Neuropeptide S (NPSa), the endogenous ligand of a previously orphan receptor now named NPSR, regulates various biological functions in the brain, including arousal, locomotion, anxiety, and food intake. Here we report on a focused structure-activity study of Gly 5 which has been replaced with L and D amino acids. Fifteen NPS related peptides were synthesized and pharmacologically tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPSR

Neuropeptide S (NPS) has been identified as the endogenous ligand of a previously orphan receptor now named NPSR. Previous studies demonstrated that the N-terminal sequence Phe (2)-Arg(3)-Asn(4) of the peptide is crucial for biological activity. Here we report on a focused structure-activity study of Phe(2) which has been replaced with a series of coded and noncoded amino acids. Thirty-one human NPS analogues were synthesized and pharmacologically tested for intracellular calcium mobilization by using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated the following NPS position 2 structure-activity features: (i) lipophilicity but not aromaticity is crucial, (ii) both the size of the chemical moiety and its distance from the peptide backbone are important for biological activity, and (iii) this position plays a role in both receptor binding and activation, since [4,4'-biphenyl-Ala(2)]hNPS behaved as a partial agonist.

Neuropeptide S (NPS) regulates various biological functions by activating the NPS receptor (NPSR). Previous studies demonstrated that the substitution of Gly5 with D-amino acids generates NPSR antagonists. Eleven [D-Xaa5]NPS derivatives were synthesized and pharmacologically tested measuring [Ca2+]i in HEK293mNPSR cells. The present results confirmed that the [D-Xaa5] substitution promotes antagonist activity with potency inversely related to the side chain size and allowed to identify the novel potent NPSR peptide antagonist [tBu-D-Gly5]NPS.