.

Claims

1-22. (canceled)

23. A method for identifying an antagonist of MrgprX2 or MrgprB2 comprising: contacting an isolated cell comprising a recombinant nucleic acid that expresses mas-related G-protein coupled receptor member X2 (MrgprX2) or MrgprB2 with a compound that induces a pseudo-allergic-type reaction, contacting the isolated cell with a candidate antagonist, detecting activation of MrgprX2 or MrgprB2, wherein a decrease in activation of MrgprX2 or MrgprB2 relative to the activation of MrgprX2 or MrgprB2 in the absence of the compound determines that the candidate compound is an antagonist.

24. The method of claim 23, wherein the recombinant nucleic acid expresses MrgprX2.

25. The method of claim 23, wherein the recombinant nucleic acid expresses MrgprB2.

26. The method of claim 24, wherein the recombinant nucleic acid that expresses MrgprX2 comprises one or more mutations.

27. The method of claim 26, wherein the one or more mutations produces an MrgprX2 protein incapable of activating a signal transduction pathway.

28. The method of claim 25, wherein the recombinant nucleic acid that expresses MrgprB2 comprises one or more mutations.

29. The method of claim 28, wherein the one or more mutations produces an MrgprB2 protein incapable of activating a signal transduction pathway.

30. The method of claim 23, wherein the isolated cell comprises a human embryonic kidney 293 (HEK 293) cell.

31. The method of claim 23 wherein the MrgprB2 comprises the amino acid sequence of SEQ ID NO: 3.

32. The method of claim 23 wherein the MrgprX2 comprises the amino acid sequence of SEQ ID NO: 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIG. 1A is diagram of the mouse and human Mrgpr genomic loci, illustrating the expanded gene family in mice and their identified human orthologues based on similar expression patterns and agonist specificity. The mouse Mrgpr genes (shown as vertical bars with names on top) are clustered on chromosome 7. Mouse MrgprA3 (A3) and MrgprC11 (C11) are the orthologues of human MrgprX1 (X1) and are expressed specifically in dorsal root ganglion (DRG) neurons, and function as itch receptors mediating chloroquine (CQ) and BAMS-22 (BAM) induced itch. The expression and agonist specificity of mouse MrgprB2 (B2), the orthologue of human MrgprX2 (X2), are described below. FIG. 1B shows results from a stringent reverse transcription polymerase chain reaction (RT-PCR) screen that identified expression of MrgprB2 transcript (arrow) in mouse peritoneal mast cells. The Mrgpr gene names are indicated on the top of the gel pictures. No band was seen when reverse transcriptase was omitted from the cDNA synthesis reaction (Neg.). FIG. 1C shows exemplary traces of intracellular calcium concentrations [Ca.sup.2+]i, as measured by ratiometric Fura-2 imaging, from HEK293 cells transiently transfected with a plasmid driving expression of MrgprB2 or MrgprX2 and exposed to bath application of 20 M PAMP(9-20) (duration indicated by black line on top). Each trace is a response from a unique cell. FIG. 1D is a series of photomicrographs showing shows representative confocal images from BAC transgenic mouse tissues. BAC mice expressing eGFP-Cre in the MrgprB2 open reading frame were mated to Rosa26-loxP-STOP-loxP-tdTomato reporter mice. Therefore, the expression of tdTomato is determined by the expression pattern of Cre, which is under control of MrgprB2 promoter. tdTomato (red) expression was compared to avidin staining (green), a marker for mast cells. Nearly 100% overlap between the two markers suggests that MrgprB2 is specifically contained in mast cells. Three mice were examined for all tissues except heart, where two mice were examined Percentages of avidin-positive mast cells that also were tdTomato-positive: glabrous skin, 97.5%; hairy skin, 90.1%; trachea, 97.2%; heart, 87.1%. Percentages of tdTomato-positive cells that also were avidin-positive: glabrous skin, 99.2%; hairy skin, 100%; trachea, 98.3%; heart, 99%. Total number of cells counted in each tissue was over 300, except for heart which was over 100. Heart mast cells were examined near cavities because the density was much higher than elsewhere in the tissue; avidin-positive cells that were negative for tdTomato were observed embedded in muscle tissue in very low numbers, but their identity was unclear. Scale bar is 20 m.

[0084] FIG. 2A (left) is a series of photomicrographs showing representative heat map images of mouse peritoneal mast cells showing changes in [Ca.sup.2+]i, as assayed by Fluo-4 imaging, induced by bath application of anti-IgE (5 g/ml) or Compound 48/80 (10 g/ml). FIG. 2A (Middle) shows representative imaging traces. Each color line represents an individual cell. Black lines in anti-IgE panels are average traces for each genotype. Note: [Ca2+]i traces are similar between WT and MUT groups. FIG. 2A (right) shows quantification of percentage of responding cells. Cells were identified as responding if the [Ca.sup.2+]i rose by at least 50% for at least 10 seconds, which clearly distinguishes a ligand-induced response from random flickering events. Group data for these and all other experiments are expressed as meanstandard error of the mean. One-tailed unpaired Student's t-test was used to determine significance in statistical comparisons, and differences were considered significant at p<0.05. **, p<0.01 (n=3 for each genotype; over 150 cells counted for each condition). Anti-IgE responses were not significantly different. Scale bar is 10 82 m. FIG. 2B is a bar graph that shows histamine release into the supernatant from trachea and abdominal skin from WT and MrgprB2.sup.MUT mice after exposure to 48/80 (30 g/ml) for 30 minutes at 37 C. The amount of histamine released into the supernatant was quantified and expressed ng/mg tissue (wet weight). **, p<0.01 (n=5 for trachea, n=8 for skin). FIG. 2C is a series of graphs. FIG. 2C (top) shows representative traces showing contractions of trachea isolated from WT and MrgprB2.sup.MUT mice (previously sensitized to ovalbumin (ova)), in response to 48/80 (30 g/ml) or ova (10 g/ml; i.e. IgE-dependent). FIG. 2C (bottom) shows the average data of maximum total contraction determined as response to 10 M carbamycholine added at the end of the experiment. n=5 for 48/80 WT, 3 for 48/80 MrgprB2.sup.MUT. FIG. 2D is a series of photographs and a bar chart showing (left) representative images of Evans Blue extravasation 15 minutes after intraplantar injection of 48/80 (right, arrow, 10 g/ml, 5 l in saline) or saline (left). FIG. 2D (right) shows quantification of Evans Blue leakage into the paw and paw thickness increase after 15 minutes. *, p<0.02 (n=5/WT, n=6/MrgprB2.sup.MUT). Differences after saline injection were not significant. FIG. 2E is a bar chart showing quantification of WT and MrgprB2.sup.MUT mast cell responsiveness to MrgprX2 ligands and basic secretagogues, assayed using Fluo-4 imaging. Concentrations of substances (in M): PAMP(9-20), 20; cortistatin-14 (cort.), 20; Substance P (sub P), 200; kallidin, 200; mastoparan (masto., a component of wasp venom), 20; vespid mastoparan, 20. n=3/genotype; >150 cells counted/secretagogue. Data are presented as meanstandard error of mean (SEM). Two-tailed unpaired Student's t test was used to determine significance in statistical comparisons, and differences were considered significant at p<0.05. *, p<0.05. **, p<0.01 unless noted.

[0085] FIG. 3A is a bar chart showing that MrgprB2 mediates mast cell responsiveness and side effects of peptidergic therapeutic drugs. FIG. 3A shows the percentage of responding cells from WT and MrgprB2.sup.MUT peritoneal mast cells after drug application, assayed using Fluo-4 imaging. Concentrations of drugs (in g/ml): icatibant, 50; cetrorelix, 20; leuprolide, 100; octreotide, 10; sermorelin, 60; insulin, 80. n=3/genotype; >150 cells counted/substance, except>100 cells counted for insulin. Difference between insulin responsiveness was not significant. FIG. 3B (left) shows representative images of Evans Blue extravasation 15 minutes after intraplantar injection of icatibant (right, arrow, 10 mg/ml, 5 l in saline) or saline (left). FIG. 3B (right) shows quantification of Evans Blue leakage into the paw after 15 minutes. **, p<0.01 (n=6 for each genotype). Difference after saline injection was not significant. FIG. 3C is a series of dot plots showing total histamine release from WT (red diamonds) and MrgprB2.sup.MUT (black squares) mice after incubation with named substances. No significant difference between WT and MrgprB2.sup.MUT cells was found at any dose of anti-IgE antibody. Experiments were repeated >3 times. Data are presented as meanSEM. Two-tailed unpaired Student's t test: *, p<0.05. **, p<0.01.

[0086] FIG. 4A is a series of structures of Compound 48/80 and a cyclized variant, which is reported to be more potent. The tetrahydroisoquinoline (THIQ) motif is highlighted in blue. FIG. 4B is a series of structures of representative members of all NMBD classes. THIQ motifs are highlighted in blue. Note that only succinylcholine lacks a bulky hydrophobic group. FIG. 4C is a bar chart showing that MrgprB2 mediates mast cell responsiveness and side effects of small molecule therapeutic drugs. FIG. 4C shows the percentage of responding cells from WT and MrgprB2.sup.MUT peritoneal mast cells after application of various NMBDs, assayed using Fluo-4 imaging. Concentrations of drugs (in g/ml): atracurium, 50; mivacurium, 20; tubocurarine, 30; rocuronium, 500. n=3 mice/genotype; >150 cells counted/substance. FIG. 4D is a depiction of the structure of ciprofloxacin, with the motif common to all fluoroquinolones highlighted in blue. Note nitrogens close to the quinolone motifs. FIG. 4E is a bar chart showing the percentage of responding cells from WT and MrgprB2.sup.MUT peritoneal mast cells after fluoroquinolone application, assayed using Fluo-4 imaging. Concentrations of drugs (in g/ml): ciprofloxacin, 200; levofloxacin, 500; moxifloxacin, 160; ofloxacin, 400. n=3 mice/genotype; >150 cells counted/substance. FIG. 4F is a line graph showing changes in body temperature after intravenous injection of ciprofloxacin (1.5 mg in 125 l saline) at time 0. n=4 mice/genotype. Data are presented as meanSEM. Two-tailed unpaired Student's t test: *, p<0.05. **, p<0.01.

[0087] FIG. 5A is a photograph of a blot showing that MrgprX1 orthologues are not expressed at relevant levels in mast cells under naive conditions. FIG. 5A shows results from a low-stringency RT-PCR screen in peritoneal mast cells for expression of the MrgprX1 orthologues MrgprA3 and MrgprC11. Arrow points to expected band sizes. FIG. 5B is a bar chart showing the percentages of peritoneal mast cells responding to the MrgprX1 and MrgprC11 agonist Bovine Adrenal Medulla derived peptide, fragment 8-22 (BAMS-22, 500 nM). Activation was assayed by measuring rises in intracellular calcium, using imaging of the Fluo-4 dye. Differences are not significant (p=0.39). Group data are expressed as meanstandard error of the mean. Two-tailed unpaired Student's t test was used to determine significance in statistical comparisons. FIG. 5C is a chart summarizing responses to MrgprX2 ligands and the MrgprX1 ligand chloroquine (CQ) by HEK293 cells transiently transfected with plasmids driving expression of MrgprX2, MrgprB2, and other mouse Mrgprs (i.e. MrgprB1, B10, and B11) most closely related to MrgprB2. Positive and negative responses are indicated as checks and crosses, respectively. Responses were considered positive if at least half of the transfected cells showed a 50% increase in [Ca2+]i. No cells transfected with MrgprB1, B10, and B11 responded to any listed drug.

[0088] FIG. 6A is a series of line graphs that show basic secretagogues and drugs that induce pseudo-allergic reactions activate mouse MrgprB2 and human MrgprX2 expressed in HEK293 cells. FIG. 6A shows example traces showing changes in [Ca2+]i, as measured by Fluo-4 imaging, from HEK293 cells expressing MrgprB2 and G15. FIG. 6B shows example traces showing changes in [Ca2+]i, as measured by Fluo-4 imaging, from HEK293 cells expressing MrgprX2 and G15. Substances were perfused from the 30 to 90 second time period, except for ciprofloxacin, which was perfused between the 30 and 60 second time periods to minimize exposure to the low pH solutions it was dissolved in. Insulin was used as a negative control. FIG. 6C is a table of EC50s of basic secretagogues and drugs associated with pseudo-allergic reactions to activate MrgprB2 and MrgprX2-expressing HEK293 cells. The EC50s were determined from dose response studies which were repeated three times. Data are expressed as meanSEM.

[0089] FIG. 7 is a series of photomicrographs showing that multiple lines of BAC transgenic mice confirm mast cell specific MrgprB2 expression. FIG. 7 shows representative confocal images from two other BAC transgenic mouse lines. BAC mice expressing eGFP-Cre in the MrgprB2 open reading frame were mated to tdTomato reporter mice and tdTomato (red) expression was compared to avidin staining (green), a marker for mast cells. Scale bar is 20 m.

[0090] FIG. 8A-FIG. 8B show that MrgprB2 is not expressed in mucosal mast cells or peripheral white blood cells. FIG. 8A shows representative images of a stomach section from an MrgprB2-tdTomato mouse stained with an anti-MCPT1 (-chymase) antibody to label mucosal mast cells. White arrows indicate positive cells. No cells were double-labeled (296 Mcpt1-labeled cells and 275 tdTomato-positive cells counted, n=3 mice). Scale bar is 40 m. FIG. 8B shows representative images of a Cytospin preparation of peripheral white blood cells from an MrgprB2-tdTomato mouse doubly labeled with tdTomato for MrgprB2-expressing cells (red; left image) and Hoechst 33342 nuclear staining (blue; right image). No peripheral white blood cell expressed MrgprB2 (n=3 mice; >4000 cells examined). Scale bar is 40 m.

[0091] FIG. 9A, FIG. 9B, FIG. 9C, and FIG. 9D show that MrgprB2.sup.MUT mice are functional knockouts. FIG. 9A is an illustration of the genomic region in and around the MrgprB2 locus. Note that repetitive sequences including long interspersed elements (LINEs), short interspersed elements (SINEs), and long tandem repeats (LTRs) begin immediately after the 3 side of the MrgprB2 gene, and in addition are present within 2.5 kb of the 5 side. A BLASTN search in March 2014 using the 500 bases adjacent to the 3 end of MrgprB2 as a query turned up more than 269,000 hits in the mouse genome. FIG. 9B is a comparison of the WT and MUT genomic sequences that shows the location of the four base pair deletion in the mutant. Numbers correspond to the MrgprB2 open reading frame. FIG. 9C is a sequencing result from WT and MUT cDNA sampled from mice born 18 months after the mutant line was established. The bases missing in the mutant are highlighted in red. FIG. 9D is an amino acid translation of the MrgprB2.sup.MUT open reading frame that reveals that the deletion creates a frameshift mutation and an early termination codon (*) shortly after the first transmembrane region. Mutsite of the frameshift deletion. TM1transmembrane region 1.

[0092] FIG. 10A-FIG. 10B is a series of photomicrographs and bar charts showing that the mast cell numbers and the histamine content of tracheal and skin tissue was not different between wild type and MrgprB2.sup.MUT animals. FIG. 10A (top) shows representative pictures of avidin staining in WT and MrgprB2.sup.MUT mice. Scale bar is 40 m. FIG. 10A (bottom) shows quantification of mast cell numbers in various tissues. Differences are not significant, using a two-tailed unpaired Student's t test (n=3 mice for each genotype; over 3000 m2 and 1000 m2 counted for each genotype for hairy and glabrous skin, respectively; over 10,000 peritoneal cells counted). FIG. 10B shows that the tracheal histamine content averaged 5.90.9 and 5.51.6 ng/mg (n=5 for each genotype), respectively; the skin histamine content averaged 30.83.2 and 30.24.0 ng/mg (n=8 for each genotype), respectively. Differences were not significant. Group data are expressed as meanstandard error of the mean. Two-tailed unpaired Student's t test was used to determine significance in statistical comparisons.

[0093] FIG. 11A, FIG. 11B, and FIG. 11C are a series of photomicrographs, a line graph, and a bar chart, respectively, which show endothelin acting through the ETA GPCR1 induced comparable activation in MrgprB2.sup.MUT and wild-type mast cells. FIG. 11A shows representative heat map images of mouse peritoneal mast cells showing changes in [Ca2+]i, as assayed by Fluo-4 imaging, induced by bath application of endothelin (1 M). Scale bar is 10 m. FIG. 11B shows averages of [Ca2+]i imaging traces for WT (red line) and MrgprB2.sup.MUT (black line). The [Ca2+]i traces are similar between WT and MUT groups. Traces were averaged as described for FIG. 2A. FIG. 11 C shows quantification of percentage of responding cells. Group data are expressed as meanstandard error of the mean. Two-tailed unpaired Student's t test was used to determine significance in statistical comparisons (n=3 for each genotype; over 180 cells counted for each genotype). Endothelin-induced responses were not significantly different.

[0094] FIG. 12A and FIG. 12B show that IgE-mediated inflammation does not differ between wild type and MrgprB2.sup.MUT mice. FIG. 12A shows representative images of Evans Blue extravasation 15 minutes after intraplantar injection of anti-IgE antibody (right, arrow, 100 g/ml, 7 l in saline) or saline (left). FIG. 12B shows quantification of Evans Blue leakage into the paw after 15 minutes (n=6 for WT, n=7 for MrgprB2.sup.MUT). Differences after anti-IgE antibody (p=0.49) and saline (p=0.23) injection are not significant. Group data are expressed as meanstandard error of the mean. Two-tailed unpaired Student's t test was used to determine significance in statistical comparisons.

[0095] FIG. 13A, FIG. 13B, FIG. 13C, FIG. 13D, and FIG. 13E show that MrgprB2.sup.MUT mast cells are unresponsive to basic secretagogues and various therapeutic drugs. FIG. 13A shows example traces showing changes in [Ca2+]i, as measured by Fluo-4 imaging, from WT and MrgprB2.sup.MUT peritoneal mast cells induced by the basic secretagogues from FIG. 2E. Each trace is a response from a unique cell. FIG. 13B shows representative Fluo-4 images (left) and fluorescence traces (right) from WT (top) and MrgprB2.sup.MUT (bottom) cultured peritoneal mast cells during application of icatibant (50 g/ml). FIG. 9C shows example traces showing changes in [Ca2+]i, as measured by Fluo-4 imaging, from WT and MrgprB2.sup.MUT peritoneal mast cells induced by selected FDA-approved cationic peptidergic drugs. Each trace is a response from a unique cell. FIG. 13D is a series of photomicrographs and a line graph showing representative Fluo-4 images (left) and fluorescence traces (right) from WT (top) and MrgprB2.sup.MUT (bottom) cultured peritoneal mast cells during application of atracurium (50 g/ml). FIG. 13E is a series of photomicrographs and a line graph showing representative Fluo-4 images (left) and fluorescence traces (right) from WT (top) and MrgprB2.sup.MUT (bottom) cultured peritoneal mast cells during application of ciprofloxacin (200 g/ml).

[0096] FIG. 14A and FIG. 14B is a series of bar charts showing that human mast cells are activated by basic secretagogues and drugs associated with pseudo-allergic reactions in an MrgprX2-dependent manner FIG. 14A human LAD2 mast cells treated with different concentrations of compound 48/80, mastoparan, icatibant, atracurium, and ciprofloxacin. The activation of mast cells in response to these substances was characterized by the release of -hexosaminidase, TNF, PGD2, and histamine In addition, 0.1 g/ml streptavidin stimulation of biotin-conjugated human IgE sensitized LAD2 cells caused a robust release of -hexosaminidase (71.31.8% release), compared to untreated cells (4.10.3% release). Group data are expressed as meanstandard error of the mean. FIG. 14B shows that knockdown of human MrgprX2 significantly reduced mast cell activation evoked by basic secretagogues and drugs associated with pseudo-allergic reactions, but not by IgE. Human LAD2 mast cells were first transfected with MrgprX2 siRNA or control siRNA. Two days after the transfection, the cells were treated with compound 48/80 (0.1 g/ml), mastoparan (5 g/ml), icatibant (10 g/ml), atracurium (25 g/ml), and ciprofloxacin (75 g/ml). The activation of mast cells in response to these substances characterized by the release of -hexosaminidase was significantly reduced in MrgprX2 siRNA treated cells, compared to release in the control group. IgE-mediated mast cell degranulation was unaffected by MrgprX2 siRNA knockdown. Group data are expressed as meanstandard error of the mean. Two-tailed unpaired Student's t test was used to determine significance in statistical comparisons, and differences were considered significant at * p<0.05; ** p<0.01; *** p<0.005 (the experiments were repeated three times).

[0097] FIGS. 15A and 15B are tables of all FDA-approved therapeutic drugs under 50 amino acids. FIG. 15A shows cationic peptidergic drug familes and FIG. 15B shows other cationic peptidergic drugs, and neutral and anionic peptidergic drugs. Charges calculated by ChemAxon. ISR data for cetrorelix, ganirelix, and octreotide are from the following references: Verschraegen, C. F. et al. Gynecologic oncology 90, 552-559 (2003); Fluker, M. et al. Fertility and sterility 75, 38-45 (2001); and Tuvia, S. et al. The Journal of clinical endocrinology and metabolism 97, 2362-2369, (2012), incorporated herein by reference. All other information was supplied by the FDA.

[0098] FIG. 16 is a chart listing all classes of FDA-approved neuromuscular blocking drugs (NMBDs), and representative members.

DETAILED DESCRIPTION OF THE INVENTION

[0099] The invention features cells and methods for determining whether a compound induces a pseudo-allergic-type reaction and methods for reducing the severity of a pseudo-allergic-type reaction in a subject. The present invention is based, at least in part, on the discovery of a G protein coupled receptor, i.e., MrgprB2 in mice and MrgprX2 in humans, exclusively expressed in a type of immune cell called the mast cell, which is linked closely to allergic-type reactions to foreign substances. The inventors determined that this single receptor is activated by at least 13 different FDA-approved drugs associated with allergic-type reactions as part of their side effect profiles.

[0100] Prior to the invention described herein, the role of MrgprX2/MrgprB2 in pseudo-allergic drug reactions was completely unknown. Described herein is the use of MrgprX2/MrgprB2 expressing cell-based assays to screen for drugs that induce pseudo-allergic drug reactions, and to screen for antagonists of MrgprX2 that block these reactions. In order to make the assay work, the inventors added the GTP-binding protein alpha 15 (G15) in MrgprX2/MrgprB2 expressing cells to convert receptor-based signals to increases in intracellular calcium, making the cell lines compatible with high-throughput screening devices. MrgprX2/MrgprB2's role in pseudo-allergic drug reactions was unknown prior to the present invention. Accordingly, it is not surprising that MrgprX2/G15 cells have not been reported. While G15 expression in other isolated cell lines has been reported, it only is utilized when the receptor does not induce a rise in intracellular calcium. MrgprX2 induces such a rise which would suggest that including G15 in an isolated cell as unnecessary. However, inventors found that MrgprX2 normally induces such a rise in response to some agonists but not others, making co-expression essential for the assay to work.

[0101] The isolated cells of the present invention express the human G-protein coupled receptor (GPCR) MrgprX2 or the mouse GPCR MrgprB2, along with expression of the GTP-binding protein G15, which allows easy visualization of receptor activation in a calcium-based screening assay. These cell lines permit the screening of FDA-approved drugs and drugs in development for MrgprX2 agonist and antagonist activity. This is desirable because off-target activation of MrgprX2 induces allergic-type side effects in the body that can result in significant adverse events.

[0102] Using these cells in a cell-based assay for drug screens, a positive result (i.e., activation of the cell line as measured by, e.g., calcium release) would indicate that the drug would normally activate mast cells and potentially cause an allergic-type reaction in a patient.

[0103] Screens of drugs in development would predict their side effect profile; screens of drugs currently in use would identify a cause of the adverse effects of these drugs; screens of antagonists would lead to new therapeutic drugs that can be provided at the same time as drugs that induce allergic-type reactions, thus blocking activation of mast cells while not interfering with their intended uses.

[0104] As described in detail below, local and systemic allergic-type responses in wild-type mice in response to these drugs are abolished in mice that lack MrgprB2, the mouse orthologue of human MrgprX2. This highly unexpected finding demonstrates that MrgprX2 is an attractive drug target, since an antagonist can be co-applied with a very wide range of drugs to block their allergic-type side effects.

[0105] Described herein are cell lines that are used to screen for FDA-approved drugs and investigational compounds that activate or antagonize this receptor. These will be useful to determine whether a drug will induce allergic-type responses, and in screens to develop antagonists that block these responses. Described herein are results demonstrating that basic secretagogues activate mouse mast cells in vitro and in vivo through a single receptor, MrgprB2, the orthologue of the human G-protein coupled receptor (GPCR) MrgprX2. Secretagogue-induced histamine release, inflammation, and airway contraction are abolished in MrgprB2 null mutant mice. Further, as described in detail below, most classes of FDA-approved peptidergic drugs associated with allergic-type injection-site reactions also activate MrgprB2 and MrgprX2, and that injection-site inflammation is absent in mutant mice. The results described herein demonstrate that MrgprB2 and MrgprX2 are targets of many small molecule drugs associated with systemic pseudo-allergic, or anaphylactoid, reactions; that drug-induced symptoms of anaphylactoid responses are significantly reduced in knockout mice. Also described herein is the identification of a common chemical motif in several of these molecules that helps predict side effects of other compounds. Described in detail below is the introduction of a mouse model to study mast cell activation by basic secretagogues and identification of MrgprX2 as a therapeutic target to reduce a subset of drug-induced adverse effects.

Mast Cells

[0106] Mast cells are primary effectors in allergic reactions, and may have significant roles in diseases by secreting histamine and various inflammatory and immunomodulatory substances (Metcalfe, et al., 1997 Physiological reviews 77, 1033-1079; Galli et al., 2005 Nature immunology 6, 135-142). While classically they are activated by IgE antibodies, a unique property of mast cells is their antibody-independent responsiveness to a range of cationic substances, collectively called basic secretagogues, including inflammatory peptides and drugs associated with allergic-type reactions (Metcalfe, et al., 1997 Physiological reviews 77, 1033-1079; Lagunoff et al., 1983 Annual review of pharmacology and toxicology 23, 331-351). Roles for these substances in pathology have prompted a decades-long search for their receptor(s).

[0107] A mast cell (also known as a mastocyte or a labrocyte) is derived from the myeloid stem cell. It is a part of the immune system and contains many granules rich in histamine and heparin. Although best known for their role in allergy and anaphylaxis, mast cells play an important protective role as well, being intimately involved in wound healing, including angiogenesis, and defense against pathogens. The mast cell is very similar in both appearance and function to the basophil, another type of white blood cell. These cells differ in that mast cells are tissue resident, e.g., in mucosal tissues, while basophils are found in the blood. Both cells are granulated cells that contain histamine and heparin, an anticoagulant. Both cells also release histamine upon binding to immunoglobulin E.

[0108] Mast cells are present in most tissues characteristically surrounding blood vessels and nerves, and are especially prominent near the boundaries between the outside world and the internal milieu, such as the skin, mucosa of the lungs, and digestive tract, as well as the mouth, conjunctiva, and nose.

[0109] Mast cells play a key role in the inflammatory process. When activated, a mast cell rapidly releases its characteristic granules and various hormonal mediators into the interstitium. Mast cells can be stimulated to degranulate by direct injury (e.g., physical or chemical (such as opioids, alcohols, and certain antibiotics such as polymyxins]), cross-linking of immunoglobulin E (IgE) receptors, or complement proteins.

[0110] Mast cells express a high-affinity receptor (FcRI) for the Fc region of IgE, the least-abundant member of the antibodies. This receptor is of such high affinity that binding of IgE molecules is in essence irreversible. As a result, mast cells are coated with IgE, which is produced by plasma cells (the antibody-producing cells of the immune system).

[0111] In allergic reactions, mast cells remain inactive until an allergen binds to IgE already coated upon the cell. Other membrane activation events can either prime mast cells for subsequent degranulation or act in synergy with FcRI signal transduction. In general, allergens are proteins or polysaccharides. The allergen binds to the antigen-binding sites, which are situated on the variable regions of the IgE molecules bound to the mast cell surface. The clustering of the intracellular domains of the cell-bound Fc receptors, which are associated with the cross-linked IgE molecules, causes a complex sequence of reactions inside the mast cell that lead to its activation. The molecules released into the extracellular environment include: preformed mediators (from the granules) (e.g., serine proteases, such as tryptase, histamine (2-5 pg/cell), serotonin, proteoglycans, heparin (active as anticoagulant)), newly formed lipid mediators (eicosanoids) (i.e., thromboxane, prostaglandin D2, leukotriene C4, platelet-activating factor), and cytokines (e.g., eosinophil chemotactic factor).

[0112] Histamine dilates post-capillary venules, activates the endothelium, and increases blood vessel permeability. This leads to local edema (swelling), warmth, redness, and the attraction of other inflammatory cells to the site of release. Histamine also depolarizes nerve endings (leading to itching or pain). Cutaneous signs of histamine release include the flare and wheal-reaction. The bump and redness immediately following a mosquito bite are a good example of this reaction, which occurs seconds after challenge of the mast cell by an allergen.

MrgprX2

[0113] As described herein, mas-related G-protein coupled receptor member X2 (MrgprX2) is a mast cell-specific receptor for basic secretagogues, i.e., cationic amphiphilic drugs, as well as endo- or exogenous peptides, consisting of a basic head group and a hydrophobic core. See, McNeil B. D., 2015 Nature, 519: 237-241, incorporated herein by reference. As described in detail below, MrgprX2 recognizes and binds small molecules containing a cyclized tetrahydroisoquinoline (THIQ), such as non-steroidal neuromuscular blocking drugs (NMBDs), including tubocurarine and atracurium. As described in detail below, in response to these compounds, MrgprX2 mediates pseudo-allergic reactions characterized by histamine release, inflammation and airway contraction. As described herein, MrgprX2 also acts as a receptor for a number of other ligands, including peptides and alkaloids, such as cortistatin-14, proadrenomedullin N-terminal peptides PAMP-12 and, at lower extent, PAMP-20, antibacterial protein LL-37, PMX-53 peptide, beta-defensins, and complanadine A.

[0114] An exemplary human MrgprX2 amino acid sequence is provided below (NP_001290544.1 (GI:746816153), incorporated herein by reference (SEQ ID NO: 1)):

TABLE-US-00001 1 mdpttpawgtesttvngndqallllcgketlipvflilfi alvglvgngfvlwllgfrmr 61 rnafsvyvlslagadflflcfqiinclvylsnffcsisin fpsffttvmtcaylaglsml 121 stvsterclsvlwpiwyrcrrprhlsavvcvllwalslll silegkfcgflfsdgdsgwc 181 qtfdfitaawliflfmvlcgsslallvrilcgsrglpltr lyltilltvlvfllcglpfg 241 iqwflilwiwkdsdvlfchihpvsvvlsslnssanpiiyf fvgsfrkqwrlqqpilklal 301 qralqdiaevdhsegcfrqgtpemsrsslv

[0115] An exemplary human MrgprX2 nucleic acid sequence is provided below (NM_001303615.1 (GI:746816152), incorporated herein by reference (SEQ ID NO: 2)):

TABLE-US-00002 1 tggctacaggaaagggccaccaggcagggctatgtcctta ggtagaaaaacactgccact 61 gccaactcacagcccttcagggcgcagggagagagccagg aaatttttaaaaaatcatcc 121 cccaatctactgtcaatgtgtccctttggctgaaaaaaaa agtcaccctccaatctcctg 181 tcaatgtgtaccctttggagcctgagtgaaagacagccca ttgacgaggcacagacatgt 241 ctcctcccaggatgcaaagtgtcacttctttggactagtc tctcactatcatcataaatg 301 ccttgagaatggaatgtggttgggaaaaaaagggattggg agtacataggtactcccagc 361 tataagtacacaggggcaccagtggaggttttctgagcat ggatccaaccaccccggcct 421 ggggaacagaaagtacaacagtgaatggaaatgaccaagc ccttcttctgctttgtggca 481 aggagaccctgatcccggtcttcctgatccttttcattgc cctggtcgggctggtaggaa 541 acgggtttgtgctctggctcctgggcttccgcatgcgcag gaacgccttctctgtctacg 601 tcctcagcctggccggggccgacttcctcttcctctgctt ccagattataaattgcctgg 661 tgtacctcagtaacttcttctgttccatctccatcaattt ccctagcttcttcaccactg 721 tgatgacctgtgcctaccttgcaggcctgagcatgctgag caccgtcagcaccgagcgct 781 gcctgtccgtcctgtggcccatctggtatcgctgccgccg ccccagacacctgtcagcgg 841 tcgtgtgtgtcctgctctgggccctgtccctactgctgag catcttggaagggaagttct 901 gtggcttcttatttagtgatggtgactctggttggtgtca gacatttgatttcatcactg 961 cagcgtggctgatttttttattcatggttctctgtgggtc cagtctggccctgctggtca 1021 ggatcctctgtggctccaggggtctgccactgaccaggct gtacctgaccatcctgctca 1081 cagtgctggtgttcctcctctgcggcctgccctttggcat tcagtggttcctaatattat 1141 ggatctggaaggattctgatgtcttattttgtcatattca tccagtttcagttgtcctgt 1201 catctcttaacagcagtgccaaccccatcatttacttctt cgtgggctcttttaggaagc 1261 agtggcggctgcagcagccgatcctcaagctggctctcca gagggctctgcaggacattg 1321 ctgaggtggatcacagtgaaggatgcttccgtcagggcac cccggagatgtcgagaagca 1381 gtctggtgtagagatggacagcctctacttccatcagata tatgtggctttgagaggcaa 1441 ctttgcccctgtctgtctgatttgctgaactttctcagtc ctgattttaaaacagttaag 1501 agagtccttgtgaggattaagtgagacagtgcctatgaaa caaacactaagtgcagtgtc 1561 tctggaactgccttactcacaggcttccaccacagcccta tgagagctttgccaactctg 1621 cggtccatgactgttcccacttttaatgaatcctaccttt cgcagaaggctgaaagcagg 1681 gcagaaaagatctacatttctttggacactgcacttgata gggactcaaagaatgttata 1741 tttttaattaatttctttttctcttccgtacaatttctgt ctcaacaaaattagaagaat 1801 taaatttaaaactagctccaaaagagcagtcgtctttcat tttggcagaccttagaatat 1861 ccccctagcttaataaatctttgttgaatggcttaatgaa tgaataaactggttaatgtt 1921 taagttaaacctctgaaaagtctccatttaccagatttga gtcactaaatttattgcttt 1981 cactacttttgaattttgcaaacatgaaattaagttttat aattagataaatcaatgtca 2041 acacatatttaaagtttgaggtacactgtcttcctgtggt ttcctttcacatgccatccc 2101 ttaaaatcccagctacacgccttcccattccttccccttt gcctttgttctaatcttccc 2161 tctctgggggctctctaattcgtcctggaagtttccagtg gtcttatagactccatgttc 2221 ttggaggacaggctgtatgtcagatttaccttttattccg aagaactcggagcatttatt 2281 ttgttaattaaattgcacatatttttaaaagttacgtgtt ccacagaataaaatactaat 2341 tgtaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaa 2401 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

[0116] An exemplary mouse MrgprB2 amino acid sequence is provided below (NP_780740.2 (GI:229094244), incorporated herein by reference (SEQ ID NO: 3)):

TABLE-US-00003 1 msgdfliknlstsawktnitvingsyyidtsvcvtrnqam illsiiislvgmglnaivlw 61 flgirmhtnaftvyilnlamadflylcsqfviclliafyi fysidiniplvlyvvpifay 121 lsglsilstisierclsviwpiwyrckrprhtsaitcfvl wvmslllgllegkacgllfn 181 sfdsywcetfdvitniwsvvffgvlcgssltllvrifcgs qripmtrlyvtitltvlvfl 241 ifglpfgiywilyqwisnfyyveicnfyleilflscvnsc mnpiiyflvgsirhrrfrrk 301 tlk111qramqdtpeeeqsgnksssehpeeletvqscs

[0117] An exemplary mouse MrgprB2 nucleic acid sequence is provided below (NM_175531.4 (GI:229094243), incorporated herein by reference (SEQ ID NO: 4)):

TABLE-US-00004 1 agaggactcttctctttgtcacagaccagtttaacacttc ccataagaagaatagagcaa 61 aggaacatgagtggagatttcctaatcaagaatctaagca cctcagcctggaaaacgaac 121 atcacagtgctgaatggaagctactacatcgatacttcag tttgtgtcaccaggaaccaa 181 gccatgattttgctttccatcatcatttccctggttggga tgggactaaatgccatagtg 241 ctgtggttcctgggcatccgtatgcacacgaatgccttca ctgtctacattctcaacctg 301 gctatggctgactttctttacctgtgctctcagtttgtaa tttgtcttcttattgccttt 361 tatatcttctactcaattgacatcaacatccctttggttc tttatgttgtgccaatattt 421 gcttatctttcaggtctgagcattctcagcaccattagca ttgagcgctgcttgtctgta 481 atatggcccatttggtatcgctgtaaacgtccaagacaca catcagctatcacatgtttt 541 gtgctttgggttatgtccttattgttgggtctcctggaag ggaaggcatgtggcttactg 601 tttaatagctttgactcttattggtgtgaaacatttgatg ttatcactaatatatggtca 661 gttgttttttttggtgttctctgtgggtctagcctcaccc tgcttgtcaggatcttctgt 721 ggctcacagcgaattcctatgaccaggctgtatgtgacta ttacactcacagtcttggtc 781 ttcctgatctttggtcttccctttgggatctattggatac tctatcagtggattagcaat 841 ttttattatgttgaaatttgtaatttttatcttgagatac tattcctatcctgtgttaac 901 agctgtatgaaccccatcatttatttccttgttggctcca ttaggcaccgaaggttcagg 961 cggaagactctcaagctacttctgcagagagccatgcaag acacccctgaggaggaacaa 1021 agtggaaataagagttcttcagaacaccctgaagaactgg aaactgttcagagctgcagc 1081 tgacaactgcttgatcagacaaaaatggttttgatggaaa tactttttcttatccgtgtg 1141 gaccatttttacaacctttattcagtttgttatctcatct tcaattgtttaattaggaca 1201 ataatttttgtaaaagttgagagaaatgggtcttgtcata ctaatactgaatgtagcatt 1261 tctgaagctgtgttacttagggatttaccatctccttttc atgggactccttgtaagtat 1321 tctgtggtagagaacttctcctattgttgacaaactctcc tttagaaggcaaatggaaat 1381 acaaggaagggctgtatttctttacccactgaaatgtata atgagtacacaaatgttaca 1441 tctagcaaatattcttttagaacacccttctcaatgttta agacttaaatagaaacactt 1501 tataatcctagtccttattaatttcttcaggttataaaga atatatgaagtgatagtttt 1561 tttacgtaaattttttactaaacaaataaaatttctcaaa agaagactgttaaatctctc 1621 ttaacccagctgagtcctcactgtgaacatcaagttcact gtgtctctaatttttaaaat 1681 ttgaagagtgcacttagatttggcaatgagatccatcaaa atccatgtccacatgaaggt 1741 gaagagagtcagacttctgtgtttctcttcacaatgcctt ctttagcattccatggtcga 1801 gtgttttccctttactccctgcctttgctgtgatttctgc tctctctgactgtctaattc 1861 ttcatgagaagtttccactaggtcctctagacaatcctgt ctcaaatttaaatcaccctc 1921 agataattta ttatgtgaat ttgttacttgcattgataac aatcattgta attgaatatg 1981 aatatttttt gtaacacttt ctataaaataatatttgttt ttaagctgta ctatgtgata 2041 ttttcagttg aagcataatt aaaagagttcaaccaaaaaa aaaaaaaaa

G15

[0118] GTP-binding protein alpha 15 (G15) is a modulator or transducer in various transmembrane signaling systems.

[0119] An exemplary human G15 amino acid sequence is provided below (NP_002059.3 (GI:597709771), incorporated herein by reference (SEQ ID NO: 5)):

TABLE-US-00005 1 marsltwrccpwcltedekaaarvdqeinrilleqkkqdr gelkllllgpgesgkstfik 61 qmriihgagyseeerkgfrplvyqnifvsmramieamerl qipfsrpeskhhaslvmsqd 121 pykvttfekryaaamqwlwrdagiracyerrrefhlldsa vyylshleriteegyvptaq 181 dvirsrmpttgineycfsvqktnlrivdvggqkserkkwi hcfenvialiylaslseydq 241 cleennqenrmkeslalfgtilelpwfkstsvilflnktd ileekiptshlatyfpsfqg 301 pkqdaeaakrfildmytrmytgcvdgpegskkgarsrrlf shytcatdtqnirkvfkdvr 361 dsvlaryldeinll

[0120] An exemplary human G15 nucleic acid sequence is provided below (NM_002068.3 (GI:597709770), incorporated herein by reference (SEQ ID NO: 6)):

TABLE-US-00006 1 cagaaggaggaagaagggccctgctggtcacacaggaccc agtctgcggtgggggttttc 61 ccgccaccgccccgccctccctggggcccccacctcaccc tctcctggcacccttcaccg 121 tcaacctgtcgggccgggtctgagcaggtctggaggtggg cggggagccctggcctcccc 181 acctcctcccgtccccaccctgttcccagcactcaagcct tgccaccgccgagccgggct 241 tcctgggtgtttcaggcaaggaagtctaggtccctggggg gtgacccccaaggaaaaggc 301 agcctccctgcgcacccggttgcccggagccctctccagg gccggctgggctgggggttg 361 ccctggccagcaggggcccgggggcgatgccacccggtgc cgactgaggccaccgcacca 421 tggcccgctcgctgacctggcgctgctgcccctggtgcct gacggaggatgagaaggccg 481 ccgcccgggtggaccaggagatcaacaggatcctcttgga gcagaagaagcaggaccgcg 541 gggagctgaagctgctgcttttgggcccaggcgagagcgg gaagagcaccttcatcaagc 601 agatgcggatcatccacggcgccggctactcggaggagga gcgcaagggcttccggcccc 661 tggtctaccagaacatcttcgtgtccatgcgggccatgat cgaggccatggagcggctgc 721 agattccattcagcaggcccgagagcaagcaccacgctag cctggtcatgagccaggacc 781 cctataaagtgaccacgtttgagaagcgctacgctgcggc catgcagtggctgtggaggg 841 atgccggcatccgggcctgctatgagcgtcggcgggaatt ccacctgctcgattcagccg 901 tgtactacctgtcccacctggagcgcatcaccgaggaggg ctacgtccccacagctcagg 961 acgtgctccgcagccgcatgcccaccactggcatcaacga gtactgcttctccgtgcaga 1021 aaaccaacctgcggatcgtggacgtcgggggccagaagtc agagcgtaagaaatggatcc 1081 attgtttcgagaacgtgatcgccctcatctacctggcctc actgagtgaatacgaccagt 1141 gcctggaggagaacaaccaggagaaccgcatgaaggagag cctcgcattgtttgggacta 1201 tcctggaactaccctggttcaaaagcacatccgtcatcct ctttctcaacaaaaccgaca 1261 tcctggaggagaaaatccccacctcccacctggctaccta tttccccagtttccagggcc 1321 ctaagcaggatgctgaggcagccaagaggttcatcctgga catgtacacgaggatgtaca 1381 ccgggtgcgtggacggccccgagggcagcaagaagggcgc acgatcccgacgcctcttca 1441 gccactacacatgtgccacagacacacagaacatccgcaa ggtcttcaaggacgtgcggg 1501 actcggtgctcgcccgctacctggacgagatcaacctgct gtgacccaggccccacctgg 1561 ggcaggcggcaccggcgggcgggtgggaggtgggagtggc tgcagggacccctagtgtcc 1621 ctggtctatctctccagcctcggcccacacgcaagggagt cgggggacggacggcccgct 1681 gctggccgctctcttctctgcctctcaccaggacagccgc cccccagggtactcctgccc 1741 ttgcttgactcagtttccctcctttgaaagggaaggagca aaacggccatttgggatgcc 1801 agggtggatgaaaaggtgaagaaatcaggggattgaggac ttgggtgggtgggcatctct 1861 caggagccccatctccgggcgtgtcacctcctgggcaggg ttctgggaccctctgtgggt 1921 gacgcacaccctgggatggggctagtagagccttcaggcg ccttcgggcgtggactctgg 1981 cgcactctagtggacaggagaaggaacgccttccaggaac ctgtggactaggggtgcagg 2041 gacttccctttgcaaggggtaacagaccgctggaaaacac tgtcactttcagagctcggt 2101 ggctcacagcgtgtcctgccccggtttgcggacgagagaa atcgcggcccacaagcatcc 2161 ccccatcccttgcaggctgggggctgggcatgctgcatct taaccttttgtatttattcc 2221 ctcaccttctgcagggctccgtgcgggctgaaattaaaga tttcttagaggctgcgtcgc 2281 cagcgtcctgtttaaaaaaaaaaaaaaaaaa

[0121] An exemplary mouse Gal5 amino acid sequence is provided below (NP_034434.1 (GI:6754010), incorporated herein by reference (SEQ ID NO: 7)):

TABLE-US-00007 1 marsltwgccpwclteeektaaridqeinrilleqkkqer eelkllllgpgesgkstfik 61 qmriihgvgyseedrrafrlliyqnifvsmqamidamdrl qipfsrpdskqhaslvmtqd 121 pykvstfekpyavamqylwrdagiracyerrrefhlldsa vyylshlerisedsyiptaq 181 dvirsrmpttgineycfsvkktklrivdvggqrserrkwi hcfenvialiylaslseydq 241 cleendqenrmeeslalfstilelpwfkstsvilflnktd iledkihtshlatyfpsfqg 301 prrdaeaaksfildmyarvyascaepqdggrkgsrarrff ahftcatdtqsvrsvfkdvr 361 dsvlaryldeinll

[0122] An exemplary mouse G15 amino acid sequence is provided below (NM_010304.3 (GI:34328487), incorporated herein by reference (SEQ ID NO: 8)):

TABLE-US-00008 1 gctggagcttccaccaccgacctgtctggcgggcagggcc aggtctgggcaagttggagg 61 gggcgggaagcagcacccaggtccccgccctgtttccagc acccaggcctcttgaagccc 121 ttgcctgggctcccacaggccctaggcagggacacggagg gccctggggtgacctccacc 181 cccacctccactccatccggagaagaaagagtcccacagt tgggctctgcaggccctgtg 241 atgtcacctggtggtctgtgaagcgcccaccatggcccgg tccctgacttggggctgctg 301 tccctggtgcctgacagaggaggagaagactgccgccaga atcgaccaggagatcaacag 361 gattttgttggaacagaaaaaacaagagcgcgaggaattg aaactcctgctgttggggcc 421 tggtgagagcgggaagagtacgttcatcaagcagatgcgc atcattcacggtgtgggcta 481 ctcggaggaggaccgcagagccttccggctgctcatctac cagaacatcttcgtctccat 541 gcaggccatgatagatgcgatggaccggctgcagatcccc ttcagcaggcctgacagcaa 601 gcagcacgccagcctagtgatgacccaggacccctataaa gtgagcacattcgagaagcc 661 atatgcagtggccatgcagtacctgtggcgggacgcgggc atccgtgcatgctacgagcg 721 aaggcgtgaattccaccttctggactccgcggtgtattac ctgtcacacctggagcgcat 781 atcagaggacagctacatccccactgcgcaagacgtgctg cgcagtcgcatgcccaccac 841 aggcatcaatgagtactgcttctccgtgaagaaaaccaaa ctgcgcatcgtggatgttgg 901 tggccagaggtcagagcgtaggaaatggattcactgtttc gagaacgtgattgccctcat 961 ctacctggcctccctgagcgagtatgaccagtgcctagag gagaacgatcaggagaaccg 1021 catggaggagagtctcgctctgttcagcacgatcctagag ctgccctggttcaagagcac 1081 ctcggtcatcctcttcctcaacaagacggacatcctggaa gataagattcacacctccca 1141 cctggccacatacttccccagcttccagggaccccggcga gacgcagaggccgccaagag 1201 cttcatcttggacatgtatgcgcgcgtgtacgcgagctgc gcagagccccaggacggtgg 1261 caggaaaggctcccgcgcgcgccgcttcttcgcacacttc acctgtgccacggacacgca 1321 aagcgtccgcagcgtgttcaaggacgtgcgggactcggtg ctggcccggtacctggacga 1381 gatcaacctgctgtgacgcgggacagggaaccccaagcgc gacgcggtcgtggcgaggac 1441 atacctccccctggtggccgcgcgtggaactgcaggtcca ggagctgccaagtggggaag 1501 ccagcccacagggagagagtccctgcttcctactgggccc ccaagcccagctcccctgta 1561 atttattccctcgcccttcttctagttgttggagaaagga catgagccgggtctttaacc 1621 ccagcgctccggaggcagaggcaggaggatttctgtgagt tccaggaccatgttttcaaa 1681 aacaaacaaaaccggatagaactgtccgggaccttgtgac ttcccaggggccctgttcac 1741 atcttcctgtggggaccatttcatcttaccaaaggggaaa ccgaggtcggcaagatggct 1801 ggtgagagtgccttgccaccaagcctgacaactggacttc aggacctgttcagtggacag 1861 agagagggagcggagtcctaggagaagttctctatctcct caggcgtgcatggtggtgac 1921 acacctacccacacagataaataaatgtaatttaaaaaca aaaaaaaaaaaaaa

HEK293 Cells

[0123] Human embryonic kidney 293 cells, also often referred to as HEK 293, HEK-293, 293 cells, or less precisely as HEK cells, are a specific cell line originally derived from human embryonic kidney cells (from an aborted human embryo) grown in tissue culture and from still born animals. HEK 293 cells are very easy to grow and transfect very readily and have been widely used in cell biology research for many years. They are also used by the biotechnology industry to produce therapeutic proteins and viruses for gene therapy.

[0124] Described herein are HEK293 cells stably expressing G15 and either MrgprB2 or MrgprX2.

[0125] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1

Materials and Methods

[0126] The following materials and methods were used.

Animal Models

[0127] All experiments involving equal treatments in WT and mutant samples and animals were conducted by experimenters blind to conditions.

Analysis

[0128] Group data were expressed as meanstandard error of the mean. Two-tailed unpaired Student's t test was used to determine significance in statistical comparisons, and differences were considered significant at p<0.05. Statistical power analysis was used to justify the sample size. It was assumed that the data were normally distributed since the most outcome values were symmetrically distributed around the mean value within each group. The variance is similar between groups determined by the F test. Mast cells deemed to be damaged, either by visible lack of fibronectin adherence or by abnormally high resting calcium levels, were excluded from analysis. Otherwise, no samples or animals subjected to successful procedures and/or treatments were excluded from the analysis. No randomization was used for animal studies since it is not applicable for the studies.

Peptides and Drugs

[0129] Compound 48/80, vespid mastoparan, rocuronium, tubocurarine, ciprofloxacin, levofloxacin, moxifloxacin, and ofloxacin were from Sigma. Cortistatin was from Tocris Biosciences. PAMP (9-20) was custom synthesized and purified to 98% by Genscript. Leuprolide was from Genscript. Substance P, kallidin, mastoparan, cetrorelix, octreotide, sermorelin (growth hormone releasing factor 1-29), icatibant (HOE-140) were from Anaspec. Atracurium and mivacurium were from Santa Cruz Biotechnology. Recombinant human insulin was from Roche. Goat anti-mouse IgE (Ab9162) was from Abcam.

Drug Preparation and Storage

[0130] Atracurium, mivacurium, tubocurarine, and all fluoroquinolone solutions were prepared on the day of the experiment because the potencies of the first three were found to be susceptible to oxidation and/or freeze-thaw effects, while the solubility of the fluoroquinolones was best when prepared fresh. Propranolol also was prepared fresh on the day of the experiment to minimize the chances of a loss in potency. All fluoroquinolones except levofloxacin were dissolved into CIB adjusted to pH 3.5. All other drugs were prepared as 100-1000 aliquots and stored at 80 C. before thawing at 4 C. and diluting into calcium imaging buffer or saline.

Mrgpr RT-PCR Screen

[0131] Ribonucleic acid (RNA) was purified from 410.sup.4 mouse peritoneal mast cells with a Qiagen RNEasy Micro column, according to the manufacturer's suggestions. RNA was treated for 20 minutes with DNAse I (New England BioLabs) and re-purified on another RNEasy Micro column. 8 ng of RNA was used to generate first strand cDNA using a SuperScript III kit (Invitrogen) according to the manufacturer's instructions, using oligo dT primers and scaling the recommended 10 l reaction up to 60 l. The negative control reaction was the same except that SuperScript III reverse transcriptase was replaced by water. 25 l PCR reactions were run with 12.5 l RedTaq ReadyMix (Sigma), 0.5 l DMSO, 0.25 l each of 50 M gene-specific forward and reverse primers, 10 l water, and 2 l mixture from the cDNA or negative control synthesis reactions. All reactions used a 4 minute initial step at 95 C., 30 seconds annealing at specific temperatures (described below), 40 seconds extension at 72 C., and 25 seconds at 95 C. (with the last three steps repeated 39 times), and a final 4 minute step at 72 C. Low stringency PCR was set to 60 C. annealing; otherwise, annealing temperatures were: 62 C. for MrgprA1, MrgprA10, MrgprB2, and MrgprB6; 64 C. for MrgprA2, MrgprA3, MrgprA4, MrgprA6, MrgprA16, MrgprA18, and MrgprB11; 65 C. for MrgprA9, MrgprA19, MrgprB1, MrgprB3, MrgprB5, and MrgprB8; 66 C. for MrgprA12 and MrgprB10; 63 C. for MrgprB4; 61 C. for MrgprA14; and 65.5 C. for MrgprC11.

[0132] Primers were as follows. MrgprA1 (for atccagcaagaggaatgggg (SEQ ID NO: 9), rev tgtgacctaggaggaagaagaag (SEQ ID NO: 10)); MrgprA2 (for cctcctacacaagccagcaa (SEQ ID NO: 11), rev aagcacaagtgaaagatgatgct (SEQ ID NO: 12)); MrgprA3 (for gctacatccagcaagaggaatg (SEQ ID NO: 13), rev gcaaaaattcctttgggtagggt (SEQ ID NO: 14)); MrgprA4 (for cctgtgtgctgtgatctggt (SEQ ID NO: 15), rev tcacggttaatccagggcac (SEQ ID NO: 16)); MrgprA6 (for cattttcctcccccaacagt (SEQ ID NO: 17), rev atgcctgaatgagcccacaa (SEQ ID NO: 18)); MrgprA9 (for cagtgatctacatccagcaaaagg (SEQ ID NO: 19), rev gcgtggaagctatgatgcga (SEQ ID NO: 20)); MrgprA10 (for cagtggtccaccatctccaa (SEQ ID NO: 21), rev acaggcaagagagtcatggtt (SEQ ID NO: 22)); MrgprA12 (for tcagggatcgggtgaagcac (SEQ ID NO: 23), rev gagcatttgaaggtgttgttgga (SEQ ID NO: 24)); MrgprA14 (for ggttgcccctgtgtttcttc (SEQ ID NO: 25), rev tattgccagtcagtaagctgag (SEQ ID NO: 26)); MrgprA16 (for gccctctggttcccattact (SEQ ID NO: 27), rev gtttttggaccactgaggcatt (SEQ ID NO: 28)); MrgprA18 (for tgctctggttttctcctttgc (SEQ ID NO: 29), rev tgaggcatgtcaagtcagtca (SEQ ID NO: 30)); MrgprA19 (for caggacccagatcacgacac (SEQ ID NO: 31), tcctgggcttccgatttcac (SEQ ID NO: 32)); MrgprB1 (for attagccttcatcaggcacca (SEQ ID NO: 33), ccagcccaactaaggcaatg (SEQ ID NO: 34)); MrgprB2 (for gtcacagaccagtttaacacttcc (SEQ ID NO: 35), cagccatagccaggttgagaa (SEQ ID NO: 36)); MrgprB3 (for acctggctgtggctgatttt (SEQ ID NO: 37), rev gctgaacccacagagaacca (SEQ ID NO: 37)); MrgprB4 (for tctggctggtgctgatttctt (SEQ ID NO: 38), rev accacgaggctcaacaataga (SEQ ID NO: 39)); MrgprB5 (for ctgtggttccttctgtgtcca (SEQ ID NO: 40), rev tttccagttccccagaccttt (SEQ ID NO: 41)); MrgprB6 (for tctgtctacatcctcaacctgg (SEQ ID NO: 42)), rev attatctcatgaggaaggctcaa (SEQ ID NO: 43)); MrgprB8 (for agagaatgcaaagcatgcga (SEQ ID NO: 44), rev gaggaagtttgccccagaca (SEQ ID NO: 45)); MrgprB10 (for cactggtcacattgccaacc (SEQ ID NO: 46, rev ggggatggaatcaatgtccaaga (SEQ ID NO: 47); MrgprB11 (for accttcttgctatttttccctcca (SEQ ID NO: 48), rev aggatgagactggacccaca (SEQ ID NO: 49)); MrgprC11 (for cagcacaagtcagctcctcaa (SEQ ID NO: 50), rev atgcccatgagaaaggacagaacc (SEQ ID NO: 51)).

Expression Constructs

[0133] Mrgpr genes were cloned and inserted into the pcDNA3.1 mammalian expression plasmid using standard techniques. All mouse genes had a Kozak sequence at their N-terminus and also encoded a C-terminal FLAG tag separated from the genes by the amino acid linker DIIL.

cDNA Constructs

[0134] First strand cDNA was prepared as described for RT-PCR screens, and amplification was performed using the Q5 HotStart High Fidelity Master Mix (New England Biolabs). At least five different clones each prepared from wild type and mutant mice were sequenced to verify the presence of the deletion in the mutant and the absence of any other mutation from wild type or mutant.

Calcium Imaging in HEK293 Cells

[0135] In initial screens, HEK293 cells (not tested for mycoplasma but rapidly dividing) were transiently transfected with gene constructs including a C-terminal FLAG tag, and plated on 100 g/ml poly-D-lysine coated glass cover slips six hours after transfection. 24 hours later, cells were loaded with AM esters of the calcium indicators Fura-2 or Fluo-4 (Molecular Probes) along with 0.02% Pluronic F-127 (Molecular Probes) for 45 minutes at 37 C. Fura-2 loaded cells were imaged during 340 and 380 nm excitation, and Fluo-4 loaded cells were imaged during 488 nm excitation. Later experiments utilized cell lines stably expressing receptors along with transient or stable expression of the promiscuous G protein G15. Cells were imaged in calcium imaging buffer (CIB; NaCl 125 mM, KCl 3 mM, CaCl.sub.2 2.5 mM, MgCl.sub.2 0.6 mM, HEPES 10 mM, glucose 20 mM, NaHCO.sub.3 1.2 mM, sucrose 20 mM, brought to pH 7.4 with NaOH). Unless otherwise specified, drugs were perfused into the chamber for 45 to 60 seconds and responses were monitored at 5-second intervals for an additional 60-90 seconds.

EC.SUB.50 .Determination

[0136] HEK293 cells stably expressing Galpha15 and either MrgprB2 or MrgprX2 were plated at 410.sup.4 cells per well in 96-well plates and incubated overnight. The next day, media was removed and replaced with imaging solution from the FLIPR Calcium 5 assay kit (Molecular Devices), diluted according to manufacturer's suggestions in Hank's Balanced Salt Solution (HBSS) with 20 mM HEPES, pH 7.4. Cells were incubated at 37 C. for 60 minutes, and allowed to recover for 15 minutes at room temperature before imaging in a Flexstation 3 (Molecular Devices). Wells were imaged according to manufacturer's specifications for 120 seconds, with 50 l of test substances at 3 concentration added 30 seconds after imaging began. Responses were determined by subtracting the minimum signal from the maximum signal. Substances were tested in duplicate wells, the signals were averaged, and EC.sub.50s were determined for each trial by normalizing to the peak response to the substance in that trial. All drugs were dissolved in HBSS+HEPES solution, with the following exceptions due to solubility issues: cetrorelix acetate was dissolved in saline containing 2.5 mM CaCl.sub.2 and 0.6 mM MgCl.sub.2, and fluoroquinolones except ofloxacin were dissolved in the same solution except that the pH was adjusted with HCl to 3.5; ofloxacin required 100 g/ml of lactic acid for full solubility. Peptides sometimes lost potency after a freeze-thaw cycle, so most peptides were prepared directly from lyophilized stock.

Peritoneal Mast Cell Purification and Imaging

[0137] Adult male and female mice 2-5 months of age were sacrificed through CO.sub.2 inhalation. A total of 12 mls of ice cold mast cell dissociation media (MCDM; HBSS with 3% fetal bovine serum and 10 mM HEPES, pH 7.2) were used to make two sequential peritoneal lavages, which were combined and cells were spun down at 200 g. The pellet from each mouse was resuspended in 2 mls MCDM, layered over 4 mls of an isotonic 70% Percoll suspension (2.8 mls Percoll, 320 ls 10 HBSS, 40 l 1 M HEPES, 830 l MCDM), and spun down for 20 minutes, 500 g, 4 C. Mast cells were recovered in the pellet. Purity was 95%, as assayed by avidin staining and by morphology. Mast cells were resuspended at 510.sup.5-110.sup.6 cells/ml in DMEM with 10% fetal bovine serum and 25 ng/ml recombinant mouse stem cell factor (Sigma), and plated onto glass cover slips coated with 30 g/ml fibronectin (Sigma). For counting, instead of plating, suspended mast cells were diluted 1/10 and affixed to slides by spinning at 1000 rpm for 5 minutes at 4 C. on a CytoSpin (Thermo Scientific).

[0138] For imaging, after two hours of incubation at 37 C., 5% CO.sub.2, mast cells were loaded with Fluo-4 along with 0.02% Pluronic F-127 for 30 minutes at room temperature, washed 3 times in CIB and used immediately for imaging. Cells were used within two hours of loading. Cells were identified as responding if the [Ca.sup.2+].sub.i rose by at least 50% for at least 10 seconds, which clearly distinguishes a ligand-induced response from random flickering events. Average traces were calculated by taking the average response from each cell in a mouse, and averaging those.

BAC Transgenic Mice Generation

[0139] The BAC clone RP23-65I23 was purchased from Children's Hospital Oakland Research Institute. This clone contains the MrgprB2 locus, 60 kb of 5 genomic sequence and over 100 kb of 3 genomic sequence. Recombineering in bacteria was used to introduce eGFP-Cre and a polyA signal immediately after the MrgprB2 start codon (Metcalfe, D. D., Baram, D. & Mekori, Y. A. Mast cells. Physiological reviews 77, 1033-1079 (1997)). The BAC was linearized with NotI (New England Biolabs) and injected into pronuclei from single cell fertilized C57B1/6 eggs. Eggs were implanted into pseudopregnant females. Three BAC mouse lines were established. Though mice were already in a C57B1/6 background, they were crossed for at least four generations to WT and tdTomato reporter mice in the C57B1/6 background before use in experiments. BAC mice were mated to ROSA26.sup.Tdtomato mice purchased from Jackson Labs for imaging studies. Experiments for FIG. 1 used mice homozygous for ROSA26.sup.Tdtomato because the tdTomato signal often was heterogeneous and weak in heterozygous mice. Genotyping reactions for BAC mice were run at 61 C. annealing, and primers were: forward, tatatcatggccgacaagca; reverse, cagaccgcgcgcctgaaga. Both primers are in the eGFP-Cre reading frame but the entire gene and correct placement in the MrgprB2 locus was verified by previous sequencing.

MrgprB2 Mutant Mice Generation

[0140] mRNAs encoding zinc finger nucleases targeting MrgprB2 were purchased from Sigma. The binding sites were GTTCCTGGGCATCCG (SEQ ID NO: 52) and TGCACACGAATGCCTTCACTG (SEQ ID NO: 53), corresponding to bases 180-194 and 196-216, respectively, of the MrgprB2 open reading frame. mRNA was diluted to 2 ng/ml in 1 mm Tris-HCl buffer, pH 7.4, with 0.25 mm EDTA, and injected into the pronuclei of single cell fertilized eggs in the C57B1/6 strain. No overt signs of toxicity were observed. Embryos were implanted into pseudopregnant females. DNA flanking the binding sites was amplified from founder mice and screened for mutations using the Cel-1 assay kit (Transgenomics), according to the manufacturer's suggestions. 3 of the first 28 mice were identified and confirmed by DNA sequencing to carry small mutations, and no more screening was performed. In addition to the 4 bp mutation used in this study, a mouse carrying a 1 bp deletion and another with a 2 bp deletion were identified.

Wild Type and MrgprB2MUT Mouse Genotyping

[0141] Primers used for wild type mice were GGTTCCTGGGCATCCGTAT (SEQ ID NO: 54) and GGTTCCTGGGCATCCGTAT (SEQ ID NO: 55), and reactions were run at an annealing temperature of 62.8 C.

[0142] Primers for MrgprB2.sup.MUT mice were GTTCCTGGGCATCCGCAC (SEQ ID NO: 56) and CTTCCGCCTGAACCTTCGGT (SEQ ID NO: 57), and reactions were run at 64.0 C. annealing temperature.

Avidin Labeling of Tissue

[0143] Adult male and female mice up to 8 months of age were anesthetized with pentobarbital and perfused with 20 ml 0.1 M PBS (pH 7.4, 4 C.) followed with 25 ml of fixative (4% formaldehyde (vol/vol), 4 C.). Heart, trachea, and skin sections were dissected from the perfused mice. Tissues were post-fixed in fixative at 4 C. overnight. When skin sections were the only tissues needed, they were dissected and placed in fixative directly after asphyxiation of mice by CO.sub.2 inhalation, eliminating the perfusion step. Tissues were cryoprotected in 20% sucrose (wt/vol) for more than 24 h and were sectioned (20 m width) with a cryostat. The sections on slides were dried at 37 C. for 30 min, and fixed with 4% paraformaldehyde at 21-23 C. for 10 min. The slides were pre-incubated in blocking solution (10% normal goat serum (vol/vol), 0.2% Triton X-100 (vol/vol) in PBS, pH 7.4) for 1 or 2 h at 21-23 C., then incubated with 1/500 FITC-avidin (Sigma) or rhodamine-avidin (Vector Labs) for 45 minutes. Sections were washed three times with water or PBS and a drop of Fluoromount G (SouthernBiotech) was added before cover slips were placed on top. Heart mast cells were examined near cavities because the density was much higher than elsewhere in the tissue; avidin-positive, tdTomato-negative cells were observed embedded in muscle tissue in very low numbers, but their identity was unclear.

[0144] For avidin labeling of peritoneal mast cells, cells were plated as described in the mast cell purification section, fixed with 4% paraformaldehyde at 21-23 C. for 10 min, incubated with 1/1000 avidin in PBS for 30 minutes at 21-23 C., and washed with PBS before immediate imaging.

Stomach Section Immunocytochemistry

[0145] Adult male and female mice up to 8 months of age were anesthetized with pentobarbital and perfused with 20 ml 0.1 M PBS (pH 7.4, 4 C.) followed with 25 ml of fixative (4% formaldehyde (vol/vol), 4 C.). Stomach sections were removed, washed thoroughly, postfixed in 4% formaldehyde for two hours, and prepared for sectioning by incubation in a 30% sucrose solution for 48 hours. Tissue samples were mounted in cryoembedding media and frozen, and 14 m sections were made using a crytostat and then fixed onto slides. Slides were washed with a 0.2% Triton X-100 PBS solution, incubated for one hour in a 10% normal goat serum solution, and then incubated overnight at 4 C. with a 1:20 dilution of rat monoclonal anti-mouse MCPT1 (monoclonal antibody RF6.1, eBiosciences) in a 0.2% Triton/1% normal goat serum solution. Slides were washed with the 0.2% Triton solution and incubated for two hours at room temperature in Triton solution with a 1:500 dilution of a goat anti-rat IgG Alexa Fluor 488 conjugated antibody (Life Technologies). Slides were washed in PBS before cover slips were added with an anti-fade solution for imaging.

Peripheral White Blood Cell Preparation

[0146] Blood was collected from MrgprB2-tdTomato mice via cardiac punctures with a syringe containing PBS with 30 units/ml heparin and 5 mM EDTA, diluted 1:1 with the same solution, and allowed to cool to room temperature before layering over 6 mls of a Histopaque-1119 solution in a 15 ml conical tube. Tubes were centrifuged at 700 g for 30 minutes, and white blood cells were collected at the interface between the PBS and Histopaque solutions. Cells were washed with PBS and spun down at 500 g for 10 minutes a total of three times. Cells were spun onto poly-lysine coated slides in a Cytospin 4 (Thermo Scientific) at 600 rpm for 3-5 minutes, dried overnight on a 37 C. heating block, and incubated for 2 minutes with Hoechst 33342 diluted to 0.5 g/ml in PBS before coverslip mounting with an anti-fade solution. In parallel, cells were also stained in suspension with Hoechst 33342, spun the cells down, and mixed the resuspended cells directly in a PBS/anti-fade solution before placing directly onto slides and mounting coverslips on the suspension. No tdTomato-positive cells were seen in any preparation using either method.

Tissue Histamine Release Studies

[0147] Whole tracheae or segments of skin isolated from the abdominal aspect of shaved male and female mice up to 6 months of age (4-8 mg wet weight) were dissected and cleaned of connective tissue. After a 60 minutes in incubation period in oxygenated Kreb's bicarbonate buffer solution (37 C.), the tissue was treated with either vehicle or Compound 48/80 for 30 min. The supernatant solution was saved for histamine analysis. The tissue was then subjected to 8% percholoric acid in a 37 C.-waterbath for 15 minutes to obtain total histamine content. Histamine was assayed by the automated fluorometric technique previously described.sup.2.

Tracheal Contractions

[0148] Tracheal contractions were carried out as previously described (Lagunoff, D., Martin, T. W. & Read, G. Agents that release histamine from mast cells. Annual review of pharmacology and toxicology 23, 331-351). For allergen (ovalbumin, OVA) responses, mice were actively sensitized by injecting 0.2 mL of an OVA solution (3.75 g/mL) mixed with Al(OH).sub.3 three times at an interval of 2 days. Experiments were conducted on male and female animals 8-12 weeks of age beginning two weeks following the first injection. Trachea were cleaned of connective tissue and tracheal rings (whole or laterally divided in half), were suspended between two tungsten stirrups in 10 mL organ chambers filled with Krebs' that was warmed to 37 C. and bubbled with 95% O.sub.2-5% CO.sub.2to maintain a pH of 7.4. One stirrup was connected to a strain gauge (model FT03; Grass Instruments, Quincy, Mass.), and tension was recorded on a Grass Model 7 polygraph (Grass Instruments, Quincy, Mass.). Preparations were stretched to a resting tension of 0.2 g, and washed with fresh Krebs' buffer at 15-minute intervals during a 60 minute equilibration period. After equilibration, trachea were challenged with either OVA (10 g/mL), or Compound 48/80. At the end of each experiment, all trachea were maximally contracted with carbachol (1 M). All results are expressed as a percentage of maximum contraction.

Hindpaw Swelling and Extravasation

[0149] Adult male mice up to 8 months of age were anesthetized with an i.p. injection of 50 mg/kg pentobarbital (Sigma). 15 minutes after induction of anesthesia, mice were injected i.v. with 50 l of 12.5 mg/ml Evans Blue (Sigma) in saline. 5 minutes later, 5 l of the test substance (or 7 l of anti-IgE) was administered by intraplantar injection in one paw and saline was administered in the other paw. Paw thickness was measured by calipers immediately after injection. 15 minutes later (30 minutes after anti-IgE), paw thickness was measured again and mice were sacrificed by decapitation. Paw tissue was collected, dried for 24 hours at 50 C., and weighed. Evans Blue was extracted by a 24 hour incubation in formamide at 50 C., and the O.D. was read at 620 nm using a spectrophotometer. For studies using ketotifen, mice were injected i.p. with 25 l of a 10 mg/ml solution of ketotifen at the same time as pentobarbital.

Systemic Anaphylaxis Assay

[0150] To minimize stress, animals were transported to the procedure area the day before injections. Adult male and female mice up to 8 months of age (25 to 35 grams) were given an intraperitoneal injection of 80 g propranolol in saline (2 mg/ml) immediately after removal from their cages, and then placed back in their cages for 30 minutes before intravenous injections. The intravenous injections were performed on one mouse at a time. For each injection, a mouse was placed in a transport box and brought to a room with no other mice, to minimize stress from vocalizations during injection. The mouse was then placed in a restrainer, and the injection was performed within 4 minutes of restraint because it was observed that longer restraint times affected body core temperature independent from the injection. Tail veins were dilated by repeated wiping of tail with a tissue soaked in 100% ethanol, followed by injection of ciprofloxacin in a 0.25 ml Hamilton syringe fit with a 30.5 gauge needle (BD Biosciences). The injection was determined to be successful only when all of the criteria were met: blood appeared in the syringe after needle insertion, all tail veins were visible after injection, and the mouse bled slightly from the injection site after needle withdrawal. The injection site was swabbed until blood stopped flowing, the mouse was placed in a separate cage from its housing cage, one mouse per cage, and returned to the room it was brought from. At least one wild type and one mutant mouse were used for each experimental session. Body core temperature was measured with a rectal thermometer.

Mouse Peritoneal Mast Cell Histamine Release Assay

[0151] Mast cells were purified as with the calcium imaging assay and allowed to recover for 2 hours in DMEM with 10% FBS and 25 ng/ml mouse stem cell factor in a 37 C. incubator with 5% CO.sub.2. Cells were then spun down, resuspended in CIB, counted, and plated at 300 cells/well in 75 l CIB in 96-well plates coated with 20 g/ml fibronectin (Sigma). They were allowed to adhere to the substrate for 45 minutes at 37 C. in atmospheric conditions (i.e. CO.sub.2 levels were not adjusted) before assay. For the assays, cells were removed to room temperature and 75 l of 2 concentrations of tested substances (all in CIB except for ciprofloxacin, which was in saline with 2.5 mM CaCl.sub.2 and 0.6 mM MgCl.sub.2, pH 3.5) were added. After 5 minutes, 40 l of supernatant was aspirated, diluted with 40 l CIB and frozen at 80 C. until histamine levels were determined. Anti-IgE treatment was similar, except that cells were incubated for 30 minutes at 37 C. after anti-IgE was added before aspiration of supernatant. Histamine content was determined by using an HTRF histamine assay kit (Cisbio Assays) according to the manufacturer's instructions.

Human Mast Cell Culture

[0152] LAD2 (Laboratory of Allergic Diseases 2) human mast cells were cultured in StemPro-34 SFM medium (Life Technologies) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 50 g/ml streptomycin, and 100 ng/ml recombinant human stem cell factor (Peprotech). The cell suspensions were seeded at a density of 0.110.sup.6 cells/ml and maintained at 37 C. and 5% CO.sub.2, and periodically tested for the expression of CD117 and FcRI by flow cytometry. Cell culture medium was hemi-depleted every week with fresh medium.

LAD2 Degranulation Assay

[0153] LAD2 cells were sensitized for 20 hours with 0.5 g/ml biotin-conjugated human IgE (Abbiotec). Cells were washed, resuspended in Hepes buffer (10 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.38 mM Na.sub.2HPO.sub.4.7H.sub.2O, 5.6 mM glucose, 1.8 mM CaCl.sub.2.H.sub.2O, 1.3 mM MgSO.sub.4.7H.sub.2O, 0.4% BSA, pH 7.4) at 0.02510.sup.6 per well, and then stimulated with 0.1 g/ml streptavidin (Life Technologies) or other agonists at the indicated concentrations for 30 minutes at 37 C./5% CO.sub.2. The -hexosaminidase released into the supernatants and in cell lysates was quantified by hydrolysis of p-nitrophenyl N-acetyl--D-glucosamide (Sigma-Aldrich) in 0.1 M sodium citrate buffer (pH 4.5) for 90 minutes at 37 C. The percentage of -hexosaminidase release was calculated as a percent of total content. Agonists tested were Compound 48/80, mastoparan, icatibant, atracurium bessylate, and ciprofloxacin hydrochloride.

EIA and ELISA

[0154] LAD2 cells were washed with medium, suspended at 0.2510.sup.6 cells per well, and incubated with Compound 48/80, mastoparan, icatibant, atracurium or ciprofloxacin at the indicated concentrations for 3-24 hours at 37 C./5% CO.sub.2. Cell-free supernatants were harvested and analyzed for PGD.sub.2 release by an EIA (Cayman chemical), while TNF content was quantified using an ELISA kit (eBioscience) according to the manufacturer's instruction. The minimum detection limits were 55 pg/ml for PGD2 and 5.5 pg/ml for TNF.

Measurement of Histamine Release from LAD2 Cells

[0155] LAD2 cells were washed, suspended in BSA-free Hepes buffer at 0.110.sup.6 per well, and incubated with Compound 48/80, mastoparan, icatibant, atracurium or ciprofloxacin at the indicated concentrations for 30 minutes at 37 C./5% CO.sub.2. A histamine (Sigma-Aldrich) stock solution of 100 g/ml was prepared and stored at 20 C. The working standards of 4000 ng/ml to 7.8 ng/ml were freshly prepared using two-fold serial dilution. O-phthalaldehyde (OPT; Sigma-Aldrich) was dissolved in acetone-free methanol (10 mg/ml) and kept in dark at 4 C. Histamine standards and cell-free supernatants (60 L) were transferred to a flat bottom 96 black well microplate and mixed with 12 l 1M NaOH and 3 l OPT. After 4 minutes at room temperature, 6 l 3M HCl was added to stop the histamine-OPT reaction. Fluorescence intensity was measured using a 355 nm excitation filter and a 460 emission filter.

siRNA Transfection of LAD2 Cells

[0156] Expression of MrgprX2 was down-regulated with ON-TARGET plus SMARTpool siRNA against MrgprX2 and control siRNA from Dharmacon. LAD2 cells were washed with medium, suspended at 0.510.sup.6 cells per well, and transfected with 100 nm MrgprX2 siRNA and control siRNA in antibiotic-free StemPro medium using Lipofectamine 3000 (Life Technologies) according to the manufacturer's instruction at 37 C./5% CO.sub.2. At 48 hours, knockdown was confirmed by reverse-transcriptase PCR, and the cells were used for degranulation assays.

Example 2

MrgprB2 is the Orthologue of Human MrgprX2

[0157] Responsiveness to basic secretagogues is conserved among mammals (Halpern, B. N. & Wood, D. R. The action of promethazine (phenergan) in protecting mice against death due to histamine. British journal of pharmacology and chemotherapy 5, 510-516 (1950)), and also is found in birds (Taneike, T., Miyazaki, H., Oikawa, S. & Ohga, A. Compound 48/80 elicits cholinergic contraction through histamine release in the chick oesophagus. General pharmacology 19, 689-695 (1988)), indicating an ancient, fundamental role for its mechanism. Many basic secretagogues are endogenous peptides, often linked to inflammation; however, they activate connective tissue mast cells only at high concentrations and independent of their canonical receptors, so another mechanism of stimulation must exist (Ferry, X., Brehin, S , Kamel, R. & Landry, Y. G protein-dependent activation of mast cell by peptides and basic secretagogues. Peptides 23, 1507-1515 (2002)). Several candidates which bind polycationic compounds have been proposed as basic secretagogue receptors (Ferry, X., Brehin, S., Kamel, R. & Landry, Y. G protein-dependent activation of mast cell by peptides and basic secretagogues. Peptides 23, 1507-1515 (2002); Purcell, W. M., Doyle, K. M., Westgate, C. & Atterwill, C. K. Characterisation of a functional polyamine site on rat mast cells: association with a NMDA receptor macrocomplex. Journal of neuroimmunology 65, 49-53 (1996); Tatemoto, K. et al. Immunoglobulin E-independent activation of mast cell is mediated by Mrg receptors. Biochemical and biophysical research communications 349, 1322-1328, (2006); Sick, E., Niederhoffer, N., Takeda, K., Landry, Y. & Gies, J. P. Activation of CD47 receptors causes histamine secretion from mast cells. Cellular and molecular life sciences: CMLS 66, 1271-1282, (2009). Among these, MrgprX2 has been screened with the most compounds (Tatemoto, K. et al. Immunoglobulin E-independent activation of mast cell is mediated by Mrg receptors. Biochemical and biophysical research communications 349, 1322-1328, (2006); Robas, N., Mead, E. & Fidock, M. MrgX2 is a high potency cortistatin receptor expressed in dorsal root ganglion. The Journal of biological chemistry 278, 44400-44404, (2003); Subramanian, H., Gupta, K., Guo, Q., Price, R. & Ali, H. Mas-related gene X2 (MrgX2) is a novel G protein-coupled receptor for the antimicrobial peptide LL-37 in human mast cells: resistance to receptor phosphorylation, desensitization, and internalization. The Journal of biological chemistry 286, 44739-44749, (2011); Kashem, S. W. et al. G protein coupled receptor specificity for C3a and compound 48/80-induced degranulation in human mast cells: roles of Mas-related genes MrgX1 and MrgX2. European journal of pharmacology 668, 299-304, (2011); Subramanian, H. et al. beta-Defensins activate human mast cells via Mas-related gene X2. Journal of immunology 191, 345-352, (2013); Kamohara, M. et al. Identification of MrgX2 as a human G-protein-coupled receptor for proadrenomedullin N-terminal peptides. Biochemical and biophysical research communications 330, 1146-1152, (2005)), and siRNA knockdown studies support at least a partial role for MrgprX2 in activation by four non-canonical basic secretagogues (Subramanian, H., Gupta, K., Guo, Q., Price, R. & Ali, H. Mas-related gene X2 (MrgX2) is a novel G protein-coupled receptor for the antimicrobial peptide LL-37 in human mast cells: resistance to receptor phosphorylation, desensitization, and internalization. The Journal of biological chemistry 286, 44739-44749, (2011); Subramanian, H. et al. beta-Defensins activate human mast cells via Mas-related gene X2. Journal of immunology 191, 345-352, (2013)). However, no direct in vivo study or knockout model has been employed for any candidate. The investigation of MrgprX2 in mice is complicated because the gene cluster containing the four human MrgprX members is dramatically expanded in mice, consisting of 22 potential coding genes, many with comparable sequence identity to MrgprX2 (FIG. 1A). Therefore, a mouse MrgprX2 orthologue must be determined by expression pattern and pharmacology. A stringent RT-PCR screen in mouse primary mast cells uncovered a band for a single family member, MrgprB2 (FIG. 1B), while MrgprX1 orthologues were not expressed at relevant levels (FIG. 5A and FIG. 5B).

[0158] Functionally, HEK293 cells heterologously expressing MrgprB2 (MrgprB2-HEK) responded to the MrgprX2 agonist PAMP (9-20).sup.14 (FIG. 1C) and Compound 48/80 (48/80), a classical mast cell activator and canonical basic secretagogue (FIG. 6A, FIG. 6B, and FIG. 6C). MrgprB2-HEK cells also responded to other MrgprX2 ligands, including the basic secretagogue Substance P, but had no response to the MrgprX1 ligand chloroquine (CQ) (Liu, Q. et al. Sensory neuron-specific GPCR Mrgprs are itch receptors mediating chloroquine-induced pruritus. Cell 139, 1353-1365, (2009)); no closely related family members in mice responded to any compound (FIG. 5C, FIG. 6A, and FIG. 6C). To determine the expression of MrgprB2, MrgprB2 BAC transgenic mice in which the expression of eGFP-Cre recombinase was under the control of the MrgprB2 promoter were generated. Strikingly, Cre expression patterns indicate that MrgprB2 expression is highly specific to connective tissue mast cells (FIG. 1D, FIG. 7, FIG. 8A, and FIG. 8B). Together, the pharmacological and expression data indicate that MrgprB2 is the mouse orthologue of MrgprX2.

Example 3

MrgprB2 is the Mouse Mast Cell Basic Secretagogue Receptor

[0159] Next, it was determined whether MrgprB2 is the basic secretagogue receptor in mouse mast cells. The MrgprB2 genomic locus contains too much repetitive sequence to permit gene targeting through homologous recombination (FIG. 9A). Therefore, a zinc finger nuclease-based strategy was used to generate a mouse line with a 4 base pair deletion in the MrgprB2 coding region (MrgprB2.sup.MUT mice), resulting in a frameshift mutation and early termination shortly after the first transmembrane domain (FIG. 9B, FIG. 9C, and FIG. 9D). The mutation was stable and inheritable (FIG. 9C), so MrgprB2.sup.MUT was regarded as a functional null. Mast cell numbers were comparable in tissues of wild-type (WT) and MrgprB2.sup.MUT mice, indicating that MrgprB2 is not essential for mast cell survival or targeting to tissue (FIG. 10A). Responsiveness of peritoneal mast cells to anti-IgE antibodies (FIG. 2A) and endothelin (FIG. 11A, FIG. 11B, and FIG. 11C) also was comparable, demonstrating that MrgprB2 mutation does not globally impair IgE or GPCR-mediated mast cell signaling. However, 48/80-induced mast cell activation (FIG. 2A) and tissue histamine release essentially was abolished in mutant mast cells (FIG. 2B and FIG. 10B). Further, 48/80-evoked tracheal contraction (FIG. 2C) and hindpaw inflammation (extravasation and swelling; FIG. 2D) were almost completely absent in an MrgprB2.sup.MUT background, while antigen (FIG. 2C) and anti-IgE evoked responses (FIG. 12A and FIG. 12B) were comparable to WT mice. Finally, four additional basic secretagogues, as well as MrgprX2 agonists PAMP (9-20) and cortistatin (Robas, N., Mead, E. & Fidock, M. MrgX2 is a high potency cortistatin receptor expressed in dorsal root ganglion. The Journal of biological chemistry 278, 44400-44404, doi:10.1074/jbc.M302456200 (2003)), strongly activated WT but not MrgprB2.sup.MUT mast cells (FIG. 2E; FIG. 13A). HEK293 cells expressing MrgprB2 or MrgprX2 (MrgprX2-HEK) also responded to these secretagogues (FIG. 6A and FIG. 6B). Taken together, it was concluded that MrgprB2 is the mouse mast cell basic secretagogue receptor. It is likely that the list of small, basic peptides that activate MrgprB2 is greater than the number in this study; indeed, dozens of such peptides have been shown to activate mast cells (Lagunoff, D., Martin, T. W. & Read, G. Agents that release histamine from mast cells. Annual review of pharmacology and toxicology 23, 331-351, doi:10.1146/annurev.pa.23.040183.001555 (1983); Ferry, X., Brehin, S , Kamel, R. & Landry, Y. G protein-dependent activation of mast cell by peptides and basic secretagogues. Peptides 23, 1507-1515 (2002); Mousli, M., Hugli, T. E., Landry, Y. & Bronner, C. Peptidergic pathway in human skin and rat peritoneal mast cell activation. Immunopharmacology 27, 1-11 (1994); Pundir, P. & Kulka, M. The role of G protein-coupled receptors in mast cell activation by antimicrobial peptides: is there a connection? Immunology and cell biology 88, 632-640, doi:10.1038/icb.2010.27 (2010)). Notably, human MrgprX2 is much more sensitive to Substance P than mouse MrgprB2 (FIG. 6C), suggesting a potential species-specific role for Substance P in mast cell signaling.

[0160] Given that micromolar concentrations of these peptides are required for MrgprB2 activation, it is unclear where such a signaling event might occur. However, mast cells are present in organs like the pancreas and adrenal glands that secrete large amounts of small, cationic peptides, and it is conceivable that concentrations close to the sites of release reach these levels. Described herein are high-affinity endogenous ligand(s) for MrgprB2. Identification of endogenous MrgprB2 and MrgprX2 ligands contributes significantly to understanding how mast cells interact with other cell types in disease states.

Example 4

MrgprB2 Mediates Mast Cell Responsiveness and Side Effects of Peptidergic Therapeutic Drugs

[0161] The critical role of mast cells in allergic and pseudo-allergic (i.e., IgE-independent) reactions suggested a need for experiments demonstrating whether MrgprX2 is a factor in these events. Drug-induced reactions were addressed because many therapeutic drugs are cationic. Up to 15% of drug-induced adverse reactions appear to be allergic in nature; however, many are not well-correlated with IgE antibody titer, indicating that antibody-independent, or pseudo-allergic, mechanisms participate (Hausmann, O., Schnyder, B. & Pichler, W. J. Etiology and pathogenesis of adverse drug reactions. Chemical immunology and allergy 97, 32-46, doi:10.1159/000335614 (2012)).

[0162] First, peptidergic drugs were analyzed because most are introduced subcutaneously or intramuscularly at millimolar concentrations (FIGS. 15A-15B), high enough for cationic peptides to activate mast cells. The most frequent allergic-type response described in FDA labels of these drugs is an injection-site reaction (ISR), a local swelling and/or flare of variable size which can be accompanied by pain or pruritus. In a survey of FDA-approved peptidergic drugs, the vast majority associated with ISRs are cationic (FIGS. 15A-15B). Representative members of all common, commercially available classes of these cationic drugs activated mast cells in an MrgprB2-dependent manner, while the innocuous protein insulin had no effect (FIG. 3A, FIG. 13B, and FIG. 13C). Consistently, all of these peptides except insulin activate both MrgprB2-HEK and MrgprX2-HEK cells (FIG. 6A, FIG. 6B, and FIG. 6C). The drug icatibant was chosen for further study because it induces ISRs nearly in every patient (Lumry, W. R. et al. Randomized placebo-controlled trial of the bradykinin B(2) receptor antagonist icatibant for the treatment of acute attacks of hereditary angioedema: the FAST-3 trial. Annals of allergy, asthma & immunology: official publication of the American College of Allergy, Asthma, & Immunology 107, 529-537, (2011)). Icatibant at the clinical concentration induced extensive extravasation and swelling, similar to human ISRs, in WT mice but not in MrgprB2.sup.MUT mice (FIG. 3B). Mice pretreated with the mast cell stabilizer ketotifen also showed no inflammation (without ketotifen: 40.72.1% increase in paw thickness; with ketotifen: 3.10.6% increase; n=4 each; p=2.2e-6), strongly indicating that mast cells mediated the inflammation. Furthermore, icatibant (as well as positive controls 48/80 and mastoparan) induced histamine release from WT peritoneal mast cells, while MrgprB2.sup.MUT mast cells released substantially less (FIG. 3C). However, IgE-mediated histamine release was unaffected by MrgprB2 deletion (FIG. 3C). These data anticipate that drug-induced ISRs may be alleviated by targeting MrgprX2 or by using peptides with less potent MrgprX2 agonist properties.

Example 5

MrgprB2 Mediates Mast Cell Responsiveness and Side Effects of Small Molecule Therapeutic Drugs

[0163] Next, the possibility that MrgprB2 mediates pseudo-allergic reactions induced by small molecules was explored. The focus was on intravenously applied drugs because they often are administered rapidly and in high doses, and thus are more likely to achieve high blood concentrations and rapid tissue distribution than drugs administered through other routes. Symptoms of pseudo-allergic reactions after intravenous administration, which at the most severe are called anaphylactoid, include skin flushing or rash, changes in blood pressure or heart rate, and bronchospasms (Nel, L. & Eren, E. Peri-operative anaphylaxis. British journal of clinical pharmacology 71, 647-658, doi:10.1111/j.1365-2125.2011.03913.x (2011)). The initial search was based on the structure of 48/80. While the structure-function relationship of 48/80 as an MrgprX2 agonist was unknown, a cyclized variant containing a tetrahydroisoquinoline (THIQ) motif (FIG. 4A) is seven times more potent than 48/80 as a mast cell degranulator (Read, G. W. Compound 48-80. Structure-activity relations and poly-THIQ, a new, more potent analog. Journal of medicinal chemistry 16, 1292-1295 (1973)). A search of FDA-approved drugs containing a THIQ recovered members of the nicotinic receptor antagonist non-steroidal neuromuscular blocking drugs (NMBDs), including tubocurarine and atracurium (FIG. 4B). NMBDs are used routinely in surgery to reduce unwanted muscle movement and allow intratracheal intubation for mechanical ventilation. Intriguingly, NMBDs alone are responsible for nearly 60% of allergic reactions in a surgical setting (Mertes, P. M., Alla, F., Trechot, P., Auroy, Y. & Jougla, E. Anaphylaxis during anesthesia in France: an 8-year national survey. J Allergy Clin Immunol 128, 366-373 (2011)), and all except succinylcholine induce histamine release in humans (Koppert, W. et al. Different patterns of mast cell activation by muscle relaxants in human skin. Anesthesiology 95, 659-667 (2001)). As shown in FIG. 16, members of all NMBD families except succinylcholine activated mast cells in an MrgprB2-dependent manner at concentrations as low as 0.5% of the clinical injection concentration (FIG. 4C and FIG. 13D). Interestingly, rocuronium does not contain a THIQ but has a bulky hydrophobic group with a charged nitrogen within several angstroms (FIG. 4B), reminiscent of 48/80. Therefore, a search was performed using modifications of the THIQ motif and the 48/80 structure, including changes in cyclization and position of the positive or polar nitrogen, limiting the assay to intravenous drugs at high injection concentrations. The fluoroquinolone family of antibiotics was identified as having a similar motif (FIG. 4D). Like NMBDs, these are associated with allergic-type reactions (Kelesidis, T., Fleisher, J. & Tsiodras, S. Anaphylactoid reaction considered ciprofloxacin related: a case report and literature review. Clin Ther 32, 515-526 (2010); Blanca-Lopez, N. et al. Hypersensitivity reactions to fluoroquinolones: analysis of the factors involved. Clin Exp Allergy 43, 560-567 (2013)) and can activate mast cells (Mori, K., Maru, C. & Takasuna, K. Characterization of histamine release induced by fluoroquinolone antibacterial agents in-vivo and in-vitro. The Journal of pharmacy and pharmacology 52, 577-584 (2000); Mori, K., Maru, C., Takasuna, K. & Furuhama, K. Mechanism of histamine release induced by levofloxacin, a fluoroquinolone antibacterial agent. European journal of pharmacology 394, 51-55 (2000)). The four members approved for intravenous use activated MrgprB2-HEK and MrgprX2-HEK cells (FIG. 6A, FIG. 6B, and FIG. 6C), and mast cells in an MrgprB2-dependent manner (FIG. 4E; FIG. 13C). Correspondingly, atracurium and ciprofloxacin induced histamine release in WT peritoneal mast cells and substantially less in MrgprB2.sup.MUT mast cells (FIG. 3C). Ciprofloxacin was selected for in vivo tests of anaphylaxis, which in mice is measured most often by a drop in body temperature, likely due to changes in blood pressure and peripheral vasodilation (Doyle, E., Trosien, J. & Metz, M. Protocols for the induction and evaluation of systemic anaphylaxis in mice. Methods in molecular biology 1032, 133-138, doi:10.1007/978-1-62703-496-8_10 (2013)). Rodents nearly are immune to histamine toxicity at a systemic level, contrary to other experimental organisms (Halpern, B. N. & Wood, D. R. The action of promethazine (phenergan) in protecting mice against death due to histamine. British journal of pharmacology and chemotherapy 5, 510-516 (1950)), but can be rendered sensitive to mast cell activators and secreted products by pretreatment with beta-adrenergic blockers (Bergman, R. K. & Munoz, J. Efficacy of beta-adrenergic blocking agents in inducing histamine sensitivity in mice. Nature 217, 1173-1174 (1968); Matsumura, Y., Tan, E. M. & Vaughan, J. H. Hypersensitivity to histamine and systemic anaphylaxis in mice with pharmacologic beta adrenergic blockade: protection by nucleotides. J Allergy Clin Immunol 58, 387-394 (1976)). Under these conditions, a high dose of ciprofloxacin induced a rapid drop in body temperature that was very slow to recover, while MrgprB2.sup.MUT mice showed a much smaller drop that recovered quickly (FIG. 4F). These results establish that mast cell activation through MrgprB2 is an off-target effect of fluoroquinolones and other drugs, and corresponding MrgprX2 activation in humans might underlie much of the pseudo-allergic responses seen with these drugs.

Example 6

Drugs Associated with Pseudo-Allergies Activate Human Mast Cells Through MrgprX2

[0164] Finally, it was determined whether drugs associated with pseudo-allergies activate human mast cells through MrgprX2. Representative members of each examined drug class evoked release of histamine, TNF, PGD.sub.2, and -hexosaminidase from LAD2 cells (FIG. 14A). 48/80 and mastoparan were used as positive controls. Importantly, MrgprX2 siRNA-treated LAD2 cells exhibited significantly less -hexosaminidase release evoked by these substances, compared to responses in control siRNA-treated cells, while IgE-mediated release was comparable (FIG. 14B). The remaining release observed in MrgprX2 siRNA-treated cells are likely due to incomplete mRNA and/or protein knockdown.

[0165] As described in detail above, MrgprB2 in mice, and MrgprX2 in humans, is the basic secretagogue receptor in mast cells. Also described herein is evidence for the first known in vivo role for this receptor as a critical mediator of IgE-independent drug-induced pseudo-allergies. Thus, knowledge of the role of MrgprX2 in drug-induced pseudo-allergies expands for two reasons. First, ligand binding requirement studies enable more specific screens for drugs that cross-activate MrgprX2. Second, screening orally administered drugs uncovers more MrgprX2 ligands, since common side effects of orally administered drugs include gastrointestinal problems and headache, both which have a mast cell component.

REFERENCES

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R. The action of promethazine (phenergan) in protecting mice against death due to histamine. British journal of pharmacology and chemotherapy 5, 510-516 (1950). [0173] Han, L. et al. A subpopulation of nociceptors specifically linked to itch. Nature neuroscience 16, 174-182, (2013). [0174] Harper, N. J. et al. Suspected anaphylactic reactions associated with anaesthesia. Anaesthesia 64, 199-211, (2009). [0175] Hausmann, O., Schnyder, B. & Pichler, W. J. Etiology and pathogenesis of adverse drug reactions. Chemical immunology and allergy 97, 32-46, (2012). [0176] Kamohara, M. et al. Identification of MrgX2 as a human G-protein-coupled receptor for proadrenomedullin N-terminal peptides. Biochemical and biophysical research communications 330, 1146-1152, (2005). [0177] Kashem, S. W. et al. G protein coupled receptor specificity for C3a and compound 48/80-induced degranulation in human mast cells: roles of Mas-related genes MrgX1 and MrgX2. European journal of pharmacology 668, 299-304, (2011). [0178] Kelesidis, T., Fleisher, J. & Tsiodras, S. Anaphylactoid reaction considered ciprofloxacin related: a case report and literature review. Clin Ther 32, 515-526 (2010). [0179] Koppert, W. et al. Different patterns of mast cell activation by muscle relaxants in human skin. Anesthesiology 95, 659-667 (2001). [0180] Lagunoff, D., Martin, T. W. & Read, G. Agents that release histamine from mast cells. Annual review of pharmacology and toxicology 23, 331-351 [0181] Liu, Q. et al. Sensory neuron-specific GPCR Mrgprs are itch receptors mediating chloroquine-induced pruritus. Cell 139, 1353-1365, (2009). [0182] Lumry, W. R. et al. Randomized placebo-controlled trial of the bradykinin B(2) receptor antagonist icatibant for the treatment of acute attacks of hereditary angioedema: the FAST-3 trial. Annals of allergy, asthma & immunology: official publication of the American College of Allergy, Asthma, & Immunology 107, 529-537, (2011). [0183] Matsumura, Y., Tan, E. M. & Vaughan, J. H. Hypersensitivity to histamine and systemic anaphylaxis in mice with pharmacologic beta adrenergic blockade: protection by nucleotides. J Allergy Clin Immunol 58, 387-394 (1976). [0184] Mertes, P. M., Alla, F., Trechot, P., Auroy, Y. & Jougla, E. Anaphylaxis during anesthesia in France: an 8-year national survey. J Allergy Clin Immunol 128, 366-373 (2011). [0185] Metcalfe, D., Baram, D. & Mekori, Y. A. Mast cells. Physiological reviews 77, 1033-1079 (1997). [0186] Mori, K., Maru, C. & Takasuna, K. Characterization of histamine release induced by fluoroquinolone antibacterial agents in-vivo and in-vitro. The Journal of pharmacy and pharmacology 52, 577-584 (2000). [0187] Mori, K., Maru, C., Takasuna, K. & Furuhama, K. Mechanism of histamine releaseinduced by levofloxacin, a fluoroquinolone antibacterial agent. European journal of pharmacology 394, 51-55 (2000). [0188] Mousli, M., Hugli, T. E., Landry, Y. & Bronner, C. Peptidergic pathway in human skin and rat peritoneal mast cell activation. Immunopharmacology 27, 1-11 (1994). [0189] Nel, L. & Eren, E. Peri-operative anaphylaxis. British journal of clinical pharmacology 71, 647-658, (2011). [0190] Pundir, P. & Kulka, M. The role of G protein-coupled receptors in mast cell activation by antimicrobial peptides: is there a connection? Immunology and cell biology 88, 632-640, (2010). [0191] Purcell, W. M., Doyle, K. M., Westgate, C. & Atterwill, C. K. Characterisation of a functional polyamine site on rat mast cells: association with a NMDA receptor macrocomplex. Journal of neuroimmunology 65, 49-53 (1996). [0192] Read, G. W. Compound 48-80. Structure-activity relations and poly-THIQ, a new, more potent analog. Journal of medicinal chemistry 16, 1292-1295 (1973). [0193] Robas, N., Mead, E. & Fidock, M. MrgX2 is a high potency cortistatin receptor expressed in dorsal root ganglion. The Journal of biological chemistry 278, 44400-44404, (2003). 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[0202] Weigand, L. A., Myers, A. C., Meeker, S. & Undem, B. J. Mast cell-cholinergic nerve interaction in mouse airways. The Journal of physiology 587, 3355-3362, (2009).

Other Embodiments

[0203] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0204] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

[0205] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.