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Opioid Receptorsreceptors

Opioid Receptorsreceptors

OPIOIDOPIOID RECEPTORSRECEPTORS

defined or “classical” types of µ,dk and .

Alistair Corbett, Sandy McKnight and Graeme Genes encoding for these receptors have been cloned.5,

Henderson 6,7,8 More recently, cDNA encoding an “orphan” receptor Dr Alistair Corbett is Lecturer in the School of was identified which has a high degree of homology to Biological and Biomedical Sciences, Glasgow the “classical” opioid receptors; on structural grounds Caledonian University, Cowcaddens Road, this receptor is an and has been named

Glasgow G4 0BA, UK. ORL (opioid receptor-like).9 As would be predicted from 1 Dr Sandy McKnight is Associate Director, Parke- their known abilities to couple through pertussis toxin- Davis Research Centre, sensitive G-proteins, all of the cloned opioid receptors Cambridge University Forvie Site, Robinson possess the same general structure of an extracellular Way, Cambridge CB2 2QB, UK. N-terminal region, seven transmembrane domains and Professor Graeme Henderson is Professor of intracellular C-terminal tail structure. There is and Head of Department, pharmacological evidence for subtypes of each Department of Pharmacology, School of Medical receptor and other types of novel, less well- Sciences, University of Bristol, University Walk, characterised opioid receptors,eliz , , , , have also been Bristol BS8 1TD, UK. postulated. Thes -receptor, however, is no longer regarded as an opioid receptor.

Introduction Receptor Subtypes

Preparations of the somniferum m-Receptor subtypes have been used for many hundreds of years to relieve The MOR-1 gene, encoding for one form of them - . In 1803, Sertürner isolated a crystalline sample of receptor, shows approximately 50-70% homology to the main constituent , , which was later shown to be almost entirely responsible for the the genes encoding for thedk -(DOR-1), -(KOR-1) and orphan (ORL ) receptors. Two splice variants of the activity of crude opium. The rigid structural 1 and stereochemical requirements essential for the MOR-1 gene have been cloned, differing only in the analgesic actions of morphine and related led to presence or absence of 8 amino in the C-terminal the theory that they produce their effects by interacting tail. The splice variants exhibit differences in their rate of onset and recovery from -induced with a specific receptor.1 The concept that there is more than one type of opioid receptor arose to explain the internalization but their pharmacology does not appear dual actions of the synthetic opioid , which to differ in binding assays.10 Furthermore, in the antagonises the analgesic effect of morphine in man but MOR-1 knockout mouse, morphine does not induce also acts as an analgesic in its own right. Martin (1967) antinociception demonstrating that at least in this concluded that the analgesic action of nalorphine is species morphine’s analgesia is not mediated through mediated by a receptor, later called thek -opioid dk- or -receptors.11 Similarly morphine did not exhibit positive reinforcing properties or an ability to induce receptor, that is different from the morphine receptor.2 in the absence of the MOR-1 Evidence for multiple receptors,mk , and s , came from gene. the demonstration of different profiles of pharmacological activity in the chronic spinal dog with mmand : The mm / subdivision was proposed by the prototype morphine, and N- 12 12 Pasternak and colleagues to explain their observations, allylnormetazocine (SKF 10047).3 The existence of the d-receptor was subsequently proposed to explain the made in radioligand binding studies, that [3 H]-labelled-m , profile of activityin vitro of the (the first -dk and - ligands displayed biphasic binding endogenous opioid ), and on the basis of the characteristics.12 Each radioligand appeared to bind to the same very high affinity site (m ) as well as to the relative of the non-selective 1 to reverse endogenous opioid appropriate high affinity site (md , or k ) depending on the inhibition of the nerve-evoked contractions of the radioligand used. and were

mouse vas deferens.4 Its existence was further reported to abolish the binding of each radioligand to confirmed by radioligand binding studies using rat them -site. Furthermore, inin vivo studies it was 1 homogenates. observed that naloxazone selectively blocked morphine-induced antinociception but did not block It is now clear from work carried out in many morphine-induced respiratory or the

laboratories over the last 20 years that there are 3 well- induction of morphine dependence.13,14 Subsequent work

Tocris Cookson Ltd., UK Tocris Cookson Inc., USA Tel: + 44 (0)117 982 6551 Tel: (800) 421-3701 Fax: + 44 (0)117 982 6552 www.tocris.com Fax: (800) 483-1993 e-mail: [email protected] [email protected] e-mail: [email protected] Is there another, novel form of them -opioid or 5¢¢ -isothiocyanate (5 -NTII).18,19,20 receptor? Furthermore, while mice develop tolerance to the Several related observations suggest the existence of a antinociceptive effects of repeated injections of either novel form ofm -receptor at which analogues of DPDPE or II, this tolerance appears to be morphine with substitutions at the 6 position (e.g. homologous in that there is no cross tolerance between these ligands.21 In vivo, dd - and -receptor-induced morphine-6b -glucuronide, and 6-acetyl 12 morphine) are agonists, but with which morphine itself antinociception can be differentially antagonised by does not interact.15 In antinociception tests on mice it has blockers of different types of potassium channels.22 been reported that morphine does not exhibit cross tolerance with morphine-6b -glucuronide, heroin or 6- The best evidence fromin vitro experiments to support thedd and subdivision of d -receptors comes from acetyl morphine. Furthermore, in mice of the CXBX 12 strain morphine is a poor antinociceptive agent inhibition of activity in membranes whereas morphine-6b -glucuronide, heroin and 6-acetyl from rat brain24,25 and from thed -receptor-mediated morphine are all potently antinociceptive. The 6- elevations of intracellular Ca2+ in the ND8-47 cell line26 substituted morphine analogues do not appear to be where BNTX selectively antagonised DPDPE, and acting throughdk - or -receptors because the naltriben selectively antagonised deltorphin II. antinociception they induce is not blocked by selective Surprisingly, little selectivity was seen in radioligand dk-- or receptor antagonists, whereas 3- displacement studies.24 The converse has been methoxynaltrexone has been reported to antagonise observed in studies on neuronal cell lines. Two distinct morphine-6b -glucuronide- and heroin-induced d-receptor binding sites were observed in radioligand antinociception without affecting that induced by binding experiments on SK-N-BE cells.27 Studies on NG108-15 cells28 or the cell line, morphine, [D-Pen25 , D-Pen ] (DPDPE ,d - SH-SY5Y,29,30 have failed to find any functional evidence selective) or U50488 (k -selective).16 ford -receptor subtypes. Recently it has been reported that heroin and morphine- 6-glucuronide, but not morphine, still produce The pharmacological properties of the cloned DOR-1 antinociception in MOR-1 knockout mice in which the receptor are somewhere between those predicted for either thedd or subtypes. DPDPE and deltorphin II are disruption in the MOR-1 gene was engineered in exon- 12 both potent displacers of [3 H]- binding to 1.17 The same authors observed that in other MOR-1 knockout mice in which exon-2, not exon-1, had been mouse and human recombinant receptors, which is not consistent with either thedd or classifications.31 In disrupted, all three agonists were ineffective as 12 antinociceptive agents. They conclude that the contrast, [3 H]-diprenorphine binding to the mouse antinociceptive actions of heroin and morphine-6- recombinant receptor is more potently displaced by glucuronide in the exon-1 MOR-1 mutant mice are naltriben than BNTX, suggesting that the cloned receptor is of thed subtype. It will be of importance to mediated through a receptor produced from an 2 alternative transcript of the MOR-1 gene differing from determine in the DOR-1 knockout mouse if analgesia can still be induced by eitherdd - or -receptor selective the MOR-1 gene product, them -opioid receptor, in the 12 exon-1 region. To substantiate this conclusion they agonists. report that in RT-PCR experiments using primers spanning exons 2 and 3, a MOR-1 gene product was ddand : The dd and subdivision of d -receptors was cx ncx cx nx c still detected in MOR-1 knockout mice. based on the hypothesis that one type ofdd -receptor ( ) cx was complexed withmk -receptors (and perhaps also - d-Receptor subtypes receptors) whereas the other type ofdd -receptor ( ) was ncx The DOR-1 gene is the onlyd -receptor gene cloned to not associated with an opioid receptor complex.32 It was date. However, two, overlapping subdivisions ofd - originally observed that sub-antinociceptive doses of agonists at thed receptor (e.g. low doses of DPDPE), receptor have been proposed (dd / and dd / ) on the cx 12 cxncx basis ofin vivo and in vitro pharmacological potentiatedm -receptor-mediated analgesia, an effect experiments. which could be antagonised by 5¢ -NTII. On the other hand, at higher doses, DPDPE then acted as an agonist ddand : The subdivision of the d -receptor into dd and at thed -receptor and itself induced analgesia which 12 12 ncx subtypes was proposed primarily on the basis of in vivo was reversed by DALCE. Data obtained from pharmacological studies (Table 1). In in vivo, subsequent radioligand binding studies have been the supraspinal antinociceptive activity of DPDPE can interpreted as demonstrating the existence of further be selectively antagonised by 7-benzylidenenaltrexone subtypes of theddd receptor i.e. and . More ncxnc( x-1 ) (nx-2 c ) (BNTX) or [D-Ala25 , D-Leu ]enkephalyl-Cys (DALCE)18,19 recently it has been suggested that thed receptor is in (nx-1 c ) whereas the antinociceptive activity of [D-Ala2 ]- fact synonymous with thedd -receptor and the - 1 cx deltorphin II (deltorphin II) and [D-Ser2 , receptor synonymous with thed -receptor of the 2 Leu5 ]enkephalyl–Thr (DSLET) can be reversed by previous classification.33

Table 1. Putative ligands ford -receptor subtypes

Receptor Agonists Antagonists subtype Competitive Nonequilibrium

d DPDPE / DADLEBNTX DALCE 1

d Deltorphin II / DSLET Naltriben 5¢ -NTII 2

(bold text denotes compounds available from Tocris)

N.B. DPDPE may not in fact be a selectivedd agonist but may also be a at -sites.23 1 2

2 k-Receptor subtypes resolved two binding sites termedkk and . The ligand 1a 1b The situation regarding the proposals for subtypes of demonstrating the highest affinity, and around 30-fold thekm -receptor is rather more complex than for the - preference, for the “ka binding site” was -neo- 1a andd -receptors, perhaps because of the continuing use endorphin.42 More recently putativekk - and -sites in 1a 1b of non-selective ligands to define the putative sites. The mouse brain were identified from complex evidence for the need for sub-division of thek -receptor displacement curves against the binding of [3 H]-U- comes almost entirely from radioligand binding assays. 69,593, in an attempt to compare the pharmacology of the mousek -sites, with that at the cloned rat KOR 1 The first characterisation of ak -receptor binding site in stably expressed in a host neuroblastoma cell line.43 brain came from work using [3 H]-ethylketocyclazocine Based on the high affinity of anda -neo-

(EKC).34 Crucial to this success was the use of the endorphin, it was deemed “consistent to term the guinea-pig brain wherek -sites are present in relative cloned KOR ak subtype”. 1b abundance, and of “suppression”, or quenching of the binding of this non-selective ligand tomd - and -sites, by Rothman (1990) also reported subdivision of thek - 2 incubation with non-radioactive ligands that bound binding of [3 H]-bremazocine into 2a- and 2b-sub- selectively at these other sites. subtypes.42 Thekb -site had high affinity for -endorphin 2b and DADLE, reminiscent of the originalk -binding site of 2 Studies of [3 H]-EKC binding in guinea-pig guinea-pig spinal cord. Thekk - and -sites in guinea- 2a 2b pointed to the existence of a non-homogeneous pig brain have undergone a further subdivision (sub- population of high-affinity binding sites, and led to the sub-subtypes?) on the basis of investigations using a first proposal forkk - and -sites distinguished by their 12 combination of depletion (ofmd - and -sites) and sensitivity to DADLE.35 The DADLE-sensitivek site 2 suppression, against the binding of 6b -[125 I]-3,14- boundb -endorphin with high affinity, and was later dihydroxy-17-cyclopropylmethyl-4,5a - identified with the recognition site of thee -receptor in epoxymorphinan ([125 I]OXY). 44 So were defined the k 2a-1 brain.36 Another study using [3 H]-EKC identified ak -site andk sites, having relatively high and low affinities 2a-2 in bovine adrenal medulla, with a pharmacology similar respectively for nor-BNI and (CI-977), and k 2b-1 to that of thek -site in guinea-pig cord37 but labelling with 1 andk sites with high and low affinities for DAMGO and 2b-2 [3 H]- revealed two additional sites, one a-neo-endorphin. resemblingk that bound [Met]enkephalyl-Arg-Gly-Leu 2 with high affinity and another termed “k ” or “MRF” that 3 Definitive functional pharmacological evidence bound [Met5 ]enkephalyl-Arg-Phe with high affinity. supporting the existence of this confusing number of putative subtypes of thek -receptor is lacking, because Thekk / terminology has more recently been applied by 12 of the absence of subtype-specific antagonists. It has other groups to the putative subtypes defined in other been reported however, that pretreatment with the tissues in their hands, but it is not always clear how isothiocyanate analogue of U-50,488 called (-)-UPHIT, closely the common nomenclature reflects a common was able to produce a long-lasting block of the pharmacology. The introduction of the first selectivek - antinociceptive effect of U-69,593 in the mouse without agonist U-50,488 and its congeners (U-69,593, affecting the action of bremazocine, while treatment PDÊ117302, CI 977, ICI 197067) led to a refinement of with the non-selective antagonist WIN 44,441 the definition of the putative subtypes, but pointed to the () blocked selectively the antinociception need for careful considerations of the effect of technical with bremazocine.45,46 These findings provide obvious differences in assays and of species as a possible support for thekk - subdivision; the pharmacological 12 explanation for discrepancies. Thus a direct corollary is that (-)-UPHIT and WIN 44,441 are comparison of the binding of [3 H]-EKC in guinea-pig and antagonists with selectivity for thekk -and -subtypes 12 rat (with suppression of binding tomd - and -sites) respectively, at least in the mouse. pointed to the existence of a high affinityk -site that 1 predominated in guinea-pig brain and was selectively Correlating genes withmd -, - and k -receptor sensitive to U-69,593, and a low affinity, U-69,593- subtypes insensitivek -site that predominated in rat brain.38 2 Although there is as yet little evidence for different Others resorted to the binding of [3 H]-bremazocine to genes encoding the different subtypes ofmd -, - and k - reveal U-69,593-insensitivek -binding sites; in contrast 2 receptor these subtypes may result from different post- to thek -site originally defined in guinea-pig spinal cord, 2 translational modifications of the gene product thekk -site in brain after suppression of was insensitive (glycosylation, palmytoylation, , etc), 2 1 to DADLE.39 from receptor dimerization to form homomeric47 and

heteromeric complexes,48,49,50 or from interaction of the Subdivision of thekk -site in guinea-pig brain into and 1 1a gene product with associated proteins such as k , was proposed to resolve the complex displacement 1b RAMPs.51 of either [33 H]-EKC or [ H]-U-69,593 with B and a-neo-endorphin which both preferentially bound to the The proposedk sub-subtype.40 The same study proposed 1b the existence of ak subtype, insensitive to U-50,488, Extending the screening of genomic and cDNA 3 that was identified from the binding of [3 H]-naloxone libraries, perhaps in an effort to identify putative benzoylhydrazone. The pharmacology of this later “k - subtypes of the classical opioid receptors, resulted in 3 site” is rather different from thek /MRF site of bovine the identification of a novel receptor that bore as high a 3 adrenal medulla, and has been proposed to be the degree of homology towards the classical opioid receptor mediating the antinociceptive effect of receptor types, as they shared among each other. The nalorphine, Martin’s “N”-receptor.41 receptor was identified in three species: rat, mouse and man, with the degree of homology among the species Nomenclature differences appear to have arisen in the variants more than 90%. Although the putative receptor context of subtyping of thek -subtype. Using binding has had as many names as the number of groups who 1 surface analyses to allow highly accurate estimation of reported its identification,52 there is some consensus for the use binding parameters, the binding of [3 H]-U-69,593

3 of the original designation for the human form, paucity of safe and sure pharmacological tools may

“ORL1 ”. Workers in the field are, however, divided in partly explain some of the in the literature their preferred terminology for the endogenous regarding the effect of in tests of response 53 latency to noxious stimulation; antinociception, pro- peptide agonist for ORL1 with both “nociceptin” or “orphanin FQ”54 being used with roughly equal /hyperalgesia, , or no overt frequency. effect, have all been reported. Although the results of some studies have been Although the ORL1 receptor was accepted as a member of the “family” of opioid receptors on the interpreted as pointing to the existence of subtypes of ORL , this conclusion is so far premature in most basis of its structural homology towards the classical 1 types, there is no corresponding pharmacological cases. The most reliable pharmacological definition of homology. Even non-selective ligands that exhibit receptors is based on differences in antagonist uniformly high affinity towardsmk -, - and d -receptors, affinity, and in this context the absence of useful antagonists for ORL1 is particularly galling to have very low affinity for the ORL1 receptor, and for this reason as much as for the initial absence of an pharmacologists. Although the synthetic analogue of endogenous ligand, the receptor was called an the N-terminal tridecapeptide of nociceptin, 12 “orphan opioid receptor”. Close comparison of the [Phey (CH22 -NH)Gly ]nociceptin(1-13)NH was first deduced amino- sequences of the four reported to be a selective antagonist,59 increased use receptors highlights structural differences that may of this peptide points to it having agonist actions. explain the pharmacological anomaly. Thus there There are no grounds for saying that this peptide is an are sites near the top of each of the trans-membrane antagonist at ORL1 receptors in the periphery, but an regions, that are conserved in themk -, - and d - agonist in the brain (not least because agonist actions in the periphery, and antagonist actions in the brain receptors, but are altered in ORL1 . Work with site- have been reported) and that these differences in directed mutants of ORL1 (rat) has shown that it is possible to confer appreciable affinity on the non- point to differences in the receptors. Although 1 selective bremazocine by changing differences in theaffinity for [Phey (CH2 - 213 2 Ala in TM5 to the conserved Lys ofmk , and d , or by NH)Gly ]nociceptin(1-13)NH2 may be found between changing the Val-Gln-Val276-278 sequence of TM6 to central and peripheral sites,60 and there may indeed 55 the conserved Ile-His-Ile motif. be different “subtypes” of ORL1 in the brain and periphery, the safest conclusion for the moment is just 12 A splice variant of the ORL1 receptor from rat has that [Phey (CH22 -NH)Gly ]nociceptin(1-13)NH is a been reported (“XOR”)56 with a long form (XOR1L) partial agonist, and that the observed differences in containing an additional 28 amino acids in the third efficacy are consistent with differences in receptor extracellular loop. In the homologous receptor from reserve. mouse (also sometimes referred to as “KOR-3”) five splice variants have been reported to date.57 Very recently a peptide related to the combinatorial hexapeptide library hit acetyl-Arg-Tyr-Tyr-Arg-Trp- Lys-NH (Ac-RYYRWK-NH ; Table 2), but with ORL1 -Receptor subtypes 22 Selective high affinity ligands with which to attempt isoleucine substituting for tryptophan, was reported to block the effects of nociceptin/orphanin FQ in rat pharmacological definitions of the ORL1 receptor are few in number (Table 2). Besides the natural cortex (stimulation of GTPg35 S binding) or heptadecapeptide agonist nociceptin/orphanin FQ (positive chronotropic effect in isolated myocytes). and some closely related peptides, the only other Although this peptide, like all of its structural ligands offering high affinity and selectivity belong to homologues, was originally reported to be a potent a class of peptides obtained by a positional scanning agonist, but with somewhat less than full efficacy,58 it approach to combinatorial libraries of will be important to see if the antagonist activity of Ac- 58 hexapeptides. Being basic peptides highly Arg-Tyr-Tyr-Arg-Ile-Lys-NH22 (Ac-RYYRIK-NH ) at the susceptible to degradation, all of those agents are 61 ORL1 receptor is confirmed. chancy tools in the hands of the unwary. So the

Table 2. Selective opioid ligands

Receptor typemd -Receptor -Receptor k -Receptor ORL 1

Selective agonistsendomorphin-1 [D-Ala2 ]- enadoline nociceptin / OFQ

-2 [D-Ala2 ]-deltorphin II U-50488 Ac-RYYRWK-NH * 2 DAMGO DPDPE U-69593 SNC 80

Selective antagonists CTAPnaltrindole nor- None as yet** TIPP-y ICI 174864

Radioligands [3 H]-DAMGO[3 H]-naltrindole [3 H]-enadoline [3 H]-nociceptin

[3 H]-pCl-DPDPE [3 H]-U69593

[3 H]-SNC 121

(bold text denotes compounds available from Tocris) *Related combinatorial library hits are also selective agonists.58 **Ac-RYYRIK-NH has been proposed to be an 2 ORL antagonist61 whereas the putative antagonist [Phe12y (CH -NH)Gly ]nociceptin(1-13)NH59 appears to be a partial 1 2 2 agonist.

4 Less Well-Characterised Opioid Receptors the precursors. Nociceptin/orphaninFQ, however, has a C-terminal (F) whereas peptides derived In addition to the µ-,dk -, - and ORL -receptors, several from the other precursors all have the pentapeptide 1 other types of opioid receptor have been postulated. sequence TyrGlyGlyPheMet/Leu (YGGFM/L) at their Since the contractions of the isolated vas deferens of N-termini. These peptides vary in their affinity for µ,d - the rat are much more sensitive to inhibition byb - andk -receptors, and have negligible affinity for ORL1 - endorphin than by other opioid peptides, it was receptors, but none binds exclusively to one opioid suggested that this tissue contains a novel type of receptor type.71,53bd -endorphin is equiactive at µ-and - opioid receptor, theeb -receptor, that is specific for - receptors with much lower affinity fork -receptors; the endorphin.62 The rabbit ileum has been proposed to post-translational product, N-acetyl-b -endorphin, has possessi -receptors, for which the enkephalins have very low affinity for any of the opioid receptors.72,73 [Met]- high affinity but which are distinct fromd -receptors.63 A and [Leu]enkephalin have high affinities ford -receptors, very labilel -binding site with high affinity for 4,5 ten-fold lower affinities for µ-receptors and negligible epoxymorphinans has been found in freshly-prepared affinity fork -receptors.71 Other products of processing of rat membrane fragments64 and there is evidence that pro-enkephalin, which are N-terminal extensions of opioids inhibit growth in S20Y murine blastoma cells by [Met]enkephalin, have a decreased preference for the an action at yet another receptor type called thez - d-receptor with some products, e.g. metorphamide

71 receptor.65 Theeli -, -, -and z -receptors are poorly displaying highest affinity for the µ-receptor. The opioid characterised and wider acceptance of their existence fragments of pro-dynorphin, particularly awaits further experimental evidence, in particular and , have high affinity fork -receptors but isolation of their cDNAs. also have significant affinity for µ- andd -receptors.71

Although originally classified as such, thes -receptor Endomorphin-1 and endomorphin-2 are putative appears not to be an opioid receptor but rather the products of an as yet unidentified precursor, that have target for another class of abused , been proposed to be the endogenous ligands for the µ-

(PCP) and its analogues.66 Phencyclidine is an effective receptor where they are highly selective.74 The blocker of the ion channel associated with the N-methyl- are amidated tetrapeptides and are D-aspartate (NMDA) receptor where it binds to the structurally unrelated to the other endogenous opioid same site as MK 801.67 peptides (Table 3) . Although the study of the cellular localisation of these peptides is at an early stage, Endogenous Ligands endomorphin-2 is found in discrete regions of rat brain, some of which are known to contain high

In mammals the endogenous opioid peptides are concentrations ofm -receptors.75 Endomorphin–2 is also mainly derived from four precursors: pro- present in primary sensory neurones and the dorsal opiomelanocortin, pro-enkephalin, pro-dynorphin and horn of the spinal cord where it could function to pro-nociceptin/orphanin FQ.68,69,70,53 Nociceptin/orphanin modulate nociceptive input.76 FQ is processed from pro-nociceptin/orphanin FQ and is the endogenous ligand for the ORL -receptor; it has In comparison to the mainly non-selective mammalian 1 little affinity for the µ-,dk - and -receptors.53,54 The amino opioid peptides (the exceptions being the acid sequence of nociceptin/orphanin FQ has endomorphins), amphibian skin contains two families of homology with other opioid peptides especially the D--containing peptides that are selective for fragment dynorphin A (Table 3), µ- ord -receptors. is a µ-selective suggesting a close evolutionary relationship between heptapeptide Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH 2

Table 3. Mammalian endogenous opioid ligands

Precursor Endogenous peptide Amino acid sequence

Pro-opiomelanocortinb -Endorphin YGGFMTSEKSQTPLVTL- FKNAIIKNAYKKGE

Pro-enkephalin [Met]enkephalin YGGFM [Leu]enkephalin YGGFL YGGFMRF YGGFMRGL Metorphamide YGGFMRRV-NH 2

Pro-dynorphin Dynorphin A YGGFLRRIRPKLKWDNQ Dynorphin A(1-8) YGGFLRRI Dynorphin B YGGFLRRQFKVVT a- YGGFLRKYPK b-neoendorphin YGGFLRKYP

Pro-nociceptin / OFQNociceptin FGGFTGARKSARKLANQ

Pro-endomorphin1 Endomorphin-1 YPWF-NH 2 Endomorphin-2 YPFF-NH 2 (bold text denotes compounds available from Tocris)

1Presumed to exist, awaiting discovery

5 without significant affinity atdk - and -receptors.77 In expression of G proteins and effector systems between contrast, the - deltorphin (dermenkephalin; cell types rather than any inherent differences in the

Tyr-D-Met-Phe-His-Leu-Met-Asp-NH ), [D-Ala2 ]- properties of the receptors themselves. 2 deltorphin I and [D-Ala2 ]-deltorphin II (Tyr-D-Ala-Phe- Xaa-Val-Val-Gly-NH , where Xaa is Asp or Glu Opioid receptor activation produces a wide array of 2 respectively) - are highly selective ford -opioid cellular responses (Table 4). Although the pertussis receptors.78 toxin sensitivity has not been assessed in all instances it is highly likely that in each the first step is activation of G i Effector Mechanisms or G . The functional significance of many of these o opioid receptor-mediated effects is still unclear, but two The opioid receptor family, in common with the recent observations on changes in receptor family, is somewhat unusual in release following acute and chronic exposure to opioids that all of the cloned opioid receptor types belong to the are worthy of special mention because they provide G/G -coupled superfamily of receptors. Opioid io potential solutions to long-asked questions. receptors do not couple directly with G or G and none of sq the cloned receptors forms a ligand-gated ion channel. The periaqueductal grey region (PAG) is a major It was originally thought thatmd - and -receptors coupled anatomical locus for opioid activation of descending through G/G proteins to activate an inwardly rectifying io inhibitory pathways to the spinal cord and is thus an potassium conductance and to inhibit voltage-operated important site form -receptor-induced analgesia. calcium conductances whereask -receptors only inhibit Opioids do not excite descending fibres directly but voltage-operated calcium conductances. However it is disinhibit them by inhibiting spontaneous GABA release now known that thek -receptor is, in some cell types, from local GABAergic interneurones.80 This inhibition of also coupled to activation of an inwardly rectifying transmitter release results from activation of a potassium conductance.79 It seems highly likely, dendrotoxin-sensitive, voltage-sensitive potassium therefore, that all of the opioid receptors will share conductance. The mechanism by which the voltage- common effector mechanisms. Indeed, many papers sensitive potassium conductance is activated appears have recently appeared demonstrating that the ORL - to be through activation of phospholipase A (PLA ) with 1 22 receptor couples to the same effector systems as the subsequent of arachidonic acid along the other more extensively studied opioid receptors. It 12¢ -lipoxygenase pathway because the inhibition of should be borne in mind that, given the heterogeneity of GABA release can be inhibited by quinacrine and 4- aab, , and g subunits which may combine to form a bromo-phenacylbromide, inhibitors of PLA , and by io 2 trimeric , there may well be some subtle baicalein, an inhibitor of 12¢ -lipoxygenase. This differences in the downstream effector mechanisms to proposed mechanism of opioid action also explains the which opioid receptors are coupled if one type of opioid between opioids and non-steroidal analgesic receptor is unable to interact with a certain form of G/G io drugs (NSAIDs) in producing analgesia because in the heterotrimer. However, different responses evoked in presence of a NSAID, with the cyclo-oxygenase different cell types in response to activation of different inhibited, more of the arachidonic acid opioid receptors or even in response to activation of the produced by opioid activation of PLA can be diverted 2 same receptor are likely to reflect changes in the down the 12¢ -lipoxygenase pathway.

Table 4. Opioid receptor-evoked cellular responses

Direct G-proteinbg or a subunit-mediated effects · activation of an inwardly rectifying potassium channel · inhibition of voltage operated calcium channels (N, P,Q and R type) · inhibition of adenylyl cyclase

Responses of unknown intermediate mechanism · activation of PLA 2 ·b(bgactivation of PLC possibly direct G protein subunit activation) · activation of MAPKinase · activation of large conductance calcium-activated potassium channels · activation of L type voltage operated calcium channels · inhibition of T type voltage operated calcium channels · direct inhibition of transmitter exocytosis

Responses which are a consequence of opioid-evoked changes in other effector pathways · activation of voltage-sensitive potassium channels (activation of PLA ) 2 · inhibition of M channels (activation of PLA ) 2 · inhibition of the hyperpolarisation-activated cation channel (Ih) (reduction in cAMP levels following inhibition of adenylyl cyclase) ·b,elevation of intracellular free calcium levels (activation of PLC activation of L type voltage operated calcium conductance) · potentiation of NMDA currents (activation of protein kinase C) · inhibition of transmitter release (inhibition of adenylyl cyclase, activation of potassium channels and inhibition of voltage operated calcium channels) · decreases in neuronal excitability (activation of potassium channels) · increases in neuronal firing rate (inhibition of inhibitory transmitter release - disinhibition) · changes in gene expression (long-term changes in adenylyl cyclase activity, elevation of intracellular calcium levels, activation of cAMP response element binding protein (CREB))

6 The cellular locus of withdrawal has long been mild, through moderate to severe pain, alone or with the Holy Grail of opioid biologists. Over 20 years ago, it adjuncts. The related to include the was shown that following chronic exposure of NG108- most potent non-peptidem -agonists known, and are 15 neuroblastoma x glioma hybrid cells to , generally used peri-operatively, often for the induction withdrawal resulted in a rebound increase in adenylyl and maintenance of anaesthesia. The use of many of cyclase;81 the functional significance of this observation the (as had been found with the first of for opiate withdrawal in brain neurones has remained the “duallists” nalorphine) has been associated with obscure. Recently, Williams and colleagues82 have dysphoric and effects in man, a observed an increase in the release of the inhibitory property originally thought to be attributable to affinity at neurotransmitter GABA, in the the non-opioids -site. during opiate withdrawal. This effect could be mimicked by the adenylyl cyclase activator, forskolin, and The attractiveness of the prospect for development of inhibited by inhibitors. Therefore, as selectivek -agonists as was based on the proposed over 25 years ago by the late Harry Collier, preclinical pharmacology in animals of the 6,7- rebound adenylyl cyclase activity in withdrawal may be benzomorphans such as ketazocine and its derivatives the fundamental step in eliciting the withdrawal (Figure 1). Although those agents are not selective in behaviour.83 terms ofaffinity , their utility as pharmacological tools is based on their for thek -receptor, Development and Clinical Applications of where theirefficacy is high. Such agents produced a Opioid Ligands powerful antinociceptive effect, but did not substitute for morphine in dependent animals. A full biochemical and Among the receptors for the many that pharmacological characterisation of thek -receptor was exist in the nervous system, the opioid receptors are not possible until the discovery of highly selective unique in that there existed before the discovery of the agonists in the aryl-acetamides that appear unrelated natural agonists, an abundance of non-peptide ligands structurally to any of the morphine derivatives. The first with which the pharmacology of the receptors was compound of this class was U-50,488, but its already defined. In current terms relating to the - importance was also as a chemical lead for the discovery process, we would consider the 4,5-epoxy- attempted design of related compounds of greater methylmorphinan opioid morphine, selectivity and potency. At least two such compounds and as “natural-product hits” on which were have entered clinical trials as centrally acting based chemical programmes to design analogues with analgesics, (U-62,066) and enadoline (CI- improved pharmacology (Figure 1). The effects of 977). Although CNS-mediated, mechanism-related morphine to reduce sensitivity to pain or to inhibit side effects of and may limit the intestinal motility and secretion, have continued to be potential for development of such compounds, the exploited clinically, however the presence of other prospects for analogues with limited brain penetration undesirable effects (e.g. depression of respiration, to produce a peripherally mediated analgesic effect in tolerance/dependence, effects on mood) provided the inflammatory conditions is under exploration, with at stimulus to seek analogues that were selective in least one compound (, EMD-61753) in producing analgesia. Thus a semi-synthetic di- clinical trials for osteoarthritis. The observation of acetylated analogue of morphine was introduced in the in the mistaken belief that this compound neuroprotective properties ofk -agonists in pre-clinical (heroin) had those desired properties. More radical models of cerebral ischaemia has lead to consideration changes to the nucleus were subsequently of the possible clinical development of selectivek - explored in various synthetic programmes, in many agonists for stroke or traumatic head injury. In this early cases resulting in the development of low efficacy context the properties ofk -agonists, and even partial agonists. perhaps their characteristic action, may be advantageous. With the benefit of hindsight, it is possible to conceive an evolution of those opioid analogues, with a The discovery of the enkephalins and of thed -receptor, progressive simplification of chemical structure from led to the idea that the peptides themselves might be the epoxymorphinans (nalorphine, ) through taken as “leads” for the synthesis of a new class of the such as , and the opioid agonist that lacked the addictive properties of benzomorphans such as , to the phenyl- morphine. Although such synthetic activities produced piperidines including and the 4-anilino- many useful experimental tools, no direct benefit in the piperidines as exemplified by fentanyl (Figure 1). The form of a drug appeared, in spite of the attempted ultimate simplification of the morphine structure was in development of several enkephalin analogues. It did the class, with methadone itself and d- become clear from the work of a number of laboratories propoxyphene (Darvon). Although thebaine is virtually that activation of thed -receptor is associated with inactive, the compound itself was an important antinociception in animals, and the development of a chemical precursor in the synthesis of 14-hydroxy selective non-peptide agonist is under consideration by derivatives of morphine, most particularly the a number of commercial drug houses. In some cases antagonists naloxone and . Also derived the synthetic strategy is based directly on structural from thebaine were the derivatives, and here considerations of the first non-peptide with significant the trend of chemical “simplification” was reversed with selectivity, the 6,7- analogue of naltrexone, the introduction of an additional six-membered ring that naltrindole (Figure 1).84 Applying the “message-address” appeared to enhance biological potency. For example, concept that produced the antagonist naltrindole to a etorphine is about one thousand times more potent than novel series of octahydroisoquinoline derivatives has morphine as an analgesic, but its use is limited to been successful in producing non-peptided -selective veterinary as a sedative for large animals. agonists TAN-6785 or SB 213698.86 Similar considerations do not serve to explain the existence of For the most part, such compounds have highest affinity another series of novel derivativesd - for them -receptor, and to a greater or lesser extent agonists, BW 373U8687 or SNC 80. 88 Preclinical studies produce the full panoply of effects, good and bad, suggest thatd -agonists may have a superior profile as obtained with morphine. Depending on the level of analgesics, but this will only affinity and efficacy, such compounds have been used acutely or chronically, to provide analgesia in cases of

7 be established when such an agent is successfully dense and wide investment in the nervous system) introduced into clinical investigation; other possible must await the initial results of the drug-discovery applications of selective ligands for this receptor may process. Only with the availability of non-peptide emerge from clinical experience. selective agonists, and perhaps more particularly antagonists, will it be possible to undertake the The prospects for clinical utilities of agonists or definitive pre-clinical studies that will serve for the antagonists for the ORL receptor can only be the identification of possible clinical targets. There is some 1 subject of speculation. Elucidation of the role of the agreement that activation of the ORL receptor in the 1 nociceptin/ORL-receptor system in pain control (and in brain leads to a motor impairment, so it may be that the other areas, for the peptide and its receptor have a development of ORL agonists would be difficult. 1

Figure 1. Structures of non-peptide agonists and antagonists

a) Analgesic morphine derivatives b) Benzomorphans

N

R4

N R1

O HO R2

R3 N N RRRR 1234

O PENTAZOCINE H CHC=C(Me) Me Me H 22 2

SKF 10,047 H CHC=CH Me Me H 2 2 NN N

KETAZOCINE H C Me Me O 2

BREMAZOCINE H C (Me) Me H 222

c) Selectivekd -opioid agonists d) Selective -receptor non-peptide ligands

N OH Cl N O

N Cl Me HO O N H

O O Et 2N O H OMe N O N Me N Me

Me N

H MeN

O N N NMe OH

8 References 58.Dooley et al (1997) J.Pharmacol.Exp.Ther. 283 735. 1.Beckett and Casey (1954) J.Pharm.Pharmacol. 6 59.Guerriniet al (1998) Br.J.Pharmacol.123 163. 986. 60.Mason et al (1998) Soc. for Neurosci . 24 536.20. 2.Martin (1967) Pharmacol.Rev.19 463. 61.Berger et al (1999) Br.J.Pharmacol.126 555. 3.Martin et al (1976) J.Pharmacol.Exp.Ther. 197 62.Wuster et al (1978) Neurosci.Lett.15 193. 517. 63.Oka (1980) Br.J.Pharmacol.68 198. 4.Lord et al (1977) Nature267 495. 64.Grevel and Sadee (1983) Science221 1198. 5.Evans et al (1992) Science258 1952. 65.Zagon et al (1989) Brain Res.482 297. 6.Kieffer et al (1992) Proc.Natl.Acad.Sci.USA 89 66.Vincent et al (1979) Proc.Natl.Acad.Sci.USA 76 12048. 4578. 7.Chen et al (1993) Mol.Pharmacol.44 8. 67.Hollmann and Heinemann (1994) 8.Minami et al (1993) FEBS Lett.329 291. Ann.Rev.Neurosci.17 31. 9.Mollereau et al (1994) FEBS Lett.341 33. 68.Nakanishi et al (1979) Nature278 423. 10.Koch et al (1998) N.S. Archives of Pharmacology 69.Kakidani et al (1982) Nature298 245. 357 SS 44. 70.Noda et al (1982) Nature295 202. 11.Matthes et al (1996) Nature383 818. 71.Corbett et al (1993) In: Handbook 12.Wolozin and Pasternak (1981) Proc.Natl.Acad. Exp.Pharmacol. Ed. A. Herz104/1 p645. Sci.USA78 6181. 72.Kosterlitz and Paterson (1985) 13.Ling et al (1984) Science226 462. Philos.Trans.R.Soc.Lond.308 291. 14.Ling et al (1985) J.Pharmacol.Exp.Ther.232 149. 73.Akil et al (1981) Peptides2 289. 15.Rossi et al (1996) Neuroscience Letters216 1. 74.Zadina et al (1997) Nature386 499. 16.Brown et al (1997) J.Pharmacol.Exp.Ther. 282 75.Schreff et al (1998) Neuroreport9 1031. 1291. 76.Martin-Schild et al (1997) Peptides18 1641. 17.Schuller et al (1999) Nature Neuroscience2 151. 77.Amiche et al (1988) Int.J.Pept.Protein Res.32 50. 18.Jiang et al (1991) J.Pharmacol.Exp.Ther. 257 78.Erspamer et al (1989) Proc.Natl.Acad.Sci.USA 86 1069. 5188. 19.Sofuoglu et al (1993) Life Sci.52 769. 79.Grudt and Williams (1993) Proc.Natl.Acad.Sci. 20.Sofuoglu et al (1991) J.Pharmacol.Exp.Ther. 257 USA90 11429. 676. 80.Vaughan et al (1997) Nature390 611. 21.Mattia et al (1991) J.Pharmacol.Exp.Ther. 258 81.Sharma et al (1975) ) Proc.Natl.Acad.Sci.USA 72 583. 3092. 22.Wild et al (1991) Eur.J.Pharmacol.193 135. 82.Chieng and Williams (1998) J.Neurosci. 18 23.Vanderah et al (1994) Eur.J.Pharmacol.252 133. 7033. 24.Buzas et al (1994) Life Sci.54 PL101. 83.Collier et al (1974) Nature249 471. 25.Noble and Cox (1995) J.Neurochem.65 125. 84.Portoghese et al (1990) J.Med.Chem.43 1714. 26.Tang et al (1994) J.Pharmacol.Exp.Ther.270 40. 85.Nagase et al (1994) Jap.J.Pharamacol.64 (suppl. 27.Polastron et al (1994) J.Neurochem.62 898. 1) 35. 28.Ho et al (1997) Eur.J.Pharmacol.319 109. 86.Dondio et al (1995) Analgesia1 394. 29.Connor et al (1997) Neuropharmacology36 125. 87.Chang et al (1993) J.Pharmacol.Exp.Ther. 267 30.Toll et al (1994) Regul.Peptides54 303. 852. 31.Law et al (1994) J.Pharmacol.Exp.Ther.271 1686 88.Calderon et al (1994) J.Med.Chem.37 2125. 32.Rothman et al (1993) In: Handbook Exp. Pharmacol. Ed. A. Herz104/1 p217. 33.Xu et al (1993) Peptides14 893. 34.Kosterlitz et al (1981) Br.J.Pharmacol.73 939. 35.Attali et al (1982) Neuropeptides3 53. 36.Chang et al (1984) Mol.Pharmacol.26 484. 37.Castanas et al (1985) J.Neurochem . 45 688. 38.Zukin et al (1988 ) Proc.Natl.Acad.Sci.USA 85 4061. 39.Tiberi and Magnan (1989) Can.J.Physiol. Pharmacol. 67 1336. 40.Clark et al (1989) J.Pharmacol.Exp.Ther. 251 461. 41.Paul et al (1991) J.Pharmacol.Exp.Ther.257 1. 42.Rothman et al . (1990) Peptides11 311. 43.Lai et al (1994) Neuroreport5 2161. 44.Ni et al (1995) Peptides16 1083. 45.Horanet al (1991) J.Pharmacol.Exp.Ther. 257 1154 46.Horanet al (1993) J.Pharmacol.Exp.Ther. 266 926. 47.Cvejic and Devi (1997) J.Biol.Chem.272 26959. 48.Jones et al (1998) Nature396 674. 49.White et al (1998) Nature396 679. 50.Kaupman et al (1998)396 683. 51.McLatchie et al (1998) Nature393 333-339. 52.Henderson and McKnight (1997) TiPS18 293. 53.Meunier et al (1995) Nature377 532. 54.Reinscheid et al (1995) Science270 792. 55.Meng et al (1996) J.Biol.Chem.271 32016. 56.Wang et al (1994) FEBS Lett.348 75. 57.Pan et al (1998) FEBS Lett.435 65.

9 Opioid Receptor Ligands Available from Tocris

m Receptor agonists 1171 DAMGO Selectivem agonist 1055 Endomorphin-1 Potent and selectivem agonist 1056 Endomorphin-2 Potent and selectivem agonist antagonists 0898 Irreversiblem antagonist 0516 Etonitazenyl isothiocyanate Irreversible affinity label (m selective) 0926b -Funaltrexamine Irreversiblem -selective antagonist 0591 Naloxonazine Selectivem antagonist 1

d Receptor agonists

1180 [D-Ala2 ]-Deltorphin II Selectived agonist peptide 1170 DSLET Selectived agonist peptide 0764 SNC 80 Highly selective non-peptided agonist 1008 SNC 121 Potent analogue of (0764)

R1008 [3 H]-SNC 121ÊÊb Radiolabeled form of (1008) antagonists 0827 ICI-154,129d selective peptide antagonist 0820 ICI-174,864d selective peptide antagonist 0740 Naltrindoled selective non-peptide antagonist

R740 [3 H]-NaltrindoleÊÊb Radiolabeled form of (0740) 0899 BNTX Standardd selective antagonist 1 0754 N-Benzylnaltrindoled selective non-peptide antagonist 2 0892 Naltriben Standardd selective antagonist 2

k Receptor agonists 0699 BRL-52537 Potent and selectivek agonist 0778 ICI-199,441 Potentk agonist 0822 ICI-204,448k agonist, acts peripherally 0783 N-Methyl-N-[(1S)-1-phenyl-2-(1-pyrrolidinyl)-ethyl]phenylacetamide Selectivek agonist 0700 (±)-1-(4-Trifluoromethylphenyl)acetyl-2-(1-pyrrolidinyl)methylpiperidine Very potent and selectivek agonist 0495 (±)-U-50488 Standard selectivek agonist 0471 (+)-U-50488 Less active enantiomer of (0495) 0496 (-)-U-50488 More active enantiomer of (0495) 0498 U-54494Ak agonist antagonists 0347 nor-Binaltorphimine Standardk selective antagonist 0794 DIPPA Selective irreversiblek antagonist

Orphan Opioid Receptor Ligands 0910 Nociceptin Endogenous ORL agonist 1 1092 [Phe12y (CH -NH)Gly ]Nociceptin(1-13)NH Selective nociceptin partial agonist 2 2 1118 Nocistatin (bovine) Opposes action of nociceptin 1198 Nocistatin (human) Human putative counterpart of nocistatin 1119 Nocll Orphan

Other / Miscellaneous Opioid Compounds

0840 Opioid ligand, Ca2+ 0599 Naloxone Broad spectrum opioid antagonist 0677 Naltrexone Broad spectrum opioid antagonist

Tocris Cookson Ltd. Tocris Cookson Inc. Northpoint Fourth Way 16144 Westwoods Business Park Avonmouth BS11 8TA UK Ellisville MO 63021 USA Tel: + 44 (0)117 982 6551 Tel: (800) 421-3701 Fax: + 44 (0)117 982 6552 www.tocris.com Fax: (800) 483-1993 [email protected] [email protected] [email protected]

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