The anorexigenic neuropeptide prolactin-releasing peptide (PrRP) is involved in the regulation of food intake and energy expenditure. Lipidization of PrRP stabilizes the peptide, facilitates central effect after peripheral administration and increases its affinity for its receptor, GPR10, and for the neuropeptide FF (NPFF) receptor NPFF-R2. The two most potent palmitoylated analogs with anorectic effects in mice, palm 11-PrRP31 and palm-PrRP31, were studied in vitro to determine their agonist/antagonist properties and mechanism of action on GPR10, NPFF-R2 and other potential off-target receptors related to energy homeostasis. Palmitoylation of both PrRP31 analogs increased the binding properties of PrRP31 to anorexigenic receptors GPR10 and NPFF-R2 and resulted in a high affinity for another NPFF receptor, NPFF-R1. Moreover, in CHO-K1 cells expressing GPR10, NPFF-R2 or NPFF-R1, palm 11-PrRP and palm-PrRP significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK), protein kinase B (Akt) and cAMP-responsive element-binding protein (CREB). Palm 11-PrRP31, unlike palm-PrRP31, did not activate either c-Jun N-terminal kinase (JNK), p38, c-Jun, c-Fos or CREB pathways in cells expressing NPFF-1R. Palm-PrRP31 also has higher binding affinities for off-target receptors, namely, the ghrelin, opioid (KOR, MOR, DOR and OPR-L1) and neuropeptide Y (Y 1, Y 2 and Y 5) receptors. Palm 11-PrRP31 exhibited fewer off-target activities; therefore, it has a higher potential to be used as an anti-obesity drug with anorectic effects.
Prolactin-releasing peptide (PrRP) was discovered as an endogenous ligand of the orphan G-protein coupled receptor GPR10 (also known as hGR3) in the hypothalamus and has been suggested to stimulate prolactin secretion. However, soon after this finding, Lawrence et al. showed a reduction in food intake and body weight and an increase in energy expenditure after intracerebroventricular (ICV) PrRP injection in rats and questioned the role of PrRP in prolactin secretion. The effects of PrRP, mostly mediated through the GPR10 receptor, which is widely expressed throughout the brain mainly in areas related to the regulation of food intake and energy homeostasis, confirm GPR10 knockout (KO) mouse studies showing an increase in body weight in KO mice.
PrRP occurs in two biologically active isoforms, PrRP31 and PrRP20. Our previous studies showed the induction of central c-Fos activation of regions related to food intake after peripheral administration of PrRP31 or PrRP20 modified with either myristoyl or palmitoyl, but this central effect was not observed after peripheral administration of natural PrRP31 or PrRP20. Lipidized PrRP31 and PrRP20 analogs decrease food intake and body weight in mice, increase stability and prolong half-life compared to natural peptides. PrRP20 and PrRP31 also strongly interact with the receptor of neuropeptide FF (NPFF), NPFF-R2. Lipidization of PrRP20 and PrRP31 increases in vitro binding affinities not only to GPR10 but also to NPFF-R2. However, lipidized PrRP20 showed lower solubility and bioavailability; therefore, our further studies were focused on lipidized PrRP31 analogs.
PrRP, together with NPFF, belongs to the RF-amide peptide family, which contains a typical C-terminal amino acid sequence motif (RF-NH 2) essential for receptor activation. All RF-amide peptides have a high affinity for and activity on both NPFF receptors NPFF-R2 and NPFF-R1 and may also exert in vivo effects through these receptors. Expression of both NPFF receptors has been found in hypothalamic areas that regulate feeding and energy homeostasis. Moreover, the ability of NPFF to regulate food intake was previously demonstrated, when ICV administration of NPFF was shown to result in decreased food intake in fasted rats.
Both NPFF receptors show the ability to regulate the cardiovascular system and modulate pain perceptions. Despite the fact that antagonist of NPFF-R1 and NPFF-R2 RF9 prevents opioid-induced hyperalgesia and that NPFF induces an increase in arterial blood pressure in rats, our previous study did not prove the antagonistic activity of RF9 on NPFF-induced anorexigenic effects. Conversely, RF9 exhibits an anorectic effect after ICV or subcutaneous administration in fasted mice.
Similar to NPFF, PrRP also appears to have antinociceptive properties. Although PrRP has a high affinity for NPFF receptors, its ability to modulate pain perception through NPFF-1R and NPFF-2R has not been proven. Kalliomäki et al. studied the nociceptive properties of 1DMe, a stable NPFF analog, and PrRP in the central nervous system of rats and refuted the ability of PrRPs to regulate pain perception through NPFF receptors.
Many G-protein coupled receptors (GPCRs) share similar characteristic features. Receptors GPR10, NPFF-R1 and NPFF-R2 are members of the β-type rhodopsin GPCR family, which has important roles in the regulation of food intake and energy homeostasis. GPR10 has a high percentage of amino sequence identity, especially in the transmembrane regions, with neuropeptide Y receptors, members of the β-type rhodopsin GPCR family, which are involved in food intake regulation. Furthermore, Y receptors share a high percentage of amino sequence homology with NPFF-R1 and NPFF-R2.
The mechanism of action of PrRP is not yet fully understood. PrRP31 and PrRP20 have been shown to mobilize Ca 2+ from intracellular stores via GPR10 by activating the second messenger IP3 (inositol-1,4,5-trisphosphate), leading to an increase in cytoplasmic Ca 2+, which can subsequently activate the extracellular signal-regulated kinase (ERK) signaling cascade. PrRPs displayed the ability to activate the phosphorylation of ERK, the c-Jun N-terminal kinase (JNK) pathway, the cAMP-responsive element binding protein (CREB) pathway and the protein kinase B (Akt/PKB) pathway, which plays a key role in the regulation of protein synthesis and maintenance of glucose homeostasis.
Maixnerová et al. previously showed that the first 20 amino acids of PrRP31 are important for the preservation of full in vivo activity. This study compares the activity of two most potent PrRP31 analogs, palm 11-PrRP31 and palm-PrRP31, which contain palmitic acid attached to the N-terminus of the amino acid chain (palm-PrRP31) or to the position 11, where original Arg 11 was replaced with Lys 11 (palm 11-PrRP31). These analogs previously showed the ability to significantly decrease food intake and body weight after repeated peripheral administration, but the mechanism of action is still unclear. We aimed to identify the off-target activity of palm 11-PrRP31 and palm-PrRP31 to map the mechanism of action and to compare intracellular transduction pathways of anorexigenic receptors GPR10, NPFF-R2, and new strong target of PrRP31 analogs, NPFF-R1. GPR10 is a highly selective receptor for PrRP31 and analogs related to PrRP31. To control the selectivity of PrRP31 for GPR10s, we used NPFF and its stable analog 1DMe in this study. To determine whether the possible analgesic effect of PrRPs is caused by off-target activity, opioid receptors were investigated.
Based on previously published data, affinity for the GPR10 and NPFF-R2 of PrRP31 and its analogs was studied. PrRP31 and its two palmitoylated analogs of PrRP31 have a high binding affinity for the GPR10 and NPFF-R2 receptors, and their K i values were in the nanomolar range. Compared to natural PrRP31, palmitoylated analogs had a higher binding affinity for both of these receptors. Palm 11-PrRP31 showed a higher affinity for the receptor GPR10 than for the receptor NPFF-R2. NPFF and its stable analog 1DMe displayed negligible affinity for the GPR10 receptor. The affinities of NPFF and 1DMe to NPFF-R2 were detected to be in the nanomolar range.
To find another possible target of the two most potent palmitoylated analogs of PrRP31, binding to NPFF-R1 was tested. Membranes from CHO-K1 cells expressing the NPFF-R1 were isolated, and the K d was determined to be 0.94 ± 0.06 nM by saturation experiments using the radioligand [125 I]-1DMe. Although natural PrRP31 bound to NPFF-R1 with a lower affinity than to NPFF-R2, the binding affinity was still in the 10−8 M range. Palmitoylation increased the binding affinities of both analogs to NPFF-R1. Palm-PrRP31 showed binding affinities in the nanomolar range to both NPFF receptors compared to palm 11-PrRP31.
Several other potential off-target receptors of PrRP31 and its palmitoylated analogs were tested. The binding properties of PrRP31, palm 11-PrRP and palm-PrRP31 to receptors Y 1, Y 2, and Y 5, ghrelin receptor (also growth hormone secretagogue receptor—GHSR) and kappa-opioid receptor (KOR) were determined. The natural ligand PYY of Y receptors bound in the nanomolar range to the Y 1, Y 2, and Y 5 receptors. From saturation binding experiments with [125 I]-PYY as a radioligand, the K d for each receptor was determined. The K d for Y 1 was 1.53 ± 0.08 nM, for Y 2 was 2.18 ± 0.85 nM and for Y 5 was 1.01 ± 0.27 nM. Natural PrRP31 had no affinity to the Y 1 and Y 2 receptors in the range of measured concentrations, but it showed a very low affinity to the Y 5 receptor. Compared to palm 11-PrRP31, palm-PrRP31 exhibited a relatively high affinity for the Y 5 receptor. Both palmitoylated analogs bound to Y 1 and Y 2 with a negligible low affinity.
