Cationic host defense peptide

Study of the N-terminal part of peptidic selective NPFF2 agonists

Study of the N-terminal part of peptidic selective NPFF2 agonists

Short communication

Abstract

Neuropeptide FF (NPFF) has been shown to act as an endogenous anti-analgesic peptide. In this paper, several peptide analogs of the selective ligand dNP(NMe)AFLFQPQRF-NH 2 modified in the putative address segment, were designed to be selective NPFF 2 receptor probes, synthesized and assayed. One peptide dA(NMe)AAFLFQPQRF-NH 2 displays a very high affinity for NPFF 2 receptors transfected in CHO cells, and a high selectivity versus NPFF 1 receptors. The exact residues carried in the N-terminal part of the ligands are not decisive to obtain a high affinity only the length of the peptide in itself seems important to create selectivity.

Introduction

Neuropeptide FF (NPFF, FLFQPQRFamide) belongs to a neuropeptide system involved in the control of pain, cardiovascular functions, appetite, thirst, and body temperature but NPFF agonists are above all described as modulators of opioid activities [11]. Injection of NPFF analogs in rodents has been shown to modulate both analgesic and motivational properties of opioid drugs since in mice, i.c.v. injection of 1DMe, a stable NPFF analog, inhibits morphine induced analgesia [15] as well as the acquisition of place conditioning by morphine [6]. The NPFF system is therefore considered to be an opioid-modulating system involved in homeostasis that counteracts the action of opioids and, thus, an interesting therapeutic target for the management of opiate tolerance and dependence.

NPFF analogs act on two G-protein coupled receptors, NPFF 1 and NPFF 2 cloned in several species including human [9] and localized especially in the superficial layers of the spinal cord and in brain areas involved in pain perception [5]. There is some confusion in the literature concerning the name of NPFF peptides. Structurally, NPFF related peptides share a conserved C-terminal PQRFamide sequence and originate from two precursors: NPFF A and NPFF B (also called precursor of RFamide-related peptide). The precursor NPFF B could also produce a RFRP-1 peptide possessing a PLRFamide C-terminal sequence which is described as ortholog of avian gonadotropin-inhibitory hormone (GnIH). While peptides from both precursors bind with a nanomolar affinity to NPFF 1 and NPFF 2 receptors, peptides issued from NPFF B precursor are preferential ligand for GPR147 (NPFF 1 in the NPFF terminology). For the sake of homogeneity, we will adopt in this paper, the original nomenclature: NPFF 1 and NPFF 2 [1].

As demonstrated in a recombinant neuroblastoma clone SH-SY5Y, activation of NPFF 2 receptors inhibits the opioid modulation of Ca 2+ channels, and reproduces the cellular anti-opioid activity observed in isolated neurons [14]. This anti-opioid effect could result from an heterodimerization of NPFF and mu opioid receptors, accompanied with an heterologous phosphorylation of the mu opioid receptor [10] which could render account for an anti-opioid effect by regulating the number of stimulable opioid receptors.

In order to investigate the exact activity mediated by NPFF 2 receptors toward opioid functions, we have synthesized and screened various analogs of NPFF. We now describe the biochemical properties of these peptides and advance the idea that the residues of the N-terminal part of eleven amino-acid peptides interact with a low specificity to NPFF receptors.

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Section snippets

Chemicals

All peptides reported here were synthesized by standard solid phase peptide synthesis methods using an automated peptide synthesizer (Applied Biosystems model 433A) as previously described [17].

The peptides generally were purified to greater than 95% purity using semi-preparation reversed phase high performance liquid chromatography (HPLC). Purity of the peptides generally was assessed using analytical HPLC and high-resolution mass spectrometry.

[3 H]EYF (EYWSLAAPQRFamide) and [3 H]-NPVF

Affinity and selectivity selection of NPFF 1 and NPFF 2 receptors agonists

The ability of several synthetic peptides to displace the specific binding of [3 H]-NPVF and [3 H]-EYF on human NPFF 1 and NPFF 2 receptors, respectively, has been compared. The selectivity and the affinity for NPFF 1 and NPFF 2 receptors of the different ligands are presented in Table 1.

The major initial goal in this research was to obtain a selective peptide ligand for NPFF 2 receptor. In previous studies in our laboratory we had synthesized D.Asn-Pro-(N-Me)Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH 2

Discussion

Results previously obtained [4] overall showed that C-terminal amino-acids of NPFF would be essential for biological response while the N-terminal segments allow the formation of the appropriate conformation required for the interaction with the receptor. According to this message-address concept [4], [3], the C-terminal part of the molecule which includes the C-terminal amide function, an aromatic ring precisely oriented in an hydrophobic pocket and an oriented guanidino group, is the message

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Cited by (4)

Neuropharmacology (2017)
However, several studies have shown that neuropeptide FF, the endogenous ligand of NPFF2R, can display both pro- or anti-nociceptive actions depending on its site of administration i.t. (Gouarderes et al., 1993) versus i.c.v. (Journigan et al., 2014; Oberling et al., 1993). Thus, despite the recent design of several peptidic and non peptidic NPFF receptors ligands (Gaubert et al., 2009; Journigan et al., 2014; Lameh et al., 2010; Mazarguil et al., 2012), the development of highly selective ligands still represents an important challenge that should greatly help clarifying the respective contribution of NPFF1/2 receptor subtypes in the modulation of nociception. In this study, we also compared the in vitro binding and activity profiles of RF313 and RF9, a dipeptidic compound we identified previously as a NPFF receptor antagonist (Simonin et al., 2006).
Pharmacology and Therapeutics (2016)
NPFF per se exhibits a modest selectivity towards NPFF2 receptor (Table 2). As the C-terminus part was considered as crucial for affinity and functional potency, Mazarguil and coworkers (Mazarguil et al., 2012) introduced an alanine-based sequence at the N-terminus of NPFF to obtain the dA(NMe)AAFLFQPQRF-NH2 peptide that displayed a better NPFF2R selectivity (Ki values 0.16 nM and 12.1 nM at NPFF2R and NPFF1R, respectively) essentially due to a decrease in affinity at the NPFF1R subtype. A dipeptide derivative as small as dansyl-RF-amide was reported to suppress morphine-induced analgesia in vivo after peripheral administration, suggesting that the minimal Arg-Phe-NH2 sequence was essential for anti-opioid activity (Brussaard et al., 1989).