Synthesis and Biological Activity of Human Neuropeptide S Analogues Modified in Position 5: Identification of Potent and Pure Neuropeptide S Receptor Antagonists

Neuropeptide S (NPS), 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 Gly5, 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. The results of this study demonstrated that peptide potency is inversely related to the side chain size, while peptide efficacy strongly depends on the relative l and d configuration, with the l amino acids favoring agonist while d amino acids display antagonist pharmacological activity. [d-Val5]NPS behaved as NPSR pure antagonist in HEK293mNPSR cells showing the highest potency (pKB 7.56) among this series of peptides. The antagonist action of [d-Val5]NPS was confirmed in vivo in mice, where the peptide at a dose of 10 nmol completely blocked the stimulatory effect of 0.1 nmol NPS on locomotor activity.