Valeria Camarda

Pharmacological profile of hemokinin 1: a novel member of the tachykinin family.

Recently, the cloning of a novel preprotachykinin gene (PPT-C) has been reported. This gene codes for a novel peptide named hemokinin 1 (HK-1). In contrast with the known tachykinins, which are exclusively expressed in neuronal tissues, PPT-C mRNA was detected primarily in hematopoietic cells. In this study, we pharmacologically characterised the effects of HK-1 using three tachykinin monoreceptor systems, namely the rabbit jugular vein (rbJV) for NK(1), the rabbit pulmonary artery (rbPA) for NK(2), and rat portal vein (rPV) for NK(3) receptors. In all these preparations substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) elicited concentration dependent contractions showing similar maximal effects and the following rank order of potency: SP > NKA = NKB in the rbJV, NKA > NKB >> SP in the rbPA, and NKB > NKA > SP in the rPV. In those vessels HK-1 behaved as a full agonist displaying potencies similar (rbPA and rPV) or slightly higher (rbJV) than those of SP. In the rbJV, SR 140333, a selective NK(1) receptor antagonist, antagonised the effects of HK-1 and SP with similar high potencies (pK(B) 9.3 and 9.5, respectively). Similar results were obtained with the pseudopeptide NK(1) antagonist, MEN 11467 (pK(B) 8.8 and 8.6, respectively). Taken together, these data indicate that HK-1 behaves as a NK(1) preferring receptor agonist.

In Vitro and in Vivo Pharmacological Characterization of the Neuropeptide S Receptor Antagonist [d-Cys(tBu)5]Neuropeptide S

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 (pKB, 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.

A new ligand for the urotensin II receptor

The cyclic peptide human urotensin II (U‐II) has been recently recognized as the endogenous ligand of an orphan GPCR, subsequently named the UT receptor. No synthetic ligands are available for investigating this novel peptide‐receptor system. A novel UT receptor ligand, [Orn8]U‐II, was synthesized and evaluated in calcium functional assays performed on HEK293 cells expressing the recombinant rat and human UT receptor and in the rat aorta bioassay. [Orn8]U‐II behaves as a full agonist (pEC50≈8) at both human and rat UT receptors in the FlipR calcium assay eliciting similar maximal effects as the natural ligand U‐II. On the contrary, in the rat aorta bioassay, [Orn8]U‐II behaves as a competitive and selective antagonist (pA2=6.56) showing however a small but consistent residual agonist activity. It is therefore proposed that [Orn8]U‐II is a partial agonist at UT receptors.

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 HEK293(mNPSR) cells showing the highest potency (pK(B) 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.

Further studies on the pharmacological profile of the neuropeptide S receptor antagonist SHA 68.

Neuropeptide S (NPS) regulates various biological functions by selectively activating the NPS receptor (NPSR). Previous studies demonstrated that the non-peptide molecule SHA 68 acts as a selective NPSR antagonist. In the present study the pharmacological profile of SHA 68 has been further investigated in vitro and in vivo. In cells expressing the mouse NPSR SHA 68 was inactive per se up to 10microM while it antagonized NPS-stimulated calcium mobilization in a competitive manner showing a pA(2) value of 8.06. In the 10-50mg/kg range of doses, SHA 68 counteracted the stimulant effects elicited by NPS, but not those of caffeine, in mouse locomotor activity experiments. In the mouse righting reflex assay SHA 68 fully prevented the arousal-promoting action of the peptide. The anxiolytic-like effects of NPS were slightly reduced by SHA 68 in the mouse open field, fully prevented in the rat elevated plus maze and partially antagonized in the rat defensive burying paradigm. Finally, SHA 68 was found poorly active in antagonizing the NPS inhibitory effect on palatable food intake in rats. In all assays SHA 68 did not produce any effect per se. In conclusion, the present study demonstrated that SHA 68 behaves as a selective NPSR antagonist that can be used to characterize the in vivo actions of NPS. However the usefulness of this research tool is limited by its poor pharmacokinetic properties.

The peptidic urotensin-II receptor ligand GSK248451 possesses less intrinsic activity than the low-efficacy partial agonists SB-710411 and urantide in native mammalian tissues and recombinant cell systems

1 Several peptidic urotensin‐II (UT) receptor antagonists exert ‘paradoxical’ agonist activity in recombinant cell‐ and tissue‐based bioassay systems, likely the result of differential urotensin‐II receptor (UT receptor) signal transduction/coupling efficiency between assays. The present study has examined this phenomenon in mammalian arteries and recombinant UT‐HEK (human embryonic kidney) cells. 2 BacMam‐mediated recombinant UT receptor upregulation in HEK cells augmented agonist activity for all four peptidic UT ligands studied. The nominal rank order of relative intrinsic efficacy was U‐II>urantide ([Pen5‐DTrp7‐Orn8]hU‐II4–11)>SB‐710411 (Cpa‐c[DCys‐Pal‐DTrp‐Lys‐Val‐Cys]‐Cpa‐amide)≫GSK248451 (Cin‐c[DCys‐Pal‐DTrp‐Orn‐Val‐Cys]‐His‐amide) (the relative coupling efficiency of recombinant HEK cells was cat>human≫rat UT receptor). 3 The present study further demonstrated that the use of high signal transduction/coupling efficiency isolated blood vessel assays (primate>cat arteries) is required in order to characterize UT receptor antagonism thoroughly. This cannot be attained simply by using the rat isolated aorta, an artery with low signal transduction/coupling efficiency in which low‐efficacy agonists appear to function as antagonists. 4 In contrast to the ‘low‐efficacy agonists’ urantide and SB‐710411, GSK248451 functioned as a potent UT receptor antagonist in all native isolated tissues studied (UT receptor selectivity was confirmed in the rat aorta). Further, GSK248451 exhibited an extremely low level of relative intrinsic activity in recombinant HEK cells (4–5‐fold less than seen with urantide). Since GSK248451 (1 mg kg−1, i.v.) blocked the systemic pressor actions of exogenous U‐II in the anaesthetized cat, it represents a suitable peptidic tool antagonist for delineating the role of U‐II in the aetiology of mammalian cardiometabolic diseases.

Chimeric G Proteins in Fluorimetric Calcium Assays: Experience with Opioid Receptors

High throughput calcium mobilization assays are extensively used for pharmacological characterization of GPCR ligands. These approaches, initially developed for G(q)-coupled receptors, can be extended to G(i) coupled GPCRs using chimeric G proteins. Here we used the Gα(qi5) protein to force the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, as well as the classical opioid receptors to signal through the PLC-IP(3)-Ca(2+) pathway in CHO cells. Calcium levels were monitored using the fluorometric imaging plate reader FlexStation II and the Ca(2+) dye Fluo 4 AM. For investigating the pharmacology of the NOP receptor a panel of full and partial agonists and antagonists were assessed, while a small panel of agonists and antagonists was used for evaluating the pharmacological profile of opioid receptors. Some limitations of this assay and differences in the results obtained in comparison with those with G(i) based biochemical assays are described. Overall, the present results confirm that the chimeric G protein strategy is useful for studying the pharmacological activity of G(i) coupled receptor ligands and that the aberrant signaling does not produce any measurable change in the pharmacological profile of the receptor under study. Thus, this G protein strategy is extremely useful for setting up primary screening assays for NOP and classical opioid receptors and likely for other members of the GPCR family.

In vitro and in vivo pharmacological characterization of the novel UT receptor ligand [Pen5,DTrp7,Dab8]urotensin II(4–11) (UFP‐803)

The novel urotensin‐II (U‐II) receptor (UT) ligand, [Pen5,DTrp7,Dab8]U‐II(4–11) (UFP‐803), was pharmacologically evaluated and compared with urantide in in vitro and in vivo assays. In the rat isolated aorta, UFP‐803 was inactive alone but, concentration dependently, displaced the contractile response to U‐II to the right, revealing a competitive type of antagonism and a pA2 value of 7.46. In the FLIPR [Ca2+]i assay, performed at room temperature in HEK293hUT and HEK293rUT cells, U‐II increased [Ca2+]i with pEC50 values of 8.11 and 8.48. Urantide and UFP‐803 were inactive as agonists, but antagonized the actions of U‐II by reducing, in a concentration‐dependent manner, the agonist maximal effects with apparent pKB values in the range of 8.45–9.05. In a separate series of experiments performed at 37°C using a cuvette‐based [Ca2+]i assay and CHOhUT cells, urantide mimicked the [Ca2+]i stimulatory effect of U‐II with an intrinsic activity (α) of 0.80, while UFP‐803 displayed a small (α=0.21) but consistent residual agonist activity. When the same experiments were repeated at 22°C (a temperature similar to that in FLIPR experiments), urantide displayed a very small intrinsic activity (α=0.11) and UFP‐803 was completely inactive as an agonist. In vivo in mice, UFP‐803 (10 nmol kg−1) antagonized U‐II (1 nmol kg−1)‐induced increase in plasma extravasation in various vascular beds, while being inactive alone. In conclusion, UFP‐803 is a potent UT receptor ligand which displays competitive/noncompetitive antagonist behavior depending on the assay. While UFP‐803 is less potent than urantide, it displayed reduced residual agonist activity and as such may be a useful pharmacological tool.

[Dmt1]N/OFQ(1–13)-NH2: a potent nociceptin/orphanin FQ and opioid receptor universal agonist

Intrathecally (i.t.) administered nociceptin/orphanin FQ (N/OFQ) evokes antinociceptive effects in rodents. Recent studies in monkeys demonstrated that i.t. co‐application of N/OFQ and morphine elicits synergistic antinociceptive actions suggesting mixed N/OFQ peptide (NOP) and μ opioid receptor agonists as innovative spinal analgesics. Thus, novel N/OFQ related peptides were synthesized in order to identify and pharmacologically characterize a mixed NOP/ μ opioid receptor agonist.

In vitro and in vivo pharmacological characterization of the novel NK(1) receptor selective antagonist Netupitant.

The novel NK(1) receptor ligand Netupitant has been characterized in vitro and in vivo. In calcium mobilization studies CHO cells expressing the human NK receptors responded to a panel of agonists with the expected order of potency. In CHO NK(1) cells Netupitant concentration-dependently antagonized the stimulatory effects of substance P (SP) showing insurmountable antagonism (pK(B) 8.87). In cells expressing NK(2) or NK(3) receptors Netupitant was inactive. In the guinea pig ileum Netupitant concentration-dependently depressed the maximal response to SP (pK(B) 7.85) and, in functional washout experiments, displayed persistent (up to 5h) antagonist effects. In mice the intrathecal injection of SP elicited the typical scratching, biting and licking response that was dose-dependently inhibited by Netupitant given intraperitoneally in the 1-10mg/kg dose range. In gerbils, foot tapping behavior evoked by the intracerebroventricular injection of a NK(1) agonist was dose-dependently counteracted by Netupitant given intraperitoneally (ID(50) 1.5mg/kg) or orally (ID(50) 0.5mg/kg). In time course experiments in gerbils Netupitant displayed long lasting effects. In all the assays Aprepitant elicited similar effects as Netupitant. These results suggest that Netupitant behaves as a brain penetrant, orally active, potent and selective NK(1) antagonist. Thus this molecule can be useful for investigating the NK(1) receptor role in the control of central and peripheral functions. Netupitant has clinical potential in conditions such as chemotherapy induced nausea and vomiting, in which the blockade of NK(1) receptors has been demonstrated valuable for patients.