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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.
Neuropeptide S (NPS, human sequence SFRNGVGTGMKKTSFQRAK) is the endogenous ligand of the 7TM receptor NPSR.1 Via selective NPSR activation, NPS regulates several biological functions including wakefulness1, 2, stress and anxiety1–5, locomotor activity1–3, 6, food intake6–8, memory processes5, 9, and drug abuse10–13. To deeply investigate these NPS sensitive biological functions and to identify the therapeutic potential of drugs interacting with the NPSR, potent and selective ligands are required. With the aim to identify NPSR ligands structure activity relationship (SAR) studies were performed on the NPS sequence that allowed to demonstrate the crucial importance of the sequence Phe2-Arg3-Asn4 for receptor binding and activation14–16 and that of the sequence Gly5-Val6-Gly7 for shaping NPS into the biologically active conformation14, 16, 17. Few SAR studies were then performed on Phe2 (ref 18), Arg3 and Asn4 (ref 19). More recently, a SAR study focused on Gly5 was carried out.20 This investigation demonstrated that the introduction in NPS position 5 of a chiral center with relative configuration D, produces important changes in peptide potency and, particularly, in its efficacy. In fact, the replacement of Gly5 with D-Leu or D-Cys generated NPSR partial agonists while that with D-Met or D-Val produced pure and fairly potent NPSR antagonists. The NPSR antagonistic properties of [D-Val5]NPS were confirmed in vivo in the mouse locomotor activity assay, where the peptide at 10 nmol, blocked the stimulatory effect elicited by the supraspinal administration of 0.1 nmol NPS.20 These findings prompted us to further investigate position 5 with the aim of understanding the chemical requirements of the D-amino acid side chain that are instrumental for generating NPSR antagonism.
Eleven novel peptides (Table 1) were synthesized in good yield and with a purity grade not less than 95% following procedures previously described.18 NPS, [D-Val5]NPS (used as reference NPSR antagonist), and the novel peptides were pharmacologically evaluated in a calcium mobilization assay using HEK293 cells stably expressing the mouse NPSR (HEK293mNPSR). The protocols and the experimental conditions used in the present study have been previously illustrated in detail.20, 21 However, to facilitate drug diffusion into the wells in antagonist type experiments, the present studies were performed at 37 °C and three cycles of mixing (25 Kl from each well moved up and down 3 times) were performed immediately after antagonist injection to the wells. In addition, inhibition response curve to putative antagonists were performed against the stimulatory effect of 30 nM NPS.
NPS increased the intracellular calcium concentrations in a concentration-dependent manner with pEC50 and Emax values of 8.32 and 295% over basal, respectively (Table 1). Confirming previous findings,20 [D-Val5]NPS did not evoke any effect per se but inhibited in a concentration dependent manner the stimulatory effect of 30 nM NPS, thus behaving as NPSR antagonist. A pKB value of 6.54 was derived from these experiments. The replacement of the isopropyl group (as in Val) with sec-butyl group (compound 1 and 2) produced a similar moderate reduction of potency independently from the side chain chiral center. Similar results were obtained substituting a methyl of the isopropyl group with an oxydril function (compound 3 and 4). In this latter case, the side chain chiral center seems to exert an effect on peptide efficacy since [D-allo-Thr5]NPS behaves as a pure NPSR antagonist while [DThr5]NPS as a low efficacy partial agonist. However the difference in efficacy between 3 and 4 ([DThr5]NPS α = 0.08; [D-allo-Thr5]NPS α = 0) is too little to be meaningful. A linear three carbon atoms side chain (compound 5) produced an analogue that behaved as a NPSR low efficacy partial agonist 10-fold less potent than [D-Val5]NPS. All together these findings indicated that the isopropyl moiety is highly important for NPSR antagonist binding and that the replacement of one of its methyl groups with ethyl or oxydril functions produced a reduction of potency. Moreover, the three carbon atoms of the D-Val side chain must have a ramified (isopropyl) rather than linear (n-propyl, as in compound 5) shape; in fact the latter generates a clear reduction of peptide potency. The introduction in position 5 of a cyclohexyl or methyl-cyclohexyl moiety (compound 6 and 7) generated inactive derivatives while the introduction of a phenyl ring (compound 8) produced only a 3-fold reduction in potency compared to the isopropyl moiety of [D-Val5]NPS. These data suggest that the increase in the side chain size (as in compound 6 and 7) decreases peptide potency. This is further suggested by the results obtained with compound 8. In fact, the aromaticity of the phenyl ring of 8 reduced the side chain size and changed its shape compared with the cyclohexyl moiety of 6 and this may explain the moderate potency of [D-Phg5]NPS compared to the inactivity of [cyclohexyl-D-Gly5]NPS. Next, the effect of the insertion in the D-Val5 isopropyl moiety of a CH3 (compound 9) or SH (compound 10) group was evaluated. In both cases the chemical change did not modify the pharmacological activity of the peptides i.e. they behaved as pure antagonists, with a 3 fold increase of potency. Finally, the insertion of a carbon atom between the tBu moiety and the peptide backbone (compound 11) caused an important reduction of peptide potency associated with a clear increase in efficacy ([tBu-D-Gly5]NPS α = 0, pKB 7.06; [tBu-D-Ala5]NPS α = 0.35, pKB 6.32). Similar results were obtained with the isopropyl ([D-Val5]NPS and [D-Leu5]NPS20). Collectively these findings indicated that a short side chain favors high potency and pure receptor antagonism. In addition, the comparison of the effects of the side chain structures (tBu and isopropyl) clearly indicates that aliphatic branched moieties are better recognized by the NPSR receptor. The best results are obtained with the tBu moiety in which the substitution of a CH3 with SH does not change the biological activity.
Then, [tBu-D-Gly5]NPS was further characterized by assessing its antagonist behavior using the classical Schild protocol and its selectivity of action over eight unrelated GPCRs. Figure 1 summarizes the data obtained by performing concentration response curves to NPS in the presence of increasing concentrations (0.1 – 10 KM) of [tBu-D-Gly5]NPS. This peptide produced a concentration dependent rightward shift of the concentration response curve to NPS which was however associated with a slight but significant depression of NPS maximal effects. From these experiments a pKB values of 6.78 (CL95% 6.33 – 7.23) was derived (using the equation described at pp 117 of22). This value is close to that obtained in inhibition response curve studies (7.06, Table 1). Thus, these experiments confirmed the pure and potent antagonist properties of [tBu-D-Gly5]NPS. However these results are not compatible with a simple competitive interaction between [tBu-D-Gly5]NPS and NPS. Similar results (rightward shift associated with significant depression of agonist maximal effects) were previously reported for [D-Val5]NPS20 which was however tested at room temperature and without the 3 cycles of mixing. Under these same experimental condition [tBu-D-Gly5]NPS caused a profound depression of NPS maximal effects (down to less than 50%) in a concentration dependent manner (data not shown). This depression of Emax was strongly reduced even if not completely eliminated performing the experiments at 37 °C and introducing the 3 cycles of mixing. Collectively these findings suggest that the depression of NPS Emax caused by NPSR antagonists in the calcium mobilization assay may likely derive from hemiequilibrium conditions due to lack of stirring22 rather than from a real insurmountable type of antagonism.
In order to get information about the selectivity of action of [tBu-D-Gly5]NPS the peptide was evaluated as agonist and antagonist in calcium mobilization experiments performed using eight unrelated human G protein coupled receptors (Table 2). These included the native PAR2 receptors expressed in A549 cells and the recombinant NK-1, B2, and UT receptors expressed in CHO cells. Moreover, this investigation was extended to nociceptin/orphanin FQ peptide receptor (NOP) and classical opioid receptors forced to couple with the calcium pathway by the chimeric protein αqi523, 24. [tBu-D-Gly5]NPS up to 10 KM neither stimulated calcium mobilization in these cells nor affected the stimulatory effects elicited by the reference receptor agonists (Table 2). These results suggest that [tBu-D-Gly5]NPS behaves as a selective NPSR antagonist. However, the panel of receptors investigated is probably too limited to draw final conclusions about the compound selectivity at this time.
Collectively the present findings demonstrated that the peptide [tBu-D-Gly5]NPS behaves as pure, potent (pKB ≈ 7) and selective NPSR antagonist. This molecule should be compared to the NPSR antagonists so far available which are the moderate potency (pKB ≈ 6.5) peptide antagonists [D-Cys(tBu)5]NPS21 and [D-Val5]NPS20 and the highly potent (pKB ≈ 7.5) bicyclic piperazine derivative SHA 68.25 The antagonist properties of these molecules have been already confirmed in in vivo studies5, 20, 21, 25, 26 while the evaluation of [tBu-D-Gly5]NPS in vivo actions is under way in our laboratories.
In conclusion, the present study i) confirmed previous indications that the D relative configuration of amino acid residues at position 5 of NPS promotes antagonist activity, ii) indicated that the peptide antagonist potency is inversely related to the D-Xaa5 side chain size, and iii) demonstrated that the tBu (and its sulfhydryl derivative) directly linked to the Cα carbon atom is the best chemical moiety for increasing antagonist potency. [tBu-D-Gly5]NPS and [D-Pen5]NPS identified in the context of the present study represent the most potent NPSR peptide antagonists so far identified.
This work was supported by funds from the University of Ferrara (FAR grants to GC and SS), the Italian Ministry of the University (PRIN grant to GC and SS) and from the National Institute of Mental Health (grant to RKR).
Supporting Information Available: a table reporting the retention time determined by analytical HPLC analyses using two different chromatographic systems and the calculated and found molecular weight of the compounds. This material is available free of charge via the internet at http://pubs.acs.org