N-methylthioacetylation of RYYRIK-NH2 with enhanced specific binding affinity and high antagonist activity for nociceptin ORL1 receptor

Antagonists of the neuropeptide nociceptin are expected to be potential analgesic and antineuropathic drugs acting on ORL1 GPCR receptors. The peptide library-based antagonist Ac-RYYRIK-NH₂ inhibits the nociceptin activity mediated through ORL1, but preserves a considerably high level of agonist activity. We previously reported that the N-terminal acyl group is important for interaction with specific receptors, and developed isovarelyl-RYYRIK-NH₂, which exhibits strong antagonist activity with negligible agonist activity. In the present study, in order to obtain a more potent antagonist, we further modified the isovarelyl group by replacing its Cβ atom with an oxygen, nitrogen, or sulfur atom to give the methyl group improved interaction ability. The methyl group bound to such heteroatoms was expected to enhance the hydrophobic interaction between the peptide and the ORL1 receptor. The RYYRIK-NH₂ peptide with a methylthioacetyl group, CH₃SCH₂CO, revealed a higher receptor-binding affinity with strong antagonist activity, and the results suggested the presence of a receptor aromatic group as a complementary residue of this CH₃S group.     

Capturing of the free cysteine residue in the ligand-binding site by affinity labeling of the ORL1 nociceptin receptor

All of the δ, μ, and κ opioid receptors have a free thiol group of the Cys residue in the ligand-binding site, although its functional role is not yet known. In order to examine whether or not a similar Cys is also present in the ORL1 nociceptin receptor, we attempted to identify it by affinity labeling using a specific antagonist peptide. We first treated ORL1-expressing COS-7 cell membrane preparations with the thiol-alkylation reagent N-ethylmaleimide (NEM) to perform a binding assay using [³H]nociceptin as a tracer and nociceptin, an ORL1 agonist, or Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH₂, a nociceptin/ORL1 antagonist, as a competitor. It was suggested that ORL1 has a free Cys in its ligand-binding site, since the NEM treatment reduced the population of ligand-binding sites. This was further confirmed by affinity labeling using Cys(Npys)-Arg-Tyr-Tyr-Arg-Ile-Lys-NH₂ with the SNpys group that can react with a free thiol group, resulting in the formation of a disulfide bond. This affinity labeling was approximately 23 times more specific than NEM alkylation. The results revealed that the ORL1 nociceptin receptor does contain a free Cys residue in the ligand-binding site.     

N-terminal modifications leading to peptide ORL1 partial agonists and antagonists.

Nociceptin/Orphanin FQ (N/OFQ) is a 17 amino acid peptide that is the endogenous ligand for the G protein-coupled receptor (opioid receptor like 1, ORL1), a member of the opioid receptor family. Although it is clear that this receptor system is involved in a variety of physiological functions, including analgesia, the precise actions of N/OFQ remain largely uncharacterized. One reason for this has been limited high affinity ligands to ORL1, and particularly the lack of availability of useful specific antagonists. Herein we describe the pharmacological activity of a series of N-terminally modified hexapeptides with high affinity for ORL1. These compounds were tested for binding affinity using [3H]N/OFQ binding to human ORL1 in CHO cells, and functional activity by measuring stimulation of [35S]GTPgammaS binding in CHO cell membranes. The N-terminal modifications have produced compounds that maintained very high receptor affinity, but led to significant changes in intrinsic activity. One compound, pentanoyl-RYYRWR-NH2, with barely measurable agonist activity was tested in vivo. It was found to possess modest analgesic activity, but it was unable to block the morphine modulatory activity of N/OFQ.     

Peptide and nonpeptide ligands for the nociceptin/orphanin FQ receptor ORL1: research tools and potential therapeutic agents

The 17-amino acid neuropeptide nociceptin/Orphanin FQ (N/OFQ) was recently identified as the endogenous ligand for the opioid receptor-like (ORL1) receptor, a fourth member of the classical mu, delta, and kappa opioid receptor family. Although ORL1 clearly belongs to the opioid receptor family, it does not bind classical opiates and the ORL1-N/OFQ system has pharmacological actions distinct from the opioid receptor system. This new ligand-receptor system has generated active interest in the opioid community because of its wide distribution and involvement in a myriad of neurological pathways. The past two years have witnessed tremendous advances in the design and discovery of very potent and selective peptide and nonpeptide agonist and antagonist ligands at ORL1. These discoveries have facilitated the understanding of the role of the ORL1-N/OFQ system in a variety of processes such as pain modulation, anxiety, food intake, learning, memory, neurotransmitter release, reward pathways, and tolerance development. The ORL1 receptor therefore represents a new molecular target for the design of novel agents for anxiety, analgesia, and drug addiction. Indeed, there is tremendous interest in the pharmaceutical industry in the development of nonpeptide ligands such as the potent ORL1 agonist, Ro 64-6198, as anxiolytics and the ORL1 antagonist JTC-801 as novel analgesics. This review presents an overview of the various peptide and nonpeptide ORL1 ligands with an emphasis on their potential therapeutic utility in various human disorders.     

Designed modification of partial agonist of ORL1 nociceptin receptor for conversion into highly potent antagonist

Nociceptin is an endogenous agonist ligand of the ORL1 (opioid receptor-like 1) receptor, and its antagonist is a potential target of therapeutics for analgesic and antineuropathy drugs. Ac-RYYRIK-NH(2) is a hexapeptide isolated from the peptide library as an antagonist that inhibits the nociceptin activities mediated through ORL1. However, the structural elements required for this antagonist activity are still indeterminate. In the present study, we evaluated the importance of the acetyl-methyl group in receptor binding and activation, examining the peptides acyl-RYYRIK-NH(2), where acyl (R-CO) possesses a series of alkyl groups, R=C(n)H(2n+1) (n=0-5). The isovaleryl derivative with the C(4)H(9) (=(CH(3))(2)CHCH(2)-) group was found to reveal a high receptor-binding affinity and a strong antagonist nature. This peptide achieved a primary goal of eliminating the agonist activity of Ac-RYYRIK-NH(2) and producing pure antagonist activity.     

In vitro binding and functional studies of Ac-RYYRIK-ol and its derivatives, novel partial agonists of the nociceptin/orphanin F/Q receptor

Following the discovery of nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) and its endogenous ligand, an extensive search has started to find selective agonists and antagonists targeting this novel receptor-ligand system due to their therapeutic potentials. By the help of the combinatorial chemistry a series of hexapeptides with a general formula of Ac-RYY-R/K-W/I-R/K-NH(2) having high NOP receptor affinity and selectivity were identified. On the basis of this information we developed a number of novel compounds. The detailed structure-activity studies on the partial agonist Ac-RYYRIK-NH(2) are reported in this communication. Besides the modifications on N- and C-terminal, Arg-Cit exchange was performed on the template structure. The novel hexapeptides were analyzed in radioligand binding, functional biochemical [(35)S]GTPgammaS binding assays by using membranes from rat brains and Chinese hamster ovary cells expressing human NOP receptor. The agonist/antagonist properties were also tested on in the mouse vas deferens bioassay. C-terminal modification yielded a high affinity, selective and potent NOP ligand (Ac-RYYRIK-ol) with a partial agonist property. Several analogs of this compound were synthesized. The presence of the positively charged arginine residue at the first position turned out to be crucial for the biological activity of the hexapeptide. The N-terminal modifications with various acyl groups (ClAc, pivaloyl, formyl, benzoyl, mesyl) decreased the affinity of the ligand towards the receptor and the intrinsic activity for stimulating the G-protein activation was also decreased. The structure-activity studies on the hexapeptide derivatives provided some basic information on the structural requirements for receptor binding and activation.     

Structural requirements of nociceptin antagonist Ac-RYYRIK-NH2 for receptor binding.

Ac-RYYRIK-NH2 is a peptide isolated from the peptide library as an antagonist that inhibits the biological activities of nociceptin, a hyperalgesic neuropeptide. In order to clarify the structural requirements of this peptide for binding to the nociceptin receptor ORL1, systematic structure-activity studies were carried out. The result of Ala-scanning indicated that the N-terminal tripeptide RYY(= Arg-Tyr-Tyr) is crucially important for binding to the ORL1 receptor. Residual truncations from the N- or C-terminus revealed the special importance of the N-terminal Arg residue. The removal of protecting groups indicated that the N-terminal acetyl group is essential, but the C-terminal amide group is insignificant. These results indicated the conspicuous importance of acetyl-Arg at position 1 of Ac-RYYRIK-NH2 as a key structure allowing binding to the receptor. To investigate the binding site of this peptide in the ORL1 receptor, we synthesized and assayed a series of analogues of the nociceptin dibasic repeat region, residues 8-13 of RKSARK. None of the derivatives were active. Ac-RYYRIK-NH2 was inactive for the mu opioid receptor to which nociceptin binds with considerable strength. All the results suggested that the mode of binding between Ac-RYYRIK-NH2 and the ORL1 receptor is different to that between the ORL1 receptor and nociceptin, and that it may consist of interaction with the receptor site to which nociceptin(1-7) or -(14-17) binds.     

Ligands for kappa-opioid and ORL1 receptors identified from a conformationally constrained peptide combinatorial library.

We have screened a synthetic peptide combinatorial library composed of 2 x 10(7) beta-turn-constrained peptides in binding assays on four structurally related receptors, the human opioid receptors mu, delta, and kappa and the opioid receptor-like ORL1. Sixty-six individual peptides were synthesized from the primary screening and tested in the four receptor binding assays. Three peptides composed essentially of unnatural amino acids were found to show high affinity for human kappa-opioid receptor. Investigation of their activity in agonist-promoted stimulation of [(35)S]guanosine 5'-3-O-(thio)triphosphate binding assay revealed that we have identified the first inverse agonist as well as peptidic antagonists for kappa-receptors. To fine-tune the potency and selectivity of these kappa-peptides we replaced their turn-forming template by other turn mimetic molecules. This "turn-scan" process allowed the discovery of compounds with modified selectivity and activity profiles. One peptide displayed comparable affinity and partial agonist activity toward all four receptors. Interestingly, another peptide showed selectivity for the ORL1 receptor and displayed antagonist activity at ORL1 and agonist activity at opioid receptors. In conclusion, we have identified peptides that represent an entirely new class of ligands for opioid and ORL1 receptors and exhibit novel pharmacological activity. This study demonstrates that conformationally constrained peptide combinatorial libraries are a rich source of ligands that are more suitable for the design of nonpeptidal drugs.     

Pharmacological characterization of the nociceptin receptor, ORL1

A novel opioid receptor-like orphan receptor (ORL1) was cloned and identified to be homologous to classical opioid receptors but insensitive to traditional opioids. A heptadecapeptide, termed orphanin FQ or nociceptin (OFQ/N), was identified as its endogenous ligand. OFQ/N shares overlapping distribution sites in pain-processing areas and common cellular mechanisms with opioids but exerts diverse effects on nociceptive responses. Of the two reported ORL1 antagonists, [Phe(1)psi(CH(2)-NH)- Gly(2)] nociceptin-(1-13)-NH(2) (Phepsi) and naloxone benzoylhydrazone (NBZ), antagonisms were validated in the activation of inward rectifying K channels induced by OFQ/N, using the patch clamp technique in ventrolateral periaqueductal gray slices. Results showed that Phepsi acted as a partial agonist and NBZ was a weak nonselective antagonist of ORL1. It is comparable with most but not all of the findings from other tissues. Comparing all the reports supports the above inference for these two antagonists. The possible causes for the discrepancy were discussed. A brief review on the putative ORL1 antagonists, acetyl-RYYRIK-NH2, some sigma-ligands and the functional antagonist, nocistatin, is also included. It indicates that a potent and selective ORL1 antagonist is expecting to elucidate the physiological role of OFQ/N.     

Spare interactions of highly potent [Arg14,Lys15]nociceptin for cooperative induction of ORL1 receptor activation

[Arg¹⁴,Lys¹⁵]Nociceptin is a very potent for ORL1 receptor, showing a few times stronger binding activity and much more enhanced biological activity than endogenous nociceptin. This synergistic outcome has been suggested to be due to the interaction with the receptor aromatic and/or acidic amino acid residues crucial to receptor activation. In order to identify such receptor residues in the second ORL1 extracellular loop, we prepared a series of recombinant mutant receptors. The mutant receptor Gln205Ala was found to be as active as wild-type ORL1 for both nociceptin and [Arg¹⁴,Lys¹⁵]nociceptin. In contrast, Asp206Ala and Tyr207Ala exhibited considerably reduced activity for [Arg¹⁴,Lys¹⁵]nociceptin, exhibiting no synergistic activity enhancement. These results suggest that Asp206 and Tyr207 are directly involved in the interaction with nociceptin-[Arg¹⁴,Lys¹⁵]. Trp208Ala was found to bind strongly both nociceptin and [Arg¹⁴,Lys¹⁵]nociceptin, although it elicited no biological activity. All these results indicate that the consecutive amino acid residues Asp206, Tyr207, and Trp208 are critical to the activation of the ORL1 receptor, but not to nociceptin-binding.     

Inhibitory activity of nociceptin/orphanin FQ on capsaicin-induced bronchoconstriction in the guinea-pig

In vivo studies were conducted in the guinea-pig to investigate the activity of the selective ORL1 receptor agonist nociceptin/orphanin FQ against capsaicin-induced bronchoconstriction, a response mediated by the release of tachykinins from pulmonary sensory nerves. Anesthetized guinea-pigs were ventilated with a rodent ventilator and placed in a whole-body plethysmograph, and pulmonary resistance (R(L)) and dynamic lung compliance (C(Dyn)) were monitored. Intravenous administration of nociceptin/orphanin FQ (0.3 mg/kg) inhibited the capsaicin-induced bronchoconstriction. The new nonpeptide ORL1 receptor antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397) administered intravenously (1 mg/kg) produced a significant blockade of the inhibitory effect of nociceptin/orphanin FQ (0.3 mg/kg) on capsaicin-induced bronchoconstriction, whereas the nonselective opioid receptor antagonist naloxone (1 mg/kg) had no effect. Nociceptin/orphanin FQ (0.3 mg/kg) did not affect the bronchoconstriction induced exogenously by the tachykinin NK2 receptor agonist [beta-ala8]-neurokinin A (4-10). We conclude that nociceptin inhibits in vivo capsaicin-evoked tachykinin release from sensory nerve terminals in the guinea-pig by a prejunctional mechanism. This inhibitory action does not involve activation of opioid receptors.     

Designing bifunctional NOP receptor–mu opioid receptor ligands from NOP receptor-selective scaffolds. Part I.

The nociceptin receptor (NOP) and its endogenous agonist, nociceptin/orphanin FQ (N/OFQ), members of the opioid receptor and peptide families respectively, modulate the pharmacological effects of classical opioids, particularly opioid-induced reward and nociception. We hypothesized that compounds containing both NOP and opioid receptor activity in a single molecule may have useful pharmacological profiles as non-addicting analgesics or as drug abuse medications. We report here our forays into the structure–activity relationships for discovering ‘bifunctional’ NOP–mu opioid receptor (MOP) ligands, starting from our NOP-selective scaffolds. This initial SAR suggests pharmacophoric elements that may be modified to modulate/increase opioid affinity, while maintaining high affinity for the NOP receptor, to result in potent bifunctional small-molecule NOP/MOP ligands.     

Synergistic effect of basic residues at positions 14-15 of nociceptin on binding affinity and receptor activation

Nociceptin is an endogenous ligand that activates a G protein-coupled receptor ORL1 and contains two indispensable Arg-Lys (RK) dipeptide units at positions 8-9 and 12-13. By replacing an additional RK unit at positions 6-7, 10-11, 14-15, or 16-17, of the peptide we have identified the analog, [RK(14-15)]nociceptin as a superagonist. In fact, this peptide exhibits 3-fold higher binding affinity and 17-fold greater potency in a functional GTPgammaS-binding assay compared to wild-type nociceptin. Here, we have further investigated the role of basic residues in position 14-15. The replacement of three other possible basic dipeptides, KR, RR, and KK, into nociceptin at positions 14-15 resulted in similar enhancements of binding affinity (3-5-fold) and biological potency (10-12-fold in the GTPgammaS assay). However, when only a single basic residue (Arg or Lys) was replaced in either position 14 or 15, all the resulting analogs showed moderate enhancements of binding and biological activity (2-4-fold in both). These results indicate that the addition of basic charges in positions 14 and 15 enhance in a synergistic fashion the interaction of nociceptin with the receptor and only the simultaneous presence of two adjacent basic residues yields an optimal effect. This suggests that specific electrostatic interactions between both amino acids present in 14-15 and corresponding residues in the receptor are responsible for the enhancement of nociceptin activity.     

Effects of intraplantar injections of nociceptin and its N-terminal fragments on nociceptive and desensitized responses induced by capsaicin in mice

Injection of capsaicin into the hindpaw has been employed as a model of chemogenic nociception in mice. Intraplantar injection of nociceptin (30–240 pmol) produced a significant and dose-dependent antinociceptive activity in the capsaicin test. The nociceptin N-terminal fragments, (1–11) and (1–13), were also active with a potency higher than nociceptin and comparable to nociceptin, respectively. Intraplantar injection of the nociceptin (1–7) fragment had no effect on capsaicin-induced nociception. Antinociception induced by nociceptin or nociceptin (1–13) was reversed significantly by intraplantar co-injection of [Nphe¹]nociceptin (1–13)NH₂, an orphan opioid receptor-like 1 (ORL1) receptor antagonist, whereas local injection of the antagonist did not interfere with the action of nociceptin (1–11). Nociceptin (1–11) was approximately 2.0-fold more potent than naturally occurring peptide nociceptin, and 10-fold more active than intraplantar morphine. Nociceptive licking/biting response to intraplantar injection of capsaicin was desensitized by repeated injections of capsaicin at the interval of 15 min. Desensitization induced by capsaicin was attenuated significantly by co-injection of nociceptin at much lower doses than antinociceptive ED₅₀ for nociceptin. Capsaicin desensitization was also decreased by co-injection of nociceptin (1–11) and (1–13) to a similar extent. The present results indicate that not only nociceptin but also the N-terminal fragment (1–13) possesses a local peripheral antinociceptive action, which may be mediated by peripheral ORL1 receptors. In addition, the difference of the effective doses suggests that the antinociceptive action and inhibition of capsaicin-induced desenitization by nociceptin, nociceptin (1–11) and (1–13), may involve distinct mechanisms at the level of the peripheral nerve terminal.     

Direct identification of a peptide binding region in the opioid receptor-like 1 receptor by photoaffinity labeling with [Bpa(10),Tyr(14)]nociceptin

The heptadecapeptide nociceptin, also known as orphanin FQ, is the endogenous agonist of the opioid receptor-like 1 (ORL1) G protein-coupled receptor. An affinity labeling approach has been implemented to probe the interactions of the neuropeptide with the receptor using the photolabile nociceptin derivative, [p-benzoyl-l-Phe(10),Tyr(14)]nociceptin ([Bpa(10),Tyr(14)]noc). In recombinant Chinese hamster ovary cells expressing the human ORL1 receptor, [Bpa(10),Tyr(14)]noc binds the receptor with high affinity (K(i) approximately 0.7 nm) and is as potent as nociceptin in the inhibition of forskolin-induced cAMP synthesis (EC(50) approximately 0.5 nm). UV irradiation at 365 nm of the complex formed by the ORL1 receptor and radioiodinated [Bpa(10),Tyr(14)]noc results in the irreversible labeling of a glycoprotein of approximately 65 kDa, determined by SDS-polyacrylamide gel electrophoresis. Complete digestion of the partially purified 65-kDa complex with kallikrein generates a single labeled fragment (approximately 6.5 kDa) that is readily cleaved by endoproteinase Glu-C to yield a labeled fragment of approximately 3.2 kDa. Kallikrein treatment of the photoaffinity cross-linked Glu(295) --> Asp mutant receptor also yields a single labeled fragment of approximately 6.5 kDa but is resistant to further cleavage by endoproteinase Glu-C. Based upon the expected proteolytic fingerprint of the labeled receptor, the photoreactive region can be identified as ORL1-(296-302; residues Thr-Ala-Val-Ala-Ile-Leu-Arg) spanning the C terminus of extracellular loop 3 and the N terminus of transmembrane helix VII. Molecular modeling of the ORL1 receptor complex with [Bpa(10)]noc suggests that reaction of the Bpa carbonyl group may occur with the side chain of Ile(300) within the experimentally identified photoreactive region.     

Synthesis of enzymatically resistant nociceptin-related peptides containing a carbamic acid residue.

Nociceptin, a 17-amino acid peptide (FGGFTGARKSARKLANQ, N/OFQ), is the endogenous ligand of the nociceptin/orphanin FQ (NOP) receptor. This receptor-ligand system is involved in various physiological as well as pathophysiological mechanisms, but owing to the peptidic structure, it is rapidly degraded by enzymes. The enzymatic digestion of nociceptin involves mainly aminopeptidases and yields Noc(2-17)-OH and other smaller fragments. We aimed at increasing the enzymatic stability against aminopeptidases in the case of peptide Noc(1-13)-NH(2), which possesses the minimum sequence capable of interacting with the NOP receptor. Therefore we developed a new procedure for the synthesis of peptides with the carbamic acid residue [...-NH-CH(R)-CO-NH-CO-NH-CH(Q)-CO-.]. A set of four carbamic acid-nociceptin derivatives were produced. The carbamic acid residue was incorporated into the inner part of the peptides, building on solid phase, by using a suitable dipeptide fragment with carbamic acid residue produced by a simple and efficient three-step solution phase procedure. Enzymatic stability of carbamic acid peptides was studied in the presence of aminopeptidase M (AP-M) and in rat brain membrane homogenate. The receptor-binding properties were also studied by radioligand binding assay on crude rat brain membranes and the activity of the ligands were analyzed on isolated mouse vas deferens (MVD) tissues. We found that incorporation of the carbamic acid residue into the N-terminal part of nociceptin significantly increases the resistance against AP-M. We observed the decrease of binding affinities to the NOP receptor in case of the peptides modified in the N-terminal portion. Consequently, the incorporation of the carbamic acid residue into peptides can be proposed as a promising and reasonable tool for increasing enzymatic stability, where the native molecule is less sensitive for carbamic acid residue-related structural change.     

Exogenous, but not endogenous nociceptin modulates mesolimbic dopamine release in mice

The effect of nociceptin (an endogenous ligand of the ORL1 receptor) on mesolimbic dopamine release and simultaneous horizontal locomotion was studied in freely moving mice undergoing microdialysis of the nucleus accumbens. Intracerebroventricular (i.c.v.) administration of nociceptin (7 nmol) induced a long-lasting suppression of mesolimbic dopamine release and horizontal locomotion in wild-type but not ORL1 knockout mice. I.c.v. administration of the recently reported peptide nociceptin antagonist [Nphe1, Arg14, Lys15] nociceptin-NH(2) (known also as UFP-101, 5 nmol) completely abolished the suppressive effect of nociceptin on mesolimbic dopamine release. However, UFP-101 administration alone induced a mild and lasting suppression of mesolimbic dopamine release in both wild-type and ORL1 knockout mice that was magnified in ORL1 knockout mice by coadministration of nociceptin. UFP-101 administration alone suppressed locomotion in both genotypes. These results confirm that the suppressive action of nociceptin on mesolimbic dopamine release is mediated entirely by the ORL1 receptor, and that UFP-101 effectively antagonizes this action. However, the lack of a stimulatory effect of UFP-101 in wild-type mice indicates that despite being sensitive to exogenous nociceptin action, basal mesolimbic dopaminergic activity is not determined by endogenous nociceptin in mice.     

Species differences in the efficacy of compounds at the nociceptin receptor (ORL1)

Recent studies have identified compounds with reduced efficacy relative to nociceptin/orphanin FQ at the opioid-like receptor ORL1. Utilizing stimulation of [(35)S]GTPgammaS binding as in vitro assays, it was determined that both [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) and the hexapeptide Ac-RYYRIK-NH(2) act as partial agonists in CHO cells transfected with either human or mouse ORL1. Maximal activity for both [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) and Ac-RYYRIK-NH(2) was significantly greater in cells transfected with the human receptor (90% and 73% in a high expressing clone, 76% and 68% in low expressing clone) rather than the mouse receptor (37.5 and 33%), regardless of receptor number in individual clones. In vitro studies in cells transfected with exaggerated receptor numbers can lead to unreliable estimates of agonist and antagonist activity, however, these studies suggest that animal experiments on the activity of novel compounds may not always be better predictors of the ultimate activity in humans.     

Opioid activity of C8813, a novel and potent opioid analgesic

Compound trans-4-(p-bromophenyl)-4-(dimethylamino)-1-(2-thiophen-2-yl-ethyl)-cyclohexanol (C8813), structurally unrelated to morphine, is a novel analgesic. The present study examined the antinociception, opioid receptor selectivity and in vitro activity of C8813. The antinociceptive activity was evaluated using mouse hot plate and acetic acid writhing tests. In mouse hot plate test, the antinociceptive ED(50) of C8813 was 11.5 microg/kg, being 591 times and 3.4 times more potent than morphine and fentanyl respectively. In mouse writhing test, the antinociceptive ED(50) of C8813 was 16.9 microg/kg, being 55 times and 2.3 times more active than morphine and fentanyl respectively. In the opioid receptor binding assay, C8813 showed high affinity for mu-opioid receptor (K(i) = 1.37 nM) and delta-opioid receptor (K(i) = 3.24 nM) but almost no affinity for kappa-opioid receptor (at 1 microM). In the bioassay, the inhibitory effect of C8813 in the guinea-pig ileum (GPI) was 16.5 times more potent than in the mouse vas deferens (MVD). The inhibitory effects of C8813 in the GPI and MVD could be antagonized by mu-opioid receptor antagonist naloxone and delta-opioid receptor antagonist ICI174,864 respectively. However, the inhibitory effect of C8813 in the rabbit vas deferens was very weak. These results indicated that C8813 was a potent analgesic and a high affinity agonist for the mu- and delta-opioid receptors.     

Discovery of {1-[4-(2-{hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl}-1H-benzimidazol-1-yl)piperidin-1-yl]cyclooctyl}methanol,systemically potent novel non-peptide agonist of nociceptin/orphanin FQ receptor as analgesic for the treatment of neuropathic pain: Design,synthesis, and structure–activity relationships

Neuropathic pain is a serious chronic disorder caused by lesion or dysfunction in the nervous systems. Endogenous nociceptin/orphanin FQ (N/OFQ) peptide and N/OFQ peptide (NOP) receptor [or opioid-receptor-like-1 (ORL1) receptor] are located in the central and peripheral nervous systems, the immune systems, and peripheral organs, and have a crucial role in the pain sensory system. Indeed, peripheral or intrathecal N/OFQ has displayed antinociceptive activities in neuropathic pain models, and inhibitory effects on pain-related neurotransmitter releases and on synaptic transmissions of C- and Aδ-fibers. In this study, design, synthesis, and structure–activity relationships of peripheral/spinal cord-targeting non-peptide NOP receptor agonist were investigated for the treatment of neuropathic pain, which resulted in the discovery of highly selective and potent novel NOP receptor full agonist {1-[4-(2-{hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl}-1H-benzimidazol-1-yl)piperidin-1-yl]cyclooctyl}methanol 1 (HPCOM) as systemically (subcutaneously) potent new-class analgesic. Thus, 1 demonstrates dose-dependent inhibitory effect against mechanical allodynia in chronic constriction injury-induced neuropathic pain model rats, robust metabolic stability and little hERG potassium ion channel binding affinity, with its unique and potentially safe profiles and mechanisms, which were distinctive from those of N/OFQ in terms of site-differential effects.