Cathelicidin genes homologous to the human CAMP gene, coding for the host defense peptide LL-37, have been sequenced and analyzed in 20 primate species, including Great Apes, hylobatidae, cercopithecidae, callithricidae, and cebidae. The region corresponding to the putative mature antimicrobial peptide is subject to a strong selective pressure for variation, with evidence for positive selection throughout the phylogenetic tree relating the peptides, which favors alterations in the charge while little affecting overall hydrophobicity or amphipathicity. Selected peptides were chemically synthesized and characterized, and two distinct types of behavior were observed. Macaque and leaf-eating monkey RL-37 peptides, like other helical antimicrobial peptides found in insect, frog, and mammalian species, were unstructured in bulk solution and had a potent, salt and medium independent antimicrobial activity in vitro, which may be the principal function also in vivo. Human LL-37 and the orangutan, hylobates, and callithrix homologues instead showed a salt-dependent structuring and likely aggregation in bulk solution that affected antimicrobial activity and its medium dependence. The two types of peptides differ also in their interaction with host cells. The evolution of these peptides has thus resulted in distinct mechanisms of action that affect the direct antimicrobial activity and may also modulate accessory antimicrobial functions due to interactions with host cells.

Neuropeptide S (NPS) was identified as the endogenous ligand of an orphan receptor now referred to as the NPS receptor (NPSR). In the frame of a structure-activity study performed on NPS Gly5, the NPSR ligand [d-Cys(tBu)⁵]NPS was identified. [d-Cys(tBu)⁵]NPS up to 100 μM did not stimulate calcium mobilization in human embryonic kidney (HEK) 293 cells stably expressing the mouse NPSR; however, in a concentration-dependent manner, the peptide inhibited the stimulatory effects elicited by 10 and 100 nM NPS (pK_B, 6.62). In Schild analysis experiments [d-Cys(tBu)⁵]NPS (0.1–100 μM) produced a concentration-dependent and parallel rightward shift of the concentration-response curve to NPS, showing a pA₂ value of 6.44. Ten micromolar [d-Cys(tBu)⁵]NPS did not affect signaling at seven NPSR unrelated G-protein-coupled receptors. In the mouse righting reflex (RR) recovery test, NPS given at 0.1 nmol i.c.v. reduced the percentage of animals losing the RR in response to 15 mg/kg diazepam and their sleeping time. [d-Cys(tBu)⁵]NPS (1–10 nmol) was inactive per se but dose-dependently antagonized the arousal-promoting action of NPS. Finally, NPSR-deficient mice were similarly sensitive to the hypnotic effects of diazepam as their wild-type littermates. However, the arousal-promoting action of 1 nmol NPS could be detected in wild-type but not in mutant mice. In conclusion, [d-Cys(tBu)⁵]NPS behaves both in vitro and in vivo as a pure and selective NPSR antagonist but with moderate potency. Moreover, using this tool together with receptor knockout mice studies, we demonstrated that the arousal-promoting action of NPS is because of the selective activation of the NPSR protein.

Background and Purpose: Intrathecally (i.t.) administered nociceptin/orphaninFQ(N/OFQ) evokes antinociceptive effects in rodents. Recent studies in monkeys demonstrated that i.t. co‐application ofN/OFQand morphine elicits synergistic antinociceptive actions suggesting mixedN/OFQpeptide (NOP) and μ opioid receptor agonists as innovative spinal analgesics. Thus, novelN/OFQrelated peptides were synthesized in order to identify and pharmacologically characterize a mixedNOP/ μ opioid receptor agonist.Experimental Approach: The followingin vitroassays were used: calcium mobilization in cells expressing the humanNOPor classical opioid receptors and chimericGproteins, receptor and[³⁵S]‐GTPγSbinding,[³⁵S]‐GTPγSbinding in rat spinal cord membranes, guinea pig ileum bioassay.In vivoexperiments were performed in monkeys using the tail withdrawal assay.Key Results: From calcium mobilization studies[Dmt¹]N/OFQ(1–13)‐NH₂was selected as the most potent and least selective compound. The mixedNOP/opioid full agonist activity and high affinity of[Dmt¹]N/OFQ(1–13)‐NH₂was confirmed at human recombinant receptors in receptor binding, calcium mobilization and/or[³⁵S]‐GTPγSbinding studies, at rat spinal cord receptors in[³⁵S]‐GTPγSbinding experiments, and at guinea pig receptors inhibiting neurogenic contractions in the ileum.In vivoin the tail withdrawal assay in monkeys i.t.[Dmt¹]N/OFQ(1–13)‐NH₂was able to elicit robust and long‐lasting antinociceptive effects.Conclusions and Implications: Collectively, these results demonstrate that[Dmt¹]N/OFQ(1–13)‐NH₂behaves asNOP/opioid receptor universal agonist and substantiate the suggestion that such mixed ligands are worthy of development as innovative spinal analgesics.