Selective Mode of Action of Guanidine-Containing Non-Peptides at Human NPFF Receptors

Abstract

The binding pocket of both NPFF receptors was investigated, focusing on subtype-selective behavior. By using four non-peptidic compounds and the peptide mimetics RF9 and BIBP3226 agonistic and antagonistic properties were characterized. A set of Ala receptor mutants was generated, the binding pocket was narrowed down to the upper part of transmembrane helices V, VI, VII, and the extracellular loop 2. Positions 5.27 and 6.59 have been shown to have a strong impact on receptor activation and were suggested to form an acidic, negatively charged binding pocket in both NPFF receptor subtypes. Additionally, position 7.35 was identified to play an important role in functional selectivity. According to docking experiments, the aryl group of AC-216 interacts with position 7.35 in the NPFF1 but not in the NPFF2 receptor. These results provide distinct insights into the receptor specific binding pockets, which is necessary for the development of drugs to address the NPFF system.

Anxiogenic and Stressor Effects of the Hypothalamic Neuropeptide RFRP-3 Are Overcome by the NPFFR Antagonist GJ14

“…RF9 displayed no NPFF1R antagonism at any concentration in both cAMP and calcium mobilization tests, therefore our in vitro experiments indicate that achieving NPFF1R antagonism using RF9 in vivo would not be possible. While RF9's agonism at NPFF1R was a surprising result, we note that Findeisen et al, (24) have reported similar findings with RF9. To date, publications with pharmacological data demonstrating the function of RF9 at NPFF1R has been limited to Simonin et al, (22) and Findeisen et al, (24).…”

“…While RF9's agonism at NPFF1R was a surprising result, we note that Findeisen et al, (24) have reported similar findings with RF9. To date, publications with pharmacological data demonstrating the function of RF9 at NPFF1R has been limited to Simonin et al, (22) and Findeisen et al, (24). Including our current work, there is now strong pharmacological evidence that RF9 is actually a weak, full …”

“…In part, the lack of a useful receptor antagonist has made it difficult to advance the understanding of this and other roles of RFRP-3. While RF9 is claimed to be a highly specific and potent NPFF receptor antagonist (22), there have been recent suggestions that RF9 has off-target actions (23) and may even be an agonist at NPFF1R (24,25). We describe here the identification of a highly specific and potent NPFF1R antagonist with moderate antagonism at NPFF2R, named GJ14 ( Figure 1).…”

RF9 Excitation of GnRH Neurons Is Dependent Upon Kiss1r in the Adult Male and Female Mouse

“…imentally. Additionally, we note that given reported Ki values (6,20), 10-to 100-fold higher concentrations of RF9 over RFRP-3 may be required to fully assess antagonistic effects of RF9 at Npff receptors. Third, RFRP-3 inhibits GnRH neurons through opening potassium channels (8) so application of RF9 to block on-going RFRP-3 transmission in the slice should result in the closure of potassium channels.…”

“…The original studies undertaken by Simonin et al (6) reported that RF9 up to 10M did not bind Kiss1r. However, recent studies have found different binding and kinetic properties of RF9 at Npff receptors (20), and there is increasing recognition that substantial crossover exists between the different RFamide ligands and receptors (21). Nevertheless, it remains unclear how RF9 may interact with Kiss1r.…”