A novel brain penetrant NPS receptor antagonist, NCGC00185684, blocks alcohol-induced ERK-phosphorylation in the central amygdala and decreases operant alcohol self-administration in rats

Abstract

The Neuropeptide S receptor, a Gs/Gq-coupled GPCR expressed in brain regions involved in mediating drug reward, has recently emerged as a candidate therapeutic target in addictive disorders. Here, we describe the in vitro and in vivo pharmacology of a novel, selective and brain penetrant NPSR antagonist with nanomolar affinity for the NPSR, NCGC00185684. In vitro, NCGC00185684 shows biased antagonist properties, and preferentially blocks ERK-phosphorylation over intracellular cAMP or calcium responses to NPS. In vivo, systemic NCGC00185684 blocks alcohol-induced ERK-phosphorylation in the rat central amygdala, a region involved in regulation of alcohol intake. NCGC00185684 also decreases operant alcohol self-administration, and lowers motivation for alcohol reward as measured using progressive ratio responding. These effects are behaviorally specific, in that they are observed at doses that do not influence locomotor activity or reinstatement responding following extinction. Together, these data provide an initial validation of the NPSR as a therapeutic target in alcoholism.

Figure 1.

Structure of NPSR antagonist NCGC84 and its inhibitory activities in Ca²⁺, cAMP, and [¹²⁵I]Y10-NPS displacement assay. A, NCGC84 was chemically optimized from MLS001018695, an original HTS hit identified from a screen using a cAMP quantitation assay. B–D, Concentration response curves of NCGC00185686 in the intracellular Ca²⁺ assay (B); cAMP assay (C), and radioligand displacement assay (D). The IC₅₀ values of NCGC84 in the Ca²⁺, cAMP, and binding assays are 22.1 ± 1.9, 36.5 ± 6.4, and 5.0 ± 0.05 nm, respectively.

Figure 2.

Schild regression analysis of NCGC84 in the cAMP assay, compared with the known NPSR antagonist SHA68. A, With increases in the concentration of NCGC84, the NPS concentration–response curves were shifted to the right in parallel. pA₂ is 8.98 (CI: 9.03–8.94). B, SHA68 also shifted the concentration response curves of NPS to the right. C, Schild plot in which the slopes for NCGC84 as well as SHA68 are ∼1.0, indicative of a competitive antagonist.

Figure 3.

NCGC84 concentrations up to 10 μm did not affect intracellular Ca²⁺-level in cells expressing vasopressin V1b or purinergic receptors following application of EC₈₀ of their respective ligand. The NPSR has the closest protein sequence homology with the vasopressin V1b receptor (∼28%).

Figure 4.

NPS activity in the ERK phosphorylation, cAMP, and intracellular calcium assays. A, NPS-induced ERK phosphorylation in CHO cells expressing NPSR with an EC₅₀ value of 0.27 ± 0.048 nm. B, NPS-stimulated intracellular calcium response with an EC₅₀ of 0.91 ± 0.044 nm. C, NPS-increased cellular cAMP level with an EC₅₀ of 1.12 ± 0.030 nm.

Figure 5.

Inhibition of NPS-induced ERK phosphorylation in CHO cells expressing NPSR. A, The NPS-induced ERK phosphorylation was inhibited by NCGC84 with an IC₅₀ value of 9.3 ± 11.5 nm. B, ERK phosphorylation (pERK) induced by NPS was significantly inhibited by NCGC84 in Western blot analysis, while levels of nonphosphorylated ERK protein (ERK) remained constant.

Figure 6.

Alcohol-induced ERK phosphorylation in the CeA and, to a lesser extent, NAc-Sh, but not in the NAc-C nor the BNST, is blocked by pretreatment with NCGC84. Pretreatment with NCGC84 inhibited ERK phosphorylation in the central nucleus of the amygdala (A–C) and the NAc-Sh (D) induced by a 1.0 g/kg, i.p. dose of alcohol. No such effect was observed within the nucleus accumbens core or the bed nucleus of the stria terminalis (E–F).

Figure 7.

NCGC84 decreases alcohol self-administration and motivation to obtain alcohol. A, I,P. administration of 1 mg/kg NCGC84 significantly suppressed alcohol self-administration (Newman–Keuls, **p < 0.005). Values (mean ± SEM) represent total rewards received in a 30 min session at an FR3 schedule of reinforcement. Inactive rewards received were negligible and unaffected by NCGC84 (data not shown). B, Additionally, NCGC84 (1 mg/kg) significantly decreased the progressive ratio breakpoint for alcohol self-administration (t = 2.42; p = 0.03).

Figure 8.

NCGC84 does not affect stress- or cue-induced reinstatement of alcohol-seeking. A, C, Alcohol seeking was robustly reinstated both by alcohol-associated cue and stress exposure. However, NCGC84 (1 mg/kg) did not have a significant effect neither on cue- nor stress-induced reinstatement. As is typically found, inactive lever responses in the cue-induced reinstatement experiment were also somewhat increased during reinstatement but this increase was marginal (B). Similarly, in the stress-induced reinstatement test, inactive lever responses were also slightly but significantly increased during reinstatement (D).