Inhibition of natriuretic peptide receptor 1 reduces itch in mice

Abstract

There is a major clinical need for new therapies for the treatment of chronic itch. Many of the molecular components involved in itch neurotransmission are known, including the neuropeptide NPPB, a transmitter required for normal itch responses to multiple pruritogens in mice. Here, we investigated the potential for a novel strategy for the treatment of itch that involves the inhibition of the NPPB receptor NPR1 (natriuretic peptide receptor 1). Because there are no available effective human NPR1 (hNPR1) antagonists, we performed a high-throughput cell-based screen and identified 15 small-molecule hNPR1 inhibitors. Using in vitro assays, we demonstrated that these compounds specifically inhibit hNPR1 and murine NPR1 (mNPR1). In vivo, NPR1 antagonism attenuated behavioral responses to both acute itch- and chronic itch-challenged mice. Together, our results suggest that inhibiting NPR1 might be an effective strategy for treating acute and chronic itch.

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 counterstain is displayed in grey. (C) Quantification of the soma-size of NPPB- (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 co-expressed. In human and mouse DRG, NPPB is expressed in a subset of TRPV1-neurons (cyan-dotted profiles), 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 (ns P > 0.9999, Mann-Whitney, n = 4 (human) 3 (mouse)).

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; red), 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). (D) Quantification of inhibition of mNPR1-cGMP-sensor cells with A-71915 (5 minutes 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 $\pm$ SEM of triplicate (B) or duplicate (C-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, red). (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 (red). (E) Schematic depicts the time course of our qHTS assay. (F) Representative 3-axis plot of concentration-response curve profiles for compounds from the Genesis chemical library; 519,417 concentration response values are displayed in grey (1574 out-lie 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 $\pm$ SEM of duplicate (A-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-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 GTP and NP results in production of cGMP and cGMP was measured using an 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 $\pm$ SEM for triplicate (B, hNPPA), duplicate (B, SNP), and duplicate measurements (C).

Fig. 6:. hNPR1 antagonists inhibit receptor activity through a non-competitive 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 (red), and saline (black). (C) Quantification of antagonist dissociation from hNPR1. HEK-hNPR1-cGMP-sensor cells were treated with JS-8 and where either given a 5-minute washing step (red), or we not treated (black). Next, cells were stimulated with hNPPA (60 pM) to test if JS-8 dissociates rapidly from hNPR1. Data represent means $\pm$ SEM of triplicate (A, C) and duplicate (B) measurements.

Fig. 7:. NPR1 antagonist inhibits acute itch-behavior.

(A) Schematic depicting the strategy employed to test effects of JS-11 in a mouse model of acute itch. (B-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 JS-11, or vehicle and 10 minutes later injected into the nape of the neck with pruritogens (100 μg, histamine and 8.9 μg, CYM5442). (D-E) Representative images of c-FOS immunostaining in the spinal cord following intradermal calf injection of histamine (100 μg) and prior administration of JS-11 (163 μg) or vehicle (20% DMSO). (F) Quantification of the number of c-FOS-positive neurons (average from 6 sections for each animal; n = 4 mice per treatment). Significant differences were assessed using 1-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-I) Representative double ISH images of human DRG sections revealed neurons stained for NPPB (magenta H and I) and 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 co-expressed. Neurons co-expressing 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).