ELSE V IE R Neuroscience Letters 181 (1994)47-49

NEUROSCI(NCE IEIT[RS

TIPP[ U], a highly selective ligand

L . M . V i scon t i a, K . M . S t a n d i f e r a, P.W. Schi l le r c, G . W . P a s t e r n a k a'b'*

aThe Cotzias Laboratory of Neuro-Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA bDepartments of Neurology and Neuroscience and Pharmacology, Cornell University Medical College, Ithaca, NY, USA

CLaboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, Montreal, Que., Canada

Received 9 March 1994; Revised version received 1 August 1994; Accepted 17 August 1994

Abstract TIPP[~ ~] is a new tLselective opioid peptide antagonist. In the current study, we have explored its selectivity against the g and

x receptor subtypes. Against [3H]DPDPE binding, TIPP[~/'] is quite potent, with a Ki value of < 1 nM, confirming its potent activity at t~ receptors. In contrast, its K~ values against gt,/.t2, 1¢1, x2 and I¢ 3 binding sites are all >5 gM. DPDPE also is d-selective. It labels d; sites > 25-fold more potently than g~ receptors and is even more selective against the other subtypes. However, this selectivity does not compare to the d;/g~ selectivity of TIPP[~P] which exceeds 15,000. This far higher selectivity, coupled with its antagonist properties, gives TIPP[~'] a number of advantages over previously reported t~-selective compounds. We have utilized these advantages to develop an improved g~ binding assay using TIPP[~V].

Key words: Opaite receptor;/t~ Receptor; d; Antagonist; Morphine

Over the years, a number of increasingly selective t~ opioids have been developed, most of them agonists [6- 8]. Although they have proven quite useful, the few t~-selective antagonists such as naltrindole and ICI 174864 have not had the high levels of selectivity seen previously with many of the agonists [5,10]. TIPP[~q (H-Tyr- Tic~[CH2NH]Phe-Phe-OH) is a highly selective /; antagonist, with little affinity for g or x receptors [11]. However, its affinity at several opioid receptor subtypes has not been explored. We now report the selectivity of TIPP[~t'] for the / l and I¢ receptor subtypes and an im- proved assay fo r /h sites using TIPP[~'].

All radioligands and Formula 963 scintillation fluor were purchased from New England Nuclear (Boston, MA). Peptides were obtained from Peninsula Laborato- ries (Belmont, CA), the Research Technology Branch of NIDA, or synthesized in the laboratory of P.W. Schiller. Fresh calf brains were obtained locally, dissected into the appropriate brain regions and prepared as previously

*Corresponding author. Address: Department of Neurology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA. Fax: (1) (212) 794-4332.

0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0304-3940(94)00647-4

reported [3,4]. Frozen guinea pig brains were obtained from Hilltop laboratories 0.

Standard/~,/.t2, 6, Xl and I¢ 3 binding was performed as previously reported [3,4]. I¢ 2 binding was measured using a modification of published methods [12,13] with [3H]bremazocine (1 nM) in the presence o f g (DAMGO, 100 nM), 8 (DPDPE, 100 nM) and ~c~ (U50,488H, 100 nM) blockers. All determinations were performed in tri- plicate and each experiment replicated at least 3 times. Non-specific binding was determined in the presence of levallorphan (1 /IM). All values are presented as mean + S.E.M. Ki values of unlabeled compounds were calculated: Ki = (IC5o)1(1 + S) where S = (concentration of radioligand)/(KD of radioligand) [1,2]. KD and Bmax values were determined by non-linear regression analysis of saturation data.

TIPP[~ r'] is a novel, highly 8-selective opioid peptide when examined in traditional binding assays [11]. In the current studies, we find that this selectivity extends to the /t and t¢ receptor subtypes as well (Table 1). TIPP[~t~] competes for d; sites labeled with [3H]DPDPE ([D-Pen2,D - PenS]enkephalin) ~10-fold more potently than DPDPE itself. Although DPDPE is selective for d; sites, its selec- tivity ratio against gl sites is only ~25. In contrast, the

48 L.M. Visconti et al./Neuroscience Letters 181 (1994) 4~49

Table 1 Binding selectivity of TIPP[Tq and DPDPE in opioid subtype assays of calf thalamic membranes

Binding TIPP[t/'] DP DP E assay

/<1 (nM) Selectivity K~ (nM) Selectivity

fl~ >5000 >15,000 82 ± 19 28 ~2 > 5000 > 15,000 457 ± 149 158

0 .28±0 .05 1 2 , 9 ± 0 . 7 1 x~ > 5000 > 15,000 > 350 > 120 x 2 >5000 >15,000 >500 >170 x~ > 5000 > 15,000 >350 > 120

IC5o values of T I P P [ ~ were determined competitions studies using a min imum of five concentrations. K~ values were calculated as previously noted [1,2]. Results are the mean + S.E.M. of three to five determina- t ions/compound. All binding used methods previously published [3,4]. ~t~ binding was measured with [3H]DADL in the presence of DPDPE to block g sites;,u 2 binding with [3H]DAMGO in the presence of D A D L to block Pl sites; g binding with [3H]DPDPE; x~ binding with [3H]U69,593; x2 binding with [3H]bremazocine in the presence of DPDPE, D A M G O and U50,488H to block p, d~ and i¢~ sites; and x~ binding with [3H]NalBzoH. Calf s tr iatum was used for all assays, except for tq which used guinea pig cerebellum.

affinity of TIPP[Tq for p~ sites is > 5/IM, giving a selec- tivity ratio of > 15,000. Similarly, the ratios for TIPP[~ against all the other subtypes are higher than those for DPDPE. TIPP[~ u] is the most selective ~ ligand we have examined.

The current fll assay [3] exploits the selectivity of DPDPE. At low concentrations, [3H]DADLE labels pre- dominantly ~ and ~ receptors. Including DPDPE selec- tively blocks the g component, leaving Pl binding. In most brain regions, the levels of g binding far exceed/11 binding, making it difficult to compete all the ~ binding without influencing/~l binding due to the limited select±v-

ity of DPDPE. This problem can be partially overcome by using thalamic membranes which contain high levels of/.t binding and few ~ receptors. This modest level of

binding is readily competed by low DPDPE concentra- tions with minimal effects on/11 binding. However, a more selective ~ ligand would permit a more effective blockade of ~ sites. Furthermore, antagonists are better candidates since their binding is far less sensitive to changes in binding conditions [9]. The extraordinary se- lectivity of TIPP[~ makes it an excellent candidate for the/1~ assay. Detailed competition studies of TIPP[7 t] against [3H]DADL in thatamic membranes reveal a biphasic inhibition of binding (data not shown). The

component, -15% of total specific binding, is readily competed by 5 nM TIPP[71], followed by a plateau of the competition curve extending beyond 100 nM. At 50 nM, the concentration of TIPP[~ is > 100-fold higher than its K~ against g receptors and > 100-fold less than its Ki for fll receptors. When we compare fit assays in thalamic membranes using TIPP[q'] (50 riM) to traditional ones using DPDPE, we see Ki values similar to those observed with DPDPE (Table 2). Saturation studies under both conditions also reveal similar KD and Bma × values (Table 3).

The limited selectivity of DPDPE precludes the use of other brain regions in a/,tl-selective binding assay. The high selectivity of TIPP[7 t] led us to explore whether we might be able to use membranes from brain regions other than the thalamus to assess/,t~ binding. The Ki values of most of the compounds in striatal membranes are similar to those obtained in thalamus but several compounds differ (Table 2). Equally important, several compounds have low Hill coefficients, raising the possibility that more than one class of site is being labeled. The signifi- cance of these differences is not yet clear and needs to be examined further. However, these finding do indicate

Table 2 Binding affinities of various ligands in ,u~ binding assays

Competitor Thalamus

TIPP[~V] DPDPE

Striatum

TIPP[7 ~]

gi nH Ki nH

Morphine 0.34 + 0.06 0.82 + 0.1 0.53 + 0.68 1.2 + 0.2 1.07 + 0.1 D A M G O 0.09 + 0.01 1.0 + 0.2 0.5 + 0.38 0.47 + 0.13 0.9 + 0.1 U50,488H 118 + 24 0.97 + 0.1 370 + 76 171 + 19.1 0.5 + 0.1 D A D L E 0.39 + 0.08 0.90 + 0.2 0.9 _+ 0.83 2.0 ± 0.3 1.2 + 0.3 DPDPE 70.8 + 6.0 0.92 + 0.1 82 + 19 22.4 ± 2.3 0.55 + 0.1 DSLET 1.38 + 0.2 0.57 + 0.1 1.4 + 0.82 5.9 +_ 1.0 1.15 + 0.1 ,8-Endorphin 1.06 + 0.1 1.67 + 0.4 0.98 + 0.14 6.5 + 1.51 0.45 + 0.2 [o-Ala2,Glu4]deltorphin 343 + 11 1.15 + 0.1 242 + 52 0.33 + 0.1 Naloxone 0.74 + 0.01 1.4 + 0.1 1.3 + 0.5 0.62 ± 0.11 0.45 + 0.1 Diprenorphine 0.11 + 0.01 1.5 + 0.1 0.2 +_ 0.2 0.12 +_ 0.04 0.33 + 0.1

/J~ binding assays were performed with [3H]DADL (0.7 nM) in the presence of TIP[7 t] (50 nM) using either calf striatal or thalamic membranes. IC5o values of the various compounds were determined from a min imum of five concentrations for each compound and converted to K, values as previously described [1,2]. Results are the mean + S.E.M. of three to five determinations/compound. Values from the ~ binding assays in the thalamus using DPDPE as a blocker are, in part, from the literature [3,4].

L.M. Visconti et al. INeuroscience Letters 181 (1994) 47-49 49

Table 3 Saturation analysis of/~ binding

[3H]DADLE Bm~x KD (fmol/mg protein) (nM)

with TIPP[~'] (50 nM) 2.98 _+ 0.42 0.95 + 0.16 with DPDPE (10 nM) 2.87 1.2

Saturation experiments were performed with concentrations of [3H DADLE (0.1-4 nM) in the presence of either TIPP[~g] (50 nM; n = 3) or DPDPE (10 nM; n = 1). Bm~x and KD values were determined with a non-linear regression analysis. The value with DPDPE is similar to values previously published by our laboratory [3,4].

that extrapolating the Pl assay to tissues other than thal- amus must be done cautiously.

In conclusion, these results confirm the extraordinary selectivity of TIPP[~r']. Its antagonist activity is well de- scribed in the mouse vas deferens assay. Together, these properties make TIPP[~] the desired d; blocker in Pl binding assays.

We thank Drs. Posner and Rapaka for their assistance with these studies. This work was supported, in part, by Grant DA02615 and Research Scientist Development Award DA000138 from the National Institute on Drug Abuse (to G.W. Pasternak) and Core Grant CA08748 (to the Memorial Sloan-Kettering Cancer Center).

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