[3H]SNC121: A novel high affinity ligand for rat brain delta receptors: Preliminary studies

209 [3H]SNC121: A NOVEL HIGH AFFINITY LIGAND FOR RAT BRAIN DELTA RECEPTORS: PRELIMINARY STUDIES. Q. Nil, H. Xul, J.S. Partlllal, F. Porreca2,S.N. Calderon3, K.C. Rice3, L.E. Smith3, R.W. McNutt4 and R.B. Rothmanl. lCPS, IRP, NIDA, NIH, Baltimore, MD 21224. 2Departmentof Pharmacology, University of Arizona, Tucson, AZ 85724. 3LMC, NIDDK, NIH, Bethesda, MD 20892.4BurroughsWellcome Co., ResearchTriangle Park, NC 27709. Abstract. Calderon et al. (1) recently prepared the enantiomers and their methoxy precursors of the non-peptide delta agonist (+)-BW373U86 and its benzlic epimer (2). In one of these enantiomers, SNC80, the allyl function was reduced with tritium gas to the corresponding propyl derivative to yield [3H]SNC121 (SA=26.8 Ci/mmol). Experiments with non-radioactive SNC-121 indicated that it had high affinity and selectivity (>1000-fold) for rat brain delta receptors. Incubations proceeded for 4-6 hr at 25"C (equilibrium) in 50 mM TrisHCI, pH 7.4 with rat brain membranes. [3H]SNC121 labeled two binding sites: an opioid binding site and a non-opioid drug binding site. For the experiments reported here, DADL (10 I~M) was used to define non-specific binding. Under these conditions, we obtained 70% 75% specific binding. [3H]SNC121 labeled an apparent single class of binding sites with a delta-like ligand selectivity profile. GppNHp and sodium decreased [3H]SNC121 binding by altering the Kd (P < 0.05) without changing the Bmax. GppNHp (20 i~M) failed to strikingly alter the IC50 and slope factor of DPDPE, Deitorphin-II and naltrindole when displacing [3H]SNC121. Viewed collectively, [3H]SNC121 selectively labels a single class of delta opioid receptors in a manner consistent with an agonist-type interaction. Future experiments will determine the nature and/or function of the non-opioid [3H]-SNC121 binding site. Methods. Rat brain membranes were prepared with minor modifications of published procedures (3). Ligand binding assays were conducted at 250 C for 4-6 hr, in 50 mM Tds, pH 7.4 (equilibrium). [3H]SNC121 was repurified by HPLC. Membranes aliquots (0.75 ml) resuspended in 50 mM TRIS-HCI (pH 7.4) were added to test tubes prefilled with 0.10 ml drug in 50 mM TRIS-HCI, pH 7.4, and with 0.10 ml [3H]SNC121 in a protease inhibitor cocktail, and 50 p.l of 50 mM Tds-HCI, pH 7.4, yielding a final assay volume of I ml. Triplicate samples were filtered after incubation, over glass fiber filters presoaked in 2 % PEI, using a Brandell cell harvester, and washed two times with 5 ml ice-cold 10 mM TRIS-HCI, pH 7.4. Results and Discussion. Initial experiments demonstrated that whereas SNC80 and congeners displaced over 90% of [3H]SNC121 binding, other opioids displaced at most 70% to 75%. We therefore used 10 I~M DADL to define the non-specific binding. Kinetic experiments showed that [3H]SNC121 bound reversibly and that its dissociation was best-fit by a one component model with a half-life of 86 min. A Scatchard plot of [3H]SNC121 binding was linear with a Kd of 3.8 nM and a Bmax of 369 fmol/mg protein (Figure 1). The ligand-selectivity profile of [3H]SNC121 binding site was that expected of a delta receptor. We observed the following IC50 ( nM ) and slope factor values: DADL (3.0, 0.92), DPDPE (5.8, 0.76), Deltorphin-II (8.2, 0.90), Naltrindole (0.87, 1.22), BNTX (27.4, 1.14), NTB (0.66,1.35), Oxymorphindole (1.20, 1.04), DAMGO (577, 0.93), Dermorphin (347, 0.97), NOR-BNI (65.1, 1.01), SNC80 (3.06, 1.30) and U69,593 (> 1 I~M). Most test agents displaced [3H]SNC121 binding with slope factors close to 1. Recent studies showed that the binding properties of BW373U86 are not changed in the presence of guanine nucleotides or NaCI (4,5). Direct binding studies with [3H]SNC121 show that NaCI (50 mM) and GppNHp (50 I~M) decrease binding via an increase in the Kd, not a decrease in the Bmax. In contrast, 20 I~M GppNHp modestly decreased the apparent Ki values of delta agonists and antagonists (Ki minus GppNHp, Ki plus GppNHp): DPDPE (1.79 nM, 1.64 nM), Deltorphin-II (5.01 nM, 2.42 nM) and naltrindole (0.36 nM, 0.26 nM). These data indicate that [3H]SNC121 labels a

210

single class of delta receptors in a manner consistent with that expected of an agonist-type interaction. The modest effect of GppNHp on DPDPE, Deltorphin-II and naltrindole inhibition curves support observations of the non-classical effects of guanyl nucleotides on delta receptors occupied by BW373U86-type delta agonists (4,5). The lack of heterogeneity of [3H]SNC121 binding sites is in contrast to the heterogeneity of [3H]DADL binding sites (6,7). Viewed collectively, these studies indicate that [3H]SNC121 will be a useful probe for studying delta opioid receptors.

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Figure 1 REFERENCES 1. S. N. Calderon, R. B. Rothman, F. Porreca, J. Flippen-Anderson, R. W. McNutt, H. Xu, L. E. Smith, E. J. Bilsky, P. Davis and K. C. Rice (1994) J. Med. Chem. (in press). 2. K. J. Chang, G. C. Rigdon, J. L. Howard and R. W. McNutt (1993) J. Pharmacol. Exp. Ther. 267, 852-857. 3. R. B. Rothman, H. Xu, M. Seggel, A. E. Jacobson, K. C. Rice, G. A. Bdne and F. I. Carroll (1991) LJ'fe Sci. 48, PL111-PL116. 4. S. R. Childers, L. M. Fleming, D. E. Selley, R. W. McNutt and K. J. Chang (1993) Mol. Pharmacol. 44, 827-834. 5. K. D. Wild, L. Fang, R. W. McNutt, K. J. Chang, G. Toth, A. Borsodi, H. I. Yamamura and F. Porreca (1993) Eur. J. Pharmacol. 246, 289-292. 6. H. Xu, J. S. Partilla, B. R. de Costa, K. C. Rice and R. B. Rothman (1992) Peptides13, 1207-1213. 7. H. Xu, J. S. Partilla, B. R. de Costa, K. C. Rice and R. B. Rothman (1993) Peptides14, 893907.