Structure-Activity Studies On Different Modifications Of Nociceptin/Orphanin FQ And NOP Antagonist

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand of the opioid receptor-like 1 receptor (NOP), the G-protein coupled receptor which shows overall 60% homology with the classical opioid receptors. A number of studies have demonstrated N/OFQ/NOP system modulates a variety of biological functions. Further understandings of physiological and pathological roles of this system require new potent agonists and antagonists of its receptor.The past five years have witnessed tremendous advances in the design and discovery of very potent and selective peptide agonist and antagonist ligands at NOP. Several modifications in 'address' region of N/OFQ increase the potency of the agonist. Two series of N/OFQ related analogues were synthesized to investigate the relationship of these different modifications. We combined modifications including: (a) Phe⁴→(pF)Phe⁴;(b) Ala⁷,Ala¹¹â†’Aib⁷,Aib¹¹;(c) Leu¹⁴,Ala¹⁵→Arg¹⁴,Lys¹⁵. Compared with the first series, N-terminus of the second series was changed from Phe¹ to Nphe¹. All the analogues were amidated at C-terminus.These compounds were tested in binding studies on rat brain membranes and mouse vas deferens assay. the compounds of the first series showed higher affinity and potency than N/OFQ. The measured binding affinities (pKi) for peptides 2-5 ranged from 10.08 to 10.78. These peptides showed 6-²7-fold higher affinity than N/OFQ (pKi 9.33). In MVD assay the peptides 2-5 showed full NOP agonism which exhibited higher potency (pEC₅₀ 8.49⁹.37) than N/OFQ (pEC₅₀ 7.50). In particular, [(pF)Phe⁴,Aib⁷,Aib¹¹,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂ was found to be a highly potent agonist with pKi = 10.78 in binding studies on rat brain membranes and pEC₅₀ = 9.37 in MVD assay.Compounds of the second series (6-10) with exchange of Phe¹ for Nphe¹ showed antagonistic activity. In MVD assay, all these compounds competitively antagonized the effect of N/OFQ with pA₂ in the range of 7.14⁸.39. These peptides up to 10μM did not show any residual agonist activity in this preparation. In receptor binding assay, the pKi values of peptides 6-10 were 9.08⁹.99. Similarly, all of these peptides containing Nphe¹ exhibited lower affinities than those peptides of the first series and the values showed average of a 0.77 decrease in pKi. In addition, [Nphe¹,(pF)Phe⁴, Aib⁷,Aib¹¹,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂ was the best antagonist with pA₂ = 8.39 and showed high binding affinity with pKi = 9.99.Taken together the present data, indicate that [p(F)Phe⁴,Aib⁷,Aib¹¹,Arg¹⁴,Lys¹⁵] N/OFQ-NH₂ is the NOP receptor agonist more potent than the natural ligand N/OFQ in mouse colon assay. [Nphe¹,(pF)Phe⁴,Aib⁷,Aib¹¹,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂ was the antagonist in this assay.In mouse tail-withdrawal assay, comparison of the effects of [p(F)Phe ,Aib , Aibu,Arg14,Lys15]N/OFQ-NH2 with those elicited by N/OFQ under the same experimental conditions indicates that the former is more potent than the natural ligand and produces longer lasting. [Nphe1,(pF)Phe4,Aib7,Aibu,Argl4,Lys15]N/OFQ-NH2 produced a robust antinociceptive effect which was still present 60 min following i.c.v. administration of the compound.In cardiovascular system, [p(F)Phe4,Aib7,AibU,Arg14,Lys15]N/OFQ-NH2 could decrease the MAP of the anesthetized rat and more potent than N/OFQ. Although [Nphe',(pF)Phe4,Aib7,Aib11,ArgI4,Lys15]N/OFQ-NH2 had no effects on MAP, it could antagonize N/OFQ induced hypotension.A series of analogs of the NOP receptor antagonist [Nphe']N/OFQ(l-13)-NH2 was prepared and tested for agonistic and antagonistic activities in the mouse vas deferens and tested in binding studies on rat brain membranes. The purpose of this study was to determine the role of the aromatic residue at the N-terminal for antagonism and eventually identify compounds with improved potency. Results indicated that all 12 compounds are inactive as antagonists, The 12 compounds can be divided into two groups: 9 show weak agonistic potencies, whereas the other 3 compounds are inactive. [N(Bzl)Phe1]N/OFQ(l-13)-NH2 acts as the best agonist activities in all analogs(at lOuM causes -53%). This study clearly shows that the aromatic ring of Nphe is very critical for the interaction with the NOP receptor and can not be enlarged or sterically modified without significant loss of antagonistic potency. We speculated that benzyl of Nphe1 is likely to be oriented toward a gap of TM3 and TM7 and produces an effect on NOP which possibly is similar to naloxone, whereas R groups of a-carbon of N-terminal residue in N/OFQ is crucial for receptor occupation and activation.Furthermore, [Aib7,Aibn]N/OFQ-NH2 was tested in mouse colon and tail-withdrawal assay. In these assay, [Aib7,Aib"]N/OFQ-NH2 is the NOP agonist more potent than the natural ligand N/OFQ. In cardiovascular system, [Aib7,Aib"]N/OFQ-NH2 could decrease the MAP of the anesthetized rat more potent than N/OFQ.In conclusion, the present investigation we explore the relationship of combinations of these different modifications and find their contributions to binding affinity and biological activity. Our results exhibit that modifications which increase the potency of agonist have synergistic effect on biological activity and a replacement of N-terminus leads to shift of analogues from agonist to antagonist. Moreover we report several peptides which behave as highly potent agonists or antagonists of NOP. [(pF)Phe4,Aib7,Aibn,Arg14,Lys15]N/OFQ-NH2 is the NOP agonist more potent than the natural ligand N/OFQ and [Nphe1,(pF)Phe4,Aib7,AibH,Arg14,Lys15]N/OFQ-NH2 is one of the most potent antagonists for NOP. These analogues can be considered novelpharmacological tools for the investigation of the neurobiology of the N/OFQ/NOP system.