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Novel Opiate Binding Sites Selective for Benzomorphan Drugs

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Novel Opiate Binding Sites Selective for Benzomorphan Drugs Proc. NatL Acad. Sci. USA Vol. 78, No. 7, pp. 4141-4145, July 1981 Biochemistry Novel opiate binding sites selective for benzomorphan drugs (enkephalins/morphine/diprenorphine/K and a receptors/morphiceptin) KWEN-JEN CHANG, ELI HAZUM, AND PEDRO CUATRECASAS Department of Molecular Biology, The Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709 Communicated by George H. Hitchings, March 30, 1981 ABSTRACT The simultaneous addition of [D-Ala2, D-Leu5]- present studies identify such benzomorphan-selective binding enkephalin and morphiceptin at concentrations at which 98% of sites by using [3H]diprenorphine and unlabeled enkephalins enkephalin (8) and morphine (it) receptors are occupied only par- and morphiceptin to quench the 8 and u receptors, respectively. tially inhibits the binding of [3H]diprenorphine to rat brain mem- branes. These conditions, furthermore, do not affect the curves for displacement of [3H]diprenorphine binding by unlabeled di- MATERIALS AND METHODS prenorphine. These data suggest that [3H]diprenorphine binds to [3H]Diprenorphine (9 Ci/mmol; 1 Ci = 3.7 x 10'° becquerels) a third subtype of opiate binding site, which has high affinity for was purchased from Amersham. Other opiates, benzomor- iliprenorphine but verylowaffinityfor ,i and8agonists. The [3H]- phans, and enkephalins were available as for previous studies diprenorphine binding observed in the presence of morphiceptin (1, 3, 7). UM 909, 2-(2-methyl-3-furylmethyl)-2'-hydroxyl-5, and [D-Ala2, D-Leu5lenkephalin exhibits high affinity for several 9a-dimethyl-6,7-benzomorphan methanesulfonate; UM 911, benzomorphan drugs in the chemical family of6,7-benzomorphan 2-(3-methylfurfuryl)-2'-hydroxyl-5,9a-dimethyl-6,7-benzomor- (e.g., cyclazocine, ethylketocyclazocine, SKF 10047, UM 1072, oxilorphan, etc). Because ofits selectivity for most benzomorphan phan methanesulfonate; UM 1070, (+)-(LR/S, 5R/S, 9S/R, drugs, this putative receptor site is tentatively referred to as a 9S/R, 5'R/S)-5,9,(3dimethyl-2'-hydroxyl-2-tetrahydrofurfur- benzomorphan binding site. Its regional distribution in rat brain yl-6,7-benzomorphan hydrochloride and UM 1072, the 9- is similar to that of morphine (#) receptors but differs from that methyl steric conformer ofUM 1070, were very kindly provided for enkephalin (8) receptors. The content ofbenzomorphan bind- by James Woods, University of Michigan. ing sites in rat brain is only one-half to one-third that ofmorphine Preparation of rat (Sprague-Dawley) brain membranes and receptors. The relative affinities of various opioids to morphine, procedures for determining distribution have been described enkephalin, andUenzomorphan binding sites are also described. (1, 3). Brain sections were homogenized in 50 mM Tris HCl (pH 7.7) and centrifuged at 40,000 x g for 30 min. The pellets Recent receptor binding studies in brain membranes indicate were subjected to a 0.1 M NaCl-containing buffer for 1 hr to that there are at least two major subtypes of opiate receptors dissociate endogenous enkephalins and washed twice with (1-6). Enkephalin (8) receptors bind enkephalins and some of Tris HCI buffer. The membranes (2 ml) were incubated with their analogues preferentially, and morphine (IL) receptors bind [3H]diprenorphine for 60-90 min in the absence and presence morphine and related alkaloids with affinities greater than those ofunlabeled ligands, quickly filtered through GF/C glass filters for enkephalins. Naloxone binds to morphine receptors with an (Whatman), and washed twice with cold buffer (10 ml). Non- affinity that is about 20 times greater than for enkephalin re- specific binding was determined with the presence of 1 uM ceptors (1, 3). Diprenorphine binds equally well to both recep- diprenorphine. Under the same conditions, no specific binding tors (1, 4, 7). Very recently, the synthetic amide of a tetrapep- can be obtained without the addition ofbrain membranes. tide fragment of 3casein, Tyr-Pro-Phe-Pro-NH2, was found to be a potent and highly specific agonist for morphine receptors, RESULTS and was thus referred to as "morphiceptin" (8). Morphine re- ceptors are localized predominantly in the hypothalamus and Apparent Homogeneity ofDiprenorphine Binding. Equilib- thalamus, whereas enkephalin receptors are highest in the stria- rium saturation binding of [3H]diprenorphine (0.1-14 nM) tum, limbic structures, and cerebral cortex (3). In vitro light shows apparent homogeneity ofbinding with a dissociation con- microscopic autoradiographic studies further confirm and ex- stant, Kd, value ofabout 0.23 nM and a maximal binding, Bmax tend these regional distributional differences (9). of 530 fmol/mg ofprotein (Fig. 1). These results are consistent On the basis of the distinctive central pharmacologic actions with otherdataindicating that diprenorphine binds to morphine of benzomorphans, Martin and colleagues proposed the possi- and enkephalin receptors with an affinity of about 0.2 nM (4, ble existence of discrete a, K, and a receptors (10, 11). The 7). The homogeneity of diprenorphine binding was confirmed occurrence of separate K and a receptors was recently ques- by displacement experiments of diprenorphine against [3H]di- tioned on the basis of binding studies using 3H-labeled ethyl- prenorphine, which show Hill coefficients (n) of unity (Fig. 2; ketocyclazocine (7, 12) and N-allylnormetazocine (SKF 10047) Hill plots are not shown). Furthermore, ifa receptor other than (12, 13). These binding studies also indicated that both the pu- the morphine (,u) or enkephalin (8) types should exist, its affin- tative K and or agonists bind with high affinities to both mor- ity for diprenorphine must exhibit a similar, if not identical, phine and enkephalin receptors (5, 7). However, these studies value. did not exclude the possible existence of a small population of Apparent Heterogeneity of Competition of [3H]Dipre- benzomorphan-selective receptor sites, possibly with affinities norphine Binding by Morphiceptin. Morphice tin is quite po- lower than those for morphine and enkephalin receptors. The tent in inhibiting the binding ofthe pu agonist [ H]dihydromor- phine, but it is weak in inhibiting the binding of the 8 agonist The publication costs ofthis article were defrayed in part by page charge '"I-labeled [D-Ala2, D-Leu5]enkephalin (Fig. 3). The 50% in- payment. This article must therefore be hereby marked "advertise- hibitory concentration (ICw) values obtained by using these la- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. beled pu and 8 agonists are 20 nM and 50 p.M, respectively. The 4141 Downloaded by guest on September 26, 2021 4142 Biochemistry: Chang et al. Proc. Natl. Acad. Sci. USA 78 (1981) -4 0 0.5 Q E._ Q ve) 0.44 8 80 0 - 0 bB a, c- 0.3 0 0-- 40 a 20- 0.2 0.4 0.6 ._v 0.1 B, pmol/mg 10-8 10-7 10-6 10-5 10-4 Morphiceptin, M 1 2 3 4 5 6 7 FIG. 3. Binding competition curves of morphiceptin against 0.12 Free [3H]diprenorphine, nM nM [3Hldihydromorphine (o), 0.1 nM 1251-labeled [D-Ala2, D-Leu5]en- kephalin (n), and 0.5 nM [3H]diprenorphine (o). The results are ex- FIG. 1. Equilibrium saturation binding of [3Hldiprenorphine to pressed as the mean of duplicate incubations, which are ± 10% of the rat brain membranes. The binding was carried out in 2 ml of 50 mM mean. Tris-HCl buffer (pH 7.7) for 90 min at room temperature. The concen- tration of [3Hldiprenorphine varied between 0.1 and 14 nM. Values are the mean of duplicate incubations. Inset is the Scatchard plot. B and Biphasic Competition Curve of Enkephalin Against [3H]- F, bound and free [3H]diprenorphine. The calculated apparent Kd is Diprenorphine Binding in the Presence of Morphiceptin. 0.23 nM and the Bmax is 530 fmol/mg of protein. The protein concen- tration was 0.45 mg/ml. Binding competition studies ofenkephalins against [3H]dipren- orphine were carried out in the absence and presence of10 ,uM morphiceptin (Fig. 4). The biphasic characteristic is readily ap- in two assays is difference the IC50 values ofthese binding about parent in the presence of and suggests that there of of morphiceptin 2500-fold. The small inhibition binding "2I-labeled [D- may be two subtypes of opiate binding sites that are labeled by Ala2, D-Leu5]enkephalin observed with low concentrations of [3H]diprenorphine under these conditions. The first phase is morphiceptin is due to the slight crossreactivity of enkephalin nearly completely inhibited at 0.1 ,uM [D-Ala2, D-Leu5]enkeph- with receptors 3). morphine (Fig. alin with an apparent Ki value of 1.8 nM. This probably re- Binding competition curves of morphiceptin against [3H]di- flects binding to enkephalin (8) receptors, because this value are About 60% of the prenorphine biphasic (Fig. 3). [3H]di- is similar to its affinity for enkephalin receptors (1, 3). The sec- prenorphine binding can clearly be inhibited by concentrations ond phase begins above 1 AM and suggests the presence of a 1 the remaining is inhibited below puM, whereas binding only third binding site with an affinity for [D-Ala2, D-Leu5]enkeph- by concentrations greater than 20 p.M. These data suggest that alin about micromolar in value. When the binding is carried only 60% of the [3H]diprenorphine-labeled sites are morphine out in the presence of10 ,uM morphiceptin and 0.1 p.M [D-Ala2, (,u) receptors. Because 10 p.M morphiceptin is 500 times above D-Leu5]enkephalin, to eliminate the binding of [3H]diprenor- its value for morphine receptors, and it does not interact with phine to both morphine and enkephalin receptors, the remain- enkephalin receptors, 10 p.M concentrations were subse- ing binding can be inhibited only by very high [Leu]enkephalin quently used to inhibit the binding of [3H]diprenorphine to concentrations (i.e., about 5 Similarly, the agonists receptors and to its IC50 ,uM).
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