Research ArticleITCH

Inhibition of natriuretic peptide receptor 1 reduces itch in mice

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Science Translational Medicine  10 Jul 2019:
Vol. 11, Issue 500, eaav5464
DOI: 10.1126/scitranslmed.aav5464
  • Fig. 1 The NPPB-NPR1 itch signaling pathway is conserved between mice and humans.

    (A) qPCR-based quantification of expression did not show a significant difference in amounts of NPPB transcripts between human and mouse DRG (P = 0.1241, unpaired t test; n = 3). (B) Representative double ISH images of a field of human DRG with neurons stained for NPPB (cyan) and TUBB3 (magenta). NPPB-positive and NPPB-negative neurons are outlined with cyan and magenta dots, respectively. DAPI (4′,6-diamidino-2-phenylindole) counterstain is displayed in gray. (C) Quantification of the soma size of NPPB-stained (red) compared to TUBB3-stained (black) neurons (n = 4). Representative double ISH images of fields of human (D) and mouse (E) DRG reveal that NPPB (cyan) and TRPV1 (magenta) are coexpressed. In human and mouse DRG, NPPB is expressed in a subset of TRPV1 neurons (cyan dotted profiles) and single-labeled TRPV1 neurons are indicated with magenta dotted profiles. (F) qPCR-based quantification of expression did not show a significant difference in amounts of NPR1 transcripts between human and mouse spinal cord (SC) [not significant (ns), P > 0.9999, Mann-Whitney; n = 4 (human) and 3 (mouse)]. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 2 A-71915 is a partial agonist of mNPR1.

    (A) Schematic depicting our strategy to measure NPR1 activity. NPR1 is stimulated by NP to increase synthesis of cGMP; in turn, increased cGMP alters the conformation of a PDE5–Firefly luciferase–based sensor (cGMP sensor), which results in hydrolysis of GloSensor reagent and production of light. (B) Time-course experiments quantifying luminescence of HEK-293 cells transiently expressing cGMP sensor stimulated with the soluble GC activator SNP (333 μM; magenta), media (black), hNPPA (blue), hNPPB (green), and NPPC (purple) (10 nM each). (C) Quantification of activity of HEK-293 cells transiently expressing mNPR1 and cGMP sensor stimulated with mNPPB (green), mNPPA (blue), and NPPC (purple). CI, confidence interval. (D) Quantification of inhibition of mNPR1-cGMP sensor cells with A-71915 (5 min after addition of A-71915, cells were treated with 1 nM mNPPB). (E) Quantification of mNPR1-cGMP sensor cells shows partial agonist activity for A-71915. Data represent means ±SEM of triplicate (B) or duplicate (C to E) measurements.

  • Fig. 3 Cell-based screen identifies candidate small-molecule inhibitors of hNPR1.

    (A and C) Time-course experiments quantifying luminescence of stable cell lines expressing pGS-40F and hNPR1 (A) and pGS-40F alone (C) stimulated by media (black), hNPPA (blue), hNPPB (green), NPPC (purple) (10 nM each), and SNP (333 μM, magenta). (B and D) Quantification of activity of HEK-hNPR1-cGMP sensor cells (B) and HEK-cGMP sensor cells (D) with hNPPA (blue), hNPPB (green), NPPC (purple), and SNP (magenta). (E) Schematic depicts the time course of our qHTS assay. (F) Representative three-axis plot of concentration-response curve profiles for compounds from the Genesis chemical library; 519,417 concentration response values are displayed in gray (1574 outlier values were not plotted). Out of the 3.9% active compounds, 105 compounds with greatest efficacy (maximum antagonism, >90%) are displayed (black traces). Curves were fit using a four-parameter logistic regression. Data represent means ±SEM of duplicate (A to C) or triplicate (D) measurements.

  • Fig. 4 Candidate inhibitors attenuate specifically hNPR1 activity.

    Quantification of inhibition of hNPR1 activity (blue squares), Firefly luciferase activity (orange squares), SNP-induced activity (black squares), and cytotoxicity for HEK-hNPR1-cGMP sensor cells (red squares) by JS-5 (A), JS-8 (B), and JS-11 (C). Data were collected from qHTS assays. (D to F) Chemical structures of JS-5 (D), JS-8 (E), and JS-11 (F).

  • Fig. 5 Cell-free membrane cyclase assay confirms that candidate compounds are specific antagonists of hNPR1.

    (A) Schematic depicts our strategy to measure hNPR1 activity with an in vitro assay. A crude membrane fraction was prepared from HEK-hNPR1-cGMP sensor cells. Incubation of hNPR1 membranes with guanosine triphosphate (GTP) and NP results in production of cGMP, and cGMP was measured using an enzyme-linked immunosorbent assay (ELISA) test. (B) Quantifications of cGMP production by hNPR1 membranes, stimulated by hNPPA (blue) and SNP (red). (C) Quantification of inhibition of hNPPA-stimulated (1 nM) hNPR1 activity by JS-5 (blue), JS-8 (green), or JS-11 (purple). Data represent means ±SEM for triplicate (B, hNPPA), duplicate (B, SNP), and duplicate measurements (C).

  • Fig. 6 hNPR1 antagonists inhibit receptor activity through a noncompetitive mechanism.

    (A) Quantification of the inhibition of basal hNPR1 activity by antagonists, JS-5 (blue), JS-8 (green), and JS-11 (purple). (B) Quantification of hNPR1 activity to increasing concentrations of hNPPA in the presence of a fixed concentration of antagonists (5 μM): JS-5 (blue), JS-8 (green), JS-11 (purple), A-71915 (magenta), and saline (black). (C) Quantification of antagonist dissociation from hNPR1. HEK-hNPR1-cGMP sensor cells were treated with JS-8 and either were given a 5-min washing step (red) or were not treated (black). Next, cells were stimulated with hNPPA (60 pM) to test whether JS-8 dissociates rapidly from hNPR1. Data represent means ± SEM of triplicate (A and C) and duplicate (B) measurements.

  • Fig. 7 NPR1 antagonist inhibits acute itch behavior.

    (A) Schematic depicting the strategy used to test effects of JS-11 in a mouse model of acute itch. i.p., intraperitoneal; i.t., intrathecal; s.c., subcutaneous. (B and C) Quantification of scratching responses to histamine (B, n = 8) and CYM5442 (C, n = 10) (B, *P = 0.0221; C, *P = 0.0128, paired t test). Mice were intraperitoneally injected with 163 μg of JS-11 or vehicle and, 10 min later, injected into the nape of the neck with pruritogens (100 μg of histamine and 8.9 μg of CYM5442). (D and E) Representative images of c-FOS immunostaining in the spinal cord after intradermal calf injection of histamine (100 μg) and previous administration of JS-11 (163 μg) or vehicle [20% dimethyl sulfoxide (DMSO)]. (F) Quantification of the number of c-FOS–positive neurons (average from six sections for each animal; n = 4 mice per treatment). Significant differences were assessed using one-way ANOVA and Sidak’s multiple comparisons post hoc test. JS-11 reduced the number of spinal c-FOS–positive neurons ipsilateral (ipsi) to the histamine injection (*P < 0.0001) without affecting basal activity on the contralateral (contra) side (ns, P = 0.9953). Histamine significantly increased numbers of c-FOS neurons ipsilateral to the injection side in both treatment groups (JS-11, *P = 0.0058; vehicle, *P < 0.0001). (G) Quantification of the effect of intrathecal delivery of JS-11 (16.3 μg) and vehicle (20% DMSO) on numbers of scratching bouts to histamine (100 μg into the nape of the neck). Itch responses were significantly reduced by administration of JS-11 (*P = 0.0030, paired t test; n = 8). (H and I) Representative double ISH images of human DRG sections revealed neurons stained for NPPB (H and I, magenta), HRH1 (H, cyan), and MRGPRX1 (I, cyan). Neurons positive for NPPB and itch receptors are highlighted with white dotted profiles.

  • Fig. 8 NPR1 antagonism inhibits itching in a mouse model of contact dermatitis.

    (A) Representative double ISH image of a human DRG section reveals that NPPB (magenta) and IL31RA (cyan) are coexpressed. Neurons coexpressing NPPB and IL31RA are highlighted with white dotted profiles. (B) Schematic depicts the experimental strategy to examine the effects of JS-11 on a mouse model of contact hypersensitivity-induced itch. (C) Quantification of the effects of JS-11 treatment on contact dermatitis–induced changes in ear thickness. There were no significant differences between JS-11 (pink, 163 μg) and vehicle (blue, 20% DMSO) groups (n = 10). Ear thickness was analyzed using one-sample t test against a theoretical mean of 100% (vehicle: ns, P = 0.8020 and JS-11: ns, P = 0.4384), and differences between treatment groups were assessed using unpaired t test (ns, P = 0.5283). (D) Quantification of the effects of JS-11 treatment on contact dermatitis–induced changes in scratching responses. Itch behavior was significantly reduced by administration of JS-11 (pink, 163 μg) compared to vehicle (blue, 20% DMSO) (n = 10). Scratching responses were analyzed using one-sample t test against a theoretical mean of 100% (vehicle: ns, P = 0.3951 and JS-11: *P < 0.0001), and differences between treatment groups were assessed using unpaired t test (*P = 0.0193).

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/500/eaav5464/DC1

    Materials and Methods

    Fig. S1. Generation of HEK-hNPR2-cGMP sensor cells.

    Fig. S2. hNPR1 inhibitors also block hNPR2.

    Fig. S3. JS-11 does not inhibit ACs.

    Fig. S4. In vivo pharmacokinetics of JS-11.

    Fig. S5. Effects of NPR1 antagonist on general motor behavior and itch responses.

    Fig. S6. JS-11 does not cause extensive cardiovascular side effects.

    Fig. S7. JS-11 inhibits itching in a mouse model of contact dermatitis.

    Fig. S8. General plate map layout for qHTS.

    Fig. S9. qHTS concentration response curves for JS-1 through JS-15.

    Table S1. Summary of qHTS and counterscreens.

    Table S2. Corroboration of hNPR1 inhibition in screening assay.

    Table S3. SafetyScreen44-dependent test for off-targets of JS-11.

    Table S4. Inhibition of hNPR1 in membrane cyclase assay.

    Table S5. Ki values for hNPR1 and mNPR1.

    Table S6. hNPR1 antagonists also inhibit mNPR1.

    Table S7. In vitro pharmacokinetics of JS-11 and JS-8.

    Table S8. PubChem AIDs deposited and used for this study.

    Table S9. Clinical information of human DRG donors.

    Data file S1. LOPAC pilot screening data.

    Data file S2. Genesis primary qHTS screening data.

    Data file S3. qHTS follow-up screening data.

    Data file S4. Raw data.

    References (6272)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Generation of HEK-hNPR2-cGMP sensor cells.
    • Fig. S2. hNPR1 inhibitors also block hNPR2.
    • Fig. S3. JS-11 does not inhibit ACs.
    • Fig. S4. In vivo pharmacokinetics of JS-11.
    • Fig. S5. Effects of NPR1 antagonist on general motor behavior and itch responses.
    • Fig. S6. JS-11 does not cause extensive cardiovascular side effects.
    • Fig. S7. JS-11 inhibits itching in a mouse model of contact dermatitis.
    • Fig. S8. General plate map layout for qHTS.
    • Fig. S9. qHTS concentration response curves for JS-1 through JS-15.
    • Table S1. Summary of qHTS and counterscreens.
    • Table S2. Corroboration of hNPR1 inhibition in screening assay.
    • Table S3. SafetyScreen44-dependent test for off-targets of JS-11.
    • Table S4. Inhibition of hNPR1 in membrane cyclase assay.
    • Table S5. Ki values for hNPR1 and mNPR1.
    • Table S6. hNPR1 antagonists also inhibit mNPR1.
    • Table S7. In vitro pharmacokinetics of JS-11 and JS-8.
    • Table S8. PubChem AIDs deposited and used for this study.
    • Table S9. Clinical information of human DRG donors.
    • Legends for data files S1 to S4
    • References (6272)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). LOPAC pilot screening data.
    • Data file S2 (Microsoft Excel format). Genesis primary qHTS screening data.
    • Data file S3 (Microsoft Excel format). qHTS follow-up screening data.
    • Data file S4 (Microsoft Excel format). Raw data.

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