Primi studi SAR (structure activity relationship) del Neuropeptide S. Modifiche chimiche per migliorare la farmacocinetica della Nocicettina

Neuropeptide S (NPS) is the last endogenous peptide identified via the reverse pharmacology approach. The human form of NPS is a 20 residue peptide of the following primary sequence, SFRNGVGTGMKKTSFQRAKS, which is highly conserved among species. After its pairing with NPS, the previously orphan G-protein coupled receptor (GPCR) GPR154 was named the NPS receptor and abbreviated as NPSR. NPSR shows low homology to other members of the GPCR family. Cells stably expressing NPSR display a transient increase in the initial Ca2+ concentration levels in response to nanomole concentrations of NPS, thus suggesting that NPS behaves as an excitatory transmitter. When injected supraspinally in rodents, NPS produces anxiolitic-like effects associated with stimulation of wakefulness. The biological originality of this peptide–receptor system relies on its combined effects on anxiety and arousal, a behavioral profile that differs from those of all the known anxiolitic and arousal promoting agents. The development of selective ligands for NPSR is now mandatory to investigate the functions of this novel neurotransmitter system. Ala- and D-scans together with N- and C-terminal truncation studies demonstrated that the N terminal portion of NPS is crucial for bioactivity. In this work the residues Phe2-Arg3-Asn4-Gly5 were subjected to systematic replacement with coded and non coded aminoacids; these modifications may provide useful indications for the design of novel NPSR ligands. All peptides were synthesized using Fmoc/tBu chemistry. Purification of the crude peptides were achieved by preparative HPLC and the purity grade was checked by analytical HPLC and mass spectrometry. The novel peptides were pharmacologically evaluated in a calcium mobilization assay using HEK293 cells stably expressing the mouse NPSR (HEK293mNPSR) and the fluorometric imaging plate reader FlexStation II. Position 2 (Phe) plays a role in both receptor binding and activation, as demonstrated by the reduction in efficacy displayed by [Bip2]hNPS. Investigation of position 3 (Arg) revealed that the guanidine moiety and its basic character are not crucial requirements and that an aliphatic aminoacid with a linaer three carbon atom long side chain is sufficient to bind and fully activate NPSR. Position 5 (Gly) can tolerate substitutions with aminoacids characterized by small side chians while larger side chains reduce agonist potency. The chirality of aminoacid residues at position 5 of NPS has a crucial role for peptide efficacy with L aminoacids favoring agonist and D aminoacids promoting antagonist bioactive conformations. Moreover, in the frame of the present study, the first generation of NPSR peptide pure antagonists was identified and [D-Val5]NPS was pharmacologically characterized in vitro and in vivo. A further study performed on position 5 confirmed previous indications that the D relative configuration of amino acid residues at position 5 of NPS promotes antagonist activity and indicates that the peptide antagonist potency is inversely related to the D-Xaa5 side chain. [tBu-D-Gly5]NPS and [D-Pen5]NPS identified in the context of the present study represent the most potent NPSR antagonist so far identified. Position 4 (Asn) seems to be very sensitive to aminoacids replacement; in fact, all the aminoacid replacements investigated produced an important decrease of biological activity or generated inactive derivatives suggesting a pivotal role of the Asn4 side chain for NPS bioactivity.