Identification of the first biased NPS receptor agonist that retains anxiolytic and memory promoting effects with reduced levels of locomotor stimulation

Abstract

The neuropeptide S system has been implicated in a number of centrally mediated behaviors including memory consolidation, anxiolysis, and increased locomotor activity. Characterization of these behaviors has been primarily accomplished using the endogenous 20AA peptide (NPS) that demonstrates relatively equal potency for the calcium mobilization and cAMP second messenger pathways at human and rodent NPS receptors. This study is the first to demonstrate that truncations of the NPS peptide provides small fragments that retain significant potency only at one of two single polymorphism variants known to alter NPSR function (NPSR-107I), yet demonstrate a strong level of bias for the calcium mobilization pathway over the cAMP pathway. We have also determined that the length of the truncated peptide correlates with the degree of bias for the calcium mobilization pathway. A modified tetrapeptide analog (4) has greatly attenuated hyperlocomotor stimulation in vivo but retains activity in assays that correlate with memory consolidation and anxiolytic activity. Analog 4 also has a bias for the calcium mobilization pathway, at the human and mouse receptor. This suggests that future agonist ligands for the NPS receptor having a bias for calcium mobilization over cAMP production will function as non-stimulatory anxiolytics that augment memory formation.

Keywords: Anxiolytic; Biased ligand; Memory; Neuropeptide S.

Figure 1

NPS peptide truncation affords bias ligands at the hNPS-107I isoform

Figure 2. NPS-mediated hyperlocomotion

Mice were habituated to a novel environment for 1 hour, they were then administered vehicle or test compound. Mice were returned to the apparatus and locomotion was recorded. Mice receiving NPS (1 nmole) moved significantly more than mice injected with aCSF (*** p < 0.001; one way ANOVA with Dunnett’s Multiple Comparison Test). Neither dose of compound 4 produced significant locomotion, likely because of the very large NPS-mediated response masking the effect in the statistical analysis. [aCSF, n = 5; compound 4 0.1 nmole, n = 8; compound 4 1 nmole, n = 8; NPS 1 nmole, n = 4]

Fig. 3. Compound 4 mediates hyperlocomotion

New groups of mice underwent the same procedure as in Figure 1. Compound 4 mediates a significant increase in locomotion as compared to vehicle treated, and this effect is blocked by pretreatment with a selective NPSR antagonist (RTI-118, 50 mg/kg i.p.). (*** p < 0.001, ** p < 0.01, * p < 0.05; one way ANOVA with Dunnett’s Multiple Comparison Test) [aCSF, n = 10; compound 4 1 nmole, n = 11; compound 4 10 nmole, n = 10; compound 4 10 nmole+RTI-118 50 mg/kg, n = 8; RTI-118 50 mg/kg, n = 7]

Figure 4. Compound 4 increases time spent on light side

Mice were administered vehicle or test compound 5–10 minutes before placement into the Light-Dark Box apparatus. Time spent on the light-side and the number of entries into the light-side was quantified. (A) Both NPS and 4 significantly increased the time spent on the light-side (as compared to aCSF treated). However, NPS treated animals also spent significantly more time on the light-side than the compound 4 treated animals. (B) Neither NPS nor 4 significantly increased the number of entries into the light-side. However, NPS treated animals had significantly more entries than compound 4 treated animals. The number of entries is a measure of activity, and so this data is consistent to that of Figure 2. (** p < 0.01, * p < 0.05; one way ANOVA with Tukey Test). [aCSF, n = 14; compound 4 1 nmole, n = 18; NPS 1 nmole, n = 12].

Figure 5. Compound 4 decreases marble burying behavior

(A) Mice were administered vehicle or test compound twenty minutes before being placed into the test chambers. Mice were allowed to explore a novel home cage with bedding and 18 marbles. After 30 minutes the mice were removed and the number of marbles buried was quantified. Both NPS and 4 significantly decreased the number of marbles buried (as compared to aCSF treated). [aCSF, n = 13; compound 4 1 nmole, n = 13; NPS 1 nmole, n = 13]. (B) Mice were administered saline or RTI-118 (20 mg/kg) fifteen minutes before being administered aCSF or compound 4 (0.5 nmole). Then 30 minutes later the mice underwent the same procedure as in Figure 5A. When pretreated with saline, compound 4 significantly decreased the number of marbles buried but this was blocked by the pretreatment with RTI-118 (as compared to saline/aCSF treated). [saline/aCSF, n = 12; RTI-118/aCSF, n = 8; saline/compound 4 0.5 nmole, n = 8; RTI-118/compound 4 0.5 nmole, n = 9] (** p < 0.01; one way ANOVA with Dunnett’s Multiple Comparison Test).

Figure 6. Compound 4 increases the latency to enter a compartment associated with an aversive stimulus

Mice were trained to associate the dark compartment with a mild electrical shock, and then 20 minutes later were administered vehicle or test compound; 48 hours later mice were tested for latency to enter the dark compartment. NPS and 4 significantly increased the latency time, as compared to aCSF injected (** p < 0.01, * p < 0.05; one way ANOVA with Dunnett’s Multiple Comparison Test). [aCSF, n = 12; compound 4 0.01 nmole, n = 9; compound 4 0.1 nmole, n = 8; compound 4 1 nmole, n = 12; compound 4 10 nmole, n = 18; NPS 1 nmole, n = 13]

Figure 7

Proposed mechanism through which NPS and compound 4 produce disparate behavioral effects.