RF-313

Although opiates represent the most effective analgesics, their use in chronic treatments is associated with numerous side effects including the development of pain hypersensitivity and analgesic tolerance. We recently identified a novel orally active neuropeptide FF (NPFF) receptor antagonist, RF313, which efficiently prevents the development of fentanyl-induced hyperalgesia in rats. In this study, we investigated the properties of this compound into more details. We show that RF313 exhibited a pronounced selectivity for NPFF receptors, antagonist activity at NPFF1 receptor (NPFF1R) subtype both in vitro and in vivo and no major side effects when administered in mice up to 30 mg/kg. When co-administered with opiates in rats and mice, it improved their analgesic efficacy and prevented the development of long lasting opioid-induced hyperalgesia. Moreover, and in marked contrast with the dipeptidic NPFF receptor antagonist RF9, RF313 displayed negligible affinity and no agonist activity (up to 100 μM) toward the kisspeptin receptor. Finally, in male hamster, RF313 had no effect when administered alone but fully blocked the increase in LH induced by RFRP-3, while RF9 per se induced a significant increase in LH levels which is consistent with its ability to activate kisspeptin receptors. Altogether, our data indicate that RF313 represents an interesting compound for the development of therapeutic tools aiming at improving analgesic action of opiates and reducing adverse side effects associated with their chronic administration. Moreover, its lack of agonist activity at the kisspeptin receptor indicates that RF313 might be considered a better pharmacological tool, when compared to RF9, to examine the regulatory roles of RF-amide-related peptides and NPFF1R in reproduction.

GnIH receptors (GnIHRs) belong to the family of G-protein coupled receptors (GPCRs) and play a key role in the regulation of reproduction from fishes to mammals, either by inhibiting or stimulating the expression of gonadotropins. The aim of this study was to characterize GnIH receptor (GnIHR2) from Indian Major Carp, Labeo catla and its docking and simulation with GnIH antagonist RF313.

The full length sequence of GnIHR2 was obtained with RACE PCR. The docking analysis of RF313 with GnIHR2 receptor was performed with AutoDock v. 4.2.6 and molecular dynamics (MD) simulation with GROMACS 5.0.

In the present study, we cloned full-length cDNA (1733 bp) of GnIHR2 from the brain of L. catla. The phylogenetic analysis showed clustering of catla GnIHR2 with goldfish and zebrafish in the GPR147 group. L. catla GnIHR2 receptor comprised seven transmembrane domains and the 3D-structure was predicted by I-TASSER tool. The docking analysis revealed high binding affinity (-11.6 kcal/mol) of GnIH antagonist, RF313 towards GnIHR2 receptor. The primary bonds involved were alkyl and hydrogen bonds while the amino acids participated were proline 43, 210, 339, cysteine 214, leucine 211, serine 213 and phenylalanine 338. The MD simulation analysis of docked complex for 100 nano-seconds (ns) in the lipid membrane environment showed the stability of the complex with time.

Our study showed that GnIH antagonist, RF313 interact tightly with the GnIH receptor, GnIHR2 of L. catla. To the best of our knowledge, this is the first report on computational modelling and MD simulation of GnIH receptor in fishes. This will help in functional characterization studies of GnIH/GnIHR system in vertebrates.