|

Synthesis and characterization of [3h]-sn56, a novel radioligand for the σ1 receptor

Contents lists available at European Journal of Pharmacology Molecular and Cellular Pharmacology Synthesis and characterization of [3H]-SN56, a novel radioligand for the σ1 receptor James A. Fishback , Christophe Mesangeau , Jacques H. Poupaert Christopher R. McCurdy , Rae R. Matsumoto ,a Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, WV 26506, USAb Department of Pharmacology, University of Mississippi, University, MS 38677, USAc Department of Medicinal Chemistry, University of Mississippi, University, MS 38677, USAd Universite Catholique de Louvain, Avenue Emmanuel Mounier 74, B-1200, Brussels, Belgium The study of the binding characteristics of σ ligands in vivo and in vitro requires radiolabeled probes with high Received 22 May 2010 affinity and selectivity. The radioligand presently used for in vitro studies of the σ1 receptor, [3H](+)- Received in revised form 30 September 2010 pentazocine, has significant limitations; it is difficult to synthesize, has limited chemical stability, and can be Accepted 6 October 2010 problematic to obtain. Evaluation of a series of novel 2(3H)-benzothiazolone compounds revealed SN56 to Available online 2 December 2010 have sub-nanomolar and preferential affinity for the σ1 subtype, relative to σ2 and non-sigma, binding sites.
The goal of this study was to characterize the binding of [3H]-SN56 to σ 1 receptors isolated from rat brain.
Radioligand binding assay Standard in vitro binding techniques were utilized to 1) determine the specificity and affinity of binding to σ1 receptors, 2) confirm that [3H]-SN56 labels sites previously identified as σ1 by comparing binding to siteslabeled by [3H](+)-pentazocine, and 3) characterize the kinetics of binding. The results indicate that [3H]-SN56 exhibits 1) specific, saturable, and reversible binding to the σ1 receptor, with Bmax = 340 ± 10 fmol/mgand Kd = 0.069 ± 0.0074 nM, 2) competitive displacement by classical sigma compounds, yielding σ1 Ki valuesconsistent with those reported in the literature, and 3) binding kinetics compatible with a 90 min incubation,and filtration for separation of free and bound radioligand. The results of these studies suggest that [3H]-SN56may serve as a viable alternative to [3H](+)-pentazocine in radioligand binding assays.
2010 Elsevier B.V. All rights reserved.
efforts to identify novel selective compounds are ongoing. While theσ2 receptor may also represent a feasible drug development target, The σ receptor was first identified as an opioid receptor subtype further research in this area will require the discovery of additional based on behavioral studies of morphine-like drugs in dogs selective ligands for this subtype. The focus of the current work is Subsequent in vitro binding data revealed that this site therefore limited to the characterization of σ1 binding.
represents a new class of non-opioid receptor (To date, first described [3H](+)-pentazocine, a highly two subtypes of σ receptors (σ1 and σ2) have been identified based on selective radioprobe for σ1 receptors. Subsequent studies demon- differences in ligand selectivity, tissue distribution, and molecular strated that [3H](+)-pentazocine labeled a single class of sites in characterization. The σ1 receptor has been cloned from multiple guinea-pig brain that correlated with the profile observed following labeling with the prototypic σ1 probe [3H](+)-3-PPP ) and a significant ). [3H](+)-Pentazocine exhibited low levels of non-specific number of ligands with high affinity and selectivity for it are available.
binding and high affinity for σ1 receptors (Kd=4.8±0.4 nM), with The σ2 receptor is less well characterized; it has not been cloned, and N700 fold preference for the σ1 over the σ2 subtype ().
few specific ligands have been described.
[3H](+)-Pentazocine does however exhibit shortcomings, including The σ1 receptor is involved in numerous physiological processes poor chemical stability, that limit its usefulness in routine studies.
and disease states, and in vivo and in vitro studies indicate that Efforts to design new σ1 specific ligands have produced a limited modulation of σ1 receptors using σ specific ligands can affect these number of radioprobes useful for exploring the pharmacology of the σ1 receptor. Unfortunately, like [3H](+)-pentazocine, each of the ). Consequently, the σ1 receptor proposed new radioprobes exhibit limtations. Therefore, we sought to is recognized as a potential medication development target and characterize the performance of SN56, a novel σ1 selective, 2(3H)-benzothiazolone compound, as a tritiated radioligand for use in σ1competition binding experiments. SN56 exhibited sub-nanomolar ⁎ Corresponding author. West Virginia University, School of Pharmacy, P.O. Box 9500, Morgantown, WV 26506, USA. Tel.: +1 304 293 1450; fax: +1 304 293 2576.
finity (Kiσ1=0.56 nM) and N1000 fold selectivity for the σ1 subtype E-mail address: (R.R. Matsumoto).
relative to σ2 and at least 350 times greater affinity for the σ1 receptor 0014-2999/$ – see front matter 2010 Elsevier B.V. All rights reserved.
doi: J.A. Fishback et al. / European Journal of Pharmacology 653 (2011) 1–7 versus a battery of common receptors and transporters ( was dissolved in ethyl acetate and the solvent was washed with water These binding characteristics coupled with a simple and and brine, dried and evaporated. The residue was recrystallized from economical synthetic scheme suggested [3H]-SN56 may provide a toluene/dioxane (2/1) to give 2.96 g (54%) of 6-propionylbenzo[d] viable alternative to [3H](+)-pentazocine in competition binding thiazol-2(3H)-one as a white solid. 1H NMR (DMSO-d6): δ 12.23 (br s, studies of the σ1 receptor.
1H), 8.20 (s, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.17 (d, J = 8.3 Hz, 1H), 2.97(q, J = 7.1 Hz, 2H), 1.06 (t, J = 7.1 Hz, 3H). 13C NMR (DMSO-d6): δ 2. Materials and methods 198.64, 170.46, 140.13, 131.21, 126.74, 123.69, 123.04, 111.17, 30.93,8.22. MS (ESI) m/z 206 (M+-1).
2.2.2. 6-Propylbenzo[d]thiazol-2(3H)-one (2) Reagents and starting materials for the synthesis of SN56 were Triethylsilane (4.75 ml, 29.75 mmol) was added to a stirred solution obtained from commercial suppliers and were used without purifica- of 1 (2.5 g, 12.06 mmol) in trifluoroacetic acid (13 ml). The mixture was tion. Precoated silica gel GF Uniplates from Analtech (Newark, DE) were stirred vigorously for 2 h at room temperature. The trifluoroacetic acid used for thin-layer chromatography (TLC). Column chromatography was removed by evaporation and the residue was purified by was performed on silica gel 60 (Sorbent Technologies, Atlanta, GA). 1H chromatography on a silica gel column using petroleum ether/ethyl and 13C NMR spectra were obtained on a Bruker APX400 (Billerica, MA) acetate (9:1) as the eluent to give 2.06 g (88%) of 6-propylbenzo[d] at 400 and 100 MHz, respectively. High resolution mass spectra (HRMS) thiazol-2(3H)-one as a white solid. 1H NMR (DMSO-d6): δ 11.76 (br s, were recorded on a Waters Micromass Q-Tof Micro mass spectrometer 1H), 7.33 (s, 1H), 7.05 (d, J = 8.1 Hz, 1H), 6.99 (d, J = 8.1 Hz, 1H), 2.50 with a lock spray source (Milford, MA). Mass spectra (MS) were (t, J = 7.4 Hz, 2H), 1.56–1.50 (m, 2H), 0.84 (t, J=7.3 Hz, 3H). 13C NMR recorded on a Waters Acquity Ultra Performance LC (Milford, MA) with (DMSO-d6): δ 169.99, 136.68, 134.23, 126.57, 123.24, 122.12, 111.22, ZQ detector in ESI mode. Elemental analysis (C, H, N) was performed on 36.87, 24.30, 13.54. MS (ESI) m/z 194 (M++1).
a Perkin-Elmer CHN/SO Series II Analyzer (Waltham, MA). Chemicalnames were generated using ChemDraw Ultra (CambridgeSoft, version 10.0, Cambridge, MA).
Bromine (0.45 ml, 8.75 mmol) was added slowly to a solution of 2 [3H](+)-Pentazocine (29 Ci/mmol) was purchased from Perkin- (1.5 g, 7.76 mmol) in acetic acid (10 ml). The mixture was stirred 15 h Elmer (Boston, MS). (+)-Pentazocine, (−)-pentazocine, haloperidol, at room temperature, poured into water and extracted with ethyl 1,3-di-o-tolylguanidine (DTG), bovine serum albumin (BSA) fraction acetate (3 × 30 ml). The combined organic layers were washed with a V, sucrose, sodium chloride, tris(hydroxymethyl)aminomethane, 1N 10% solution of potassium carbonate followed by brine. The solution hydrochloric acid solution, and glacial acetic acid were purchased was dried over sodium sulfate and evaporated under vacuum. The from Sigma-Aldrich (St. Louis, MO). Bio–rad Protein Assay reagent residue was purified by chromatography on a silica gel column using was purchased from Bio–rad (Hercules, CA). Ecoscint scintillation petroleum ether/ethyl acetate (9:1) as the eluent to give 0.5 g (24%) fluid and Brandel GF/B filter papers, 2.25×12.25 in. were purchased of 4-bromo-6-propylbenzo[d]thiazol-2(3 H)-one as a white solid.
from Fisher Scientific (Pittsburgh, PA).
1H NMR (CDCl3): δ 9.29 (s, 1H), 7.26 (s, 1H), 7.12 (s, 1H), 2.56(t, J = 7.6 Hz, 2H), 1.62 (m, 2H), 0.93 (t, J = 7.2 Hz, 2H). 13C NMR 2.2. Synthesis of [3H]-SN56 (CDCl3): δ 170.45, 139.35, 132.22, 129.34, 124.44, 121.24, 103.90,37.37, 24.48, 13.54. MS (ESI) m/z 270 (M+−1), 272 (M++1).
The design strategy for generating [3H]-SN56 involved replacing a bromine atom on the aromatic ring of SN56 with a tritium atom (The preparation of the brominated precursor 4 is described (3H)-one hydrochloride (4) below. Compounds 1 and 2 were prepared according to previously NaHCO3 (0.51 g, 6.09 mmol) and 2-(hexamethyleneimino) described procedures with minor modifications ( ethylchloride hydrochloride (0.80 g, 4.06 mmol) were added, with ). Selective bromination of the 6-propylbenzo[d] mechanical stirring, to a solution of 3 (0.55 g, 2.03 mmol) in thiazol-2(3H)-one 2 at the C-4 position was effected with bromine in anhydrous DMF (15 ml). The reaction mixture was heated to 80 °C acetic acid at room temperature. The bromo derivative was alkylated for 1 h. After cooling, the mixture was poured into 80 ml of water, with 2-(hexamethyleneimino)ethylchloride in the presence of potas- extracted with ethyl acetate (3 × 60 ml), and the combined organic sium carbonate in DMF to yield 4. Compound 4 was radiolabeled with layers were washed with brine and dried. The solvent was removed in tritium (30 Ci/mmol) by AmBios Labs, Inc. (Newington, CT).
vacuo, and the residue was chromatographed on a silica gel columnusing ethyl acetate/petroleum ether (4:6) as the eluent. 3-(2- 2.2.1. 6-Propionylbenzo[d]thiazol-2(3H)-one (1) Dimethylformamide (5.96 ml, 76.73 mmol) was added slowly to was isolated as a hydrochloride salt (white solid, 0.44 g, 49%) by aluminium chloride (35.5 g, 264.6 mmol) with vigorous stirring. After addition of HCl/dioxane. 1H NMR (DMSO-d6): δ 10.81 (br s, 1H), 7.60 15 min, 2-hydroxybenzothiazole (5.4 g, 40 mmol) was added and the (s, 1H), 7.46 (s, 1H), 4.72 (t, J = 7.2 Hz, 2H), 3.47–3.24 (m, 6H), 2.56 mixture was heated to 45 °C. After 15 min, propionyl chloride (t, J = 7.5 Hz, 2H), 1.86 (br s, 4H), 1.67–1.55 (m, 6H), 0.88 (3.46 ml, 39.7 mmol) was added and the reaction mixture was heated (t, J = 7.2 Hz, 3H). 13C NMR (DMSO-d6): δ 170.54, 138.87, 132.87, to 85 °C for 3 h. The hot mixture was then poured on ice; the crude 132.63, 125.62, 121.79, 103.79, 56.73, 55.89, 43.14, 37.04, 28.58, product was collected by filtration, and washed with water. The solid 27.22, 24.51, 13.81. Anal. calcd for C18H26 BrClN2OS: C, 49.83; H, 6.04;N, 6.46. Found: C, 50.06; H, 5.93; N, 6.47. HRMS calcd for C18H26N2-OSBr [M + H]+ 397.0949, found 397.0945.
A synopsis of the synthetic route of the synthesis of the brominated [3H]-SN56 precursor is provided in .
2.3. Membrane preparation Crude P2 rat brain homogenates were prepared from male, Sprague Dawley rats (150–200 g) purchased from Harlan (Indianapolis, IN) as Fig. 1. Structure of SN56, [3H]-SN56 and its bromo precursor 4.
described previously (). All procedures involving J.A. Fishback et al. / European Journal of Pharmacology 653 (2011) 1–7 Fig. 2. Synthesis of brominated [3H]-SN56 precursor 4: A) propionyl chloride, AlCl3, 85 °C; B) (C2H5)3SiH, CF3COOH, rt; C) Br2, CH3COOH, rt; d) 2-(hexamethyleneimino)ethylchloridehydrochloride, K2CO3, DMF, 80 °C.
live animals were performed as approved by the Institutional Animal 2.4.1. Association and dissociation assays Care and Use Committee at the locations where the assays were Association and dissociation studies were conducted to confirm performed. Briefly, unanesthetized rats were sacrificed by decapitation; that the binding kinetics of [3H]-SN56 were appropriate for a 1–2 h brains minus cerebellum were harvested and maintained in ice cold incubation and processing by filtration. Kinetic studies were 10 mM Tris–HCl/0.9% NaCl until processed. Tissues were homogenized performed with 0.8 nM [3H]-SN56 and 100 μg membrane. For with a Potter-Elvehjem homogenizer (5–10 strokes with motor driven determination of association rates, samples were incubated for Teflon pestle) in ice-cold 10 mM Tris–sucrose buffer (0.32 M sucrose in times ranging from 5 min to 2 h prior to filtration. For determination 10 mM Tris–HCl, pH 7.4) using 10 ml buffer per gram of tissue× 3 g of dissociation rates, membranes were incubated for 120 min with tissue/batch. Homogenates from multiple batches were combined and [3H]-SN56 prior to the addition of 100 μM (final concentration) centrifuged for 10 min at 1000 ×g, at 4 °C. Supernatants were decanted, haloperidol, followed by filtration at times ranging from 30 min to 4 h combined and centrifuged for 15 min at 31,000 ×g, at 4 °C. To reduce from the addition of haloperidol. The assays were performed in levels of bound endogenous ligand(s), the material from centrifugation duplicate and repeated three times.
at 31,000 ×g was washed as follows: 1) pellets were re-suspended in10 mM Tris–HCl, pH 7.4 using 3 ml buffer per gram of tissue, 2) the 2.4.2. Saturation binding assays resulting suspension was incubated for 30 min at 25 °C, 3) following For the determination of Kd and Bmax by saturation binding, ten incubation, the suspension was centrifuged for 15 min at 31,000 ×g, at concentrations ranging from 0.01 to 0.8 nM of [3H]-SN56 were tested 4 °C. The resulting pellets were re-suspended in Tris–HCl, pH 7.4 buffer per experiment with 100 μg membrane per sample. Non-specific at a final concentration of 1 g of tissue per 1.53 ml buffer. Tissue binding was determined by the addition of haloperidol, at a 100 μM preparations were aliquoted in 1 ml portions and stored at −80 °C. The final concentration. Samples for the determination of total and non- Bradford assay was used to quantitate protein concentration specific binding for each experiment were run concurrently and 2.4.3. Competition binding assays 2.4. Radioligand binding assays For the determination of Ki for established σ ligands by competition binding, aliquots of membrane were incubated with Initial optimization of assay conditions was performed to maximize [3H]-SN56 and varying concentrations of test ligands. The following total binding and minimize non-specific binding. Parameters examined test compounds were evaluated: DTG, haloperidol, (+)-pentazocine, included evaluation of the buffer composition and pH, ratio of (−)-pentazocine. For each test compound, 10 concentrations were radioligand to membrane concentration, and determination of ligand incubated with 0.7–0.8 nM [3H]-SN56 with 100 μg membrane per and ligand concentration for defining non-specific binding. The sample. Non-specific binding was determined by the addition of following optimized conditions were used for subsequent studies haloperidol, 100 μM final concentration. Samples for the determina- reported below: 0.5 ml final sample volume, 90 min sample incubation tion of total and non-specific binding for each experiment were run at 25 °C, Tris–HCl pH 8.0 (assay buffer), and 10 μM haloperidol (to define concurrently and filtered simultaneously.
non-specific binding). Assay termination was effected by vacuumfiltration through glass fiber filters on a 24 position Brandel cell 2.5. Scintillation counting and data analysis harvester. Prior to use, filters were presoaked for 30 min in 0.5%polyethyleneimine to reduce non-specific binding. Following the initial Following washing, filters were transferred to scintillation vials filtration step, filters were washed in triplicate with 5 ml ice-cold and 5 ml scintillation cocktail was added. Filters were allowed to soak 10 mM Tris–HCl, pH 8. The conditions determined from the preliminary in cocktail for a minimum of 10 h prior to counting.
studies were consistent with those reported in the literature for the The data were analyzed using GraphPad Prism software (San analysis of σ1 receptor binding using [3H](+)–pentazocine ( Diego, CA). Saturation binding data were fit using nonlinear regression to a one site model. Association kinetics data were fit noted that it was necessary to prepare [3H]-SN56 spiking solutions in using linear regression of the plot of ln(Be − B/Be) versus time, where 1 mM HCl, to prevent non-specific binding of the radioligand to glass Be = radioligand bound at equilibrium, and B = radioligand bound at and plasticware, which was problematic with solutions prepared in the time t; the slope of the plot yielded kobs. The association rate constant assay buffer. This requirement is not unprecedented and the small (k+1) was calculated using the pseudo first-order method from the amount of acid has no impact on the final pH of the assay sample equation k+1 = (kobs − k−1)/[L], where [L]= radioligand concentra- tion. Dissociation kinetics data were fit using linear regression of the J.A. Fishback et al. / European Journal of Pharmacology 653 (2011) 1–7 plot of ln(B/B0) versus time, where B0 = specific radioligand bound at time of addition of haloperidol, and B = specific radioligand bound attime t. For competition binding data, K i values were calculated from experimentally determined IC 50 values using the Cheng–Prusoff equation using the K d for [3H]-SN56 determined from the saturation binding experiments (0.069 nM).
H]-SN56 Bound (fmole/mg)
3.1. Basic binding parameters At near saturating conditions, non-specific binding of [3H]-SN56 remained constant in the presence of 25 to 200 μg membrane, Fig. 4. Saturation curve for [3H]-SN56 in rat brain membranes. Samples contained suggesting that the observed non-specific binding is due primarily to 100 μg membrane in a total volume of 0.5 ml. Data points represent the mean ± SEM of radioligand binding to the filter. Total binding was linear from 50 to three independent determinations of duplicate samples at each concentration.
Bmax = 340 ± 10 fmol/mg, Kd = 0.069 ± 0.007 nM and r2 = 0.96.
200 μg membrane ).
3.2. Association and dissociation kinetics Characterization of a series of novel 2(3H)-benzothiazolone From the association studies, kobs=0.080 min−1 and k+1=9.05× compounds in σ receptor competition binding assays revealed SN56 107 min−1 M−1. From the dissociation studies, k−1=0.0076 min−1 and t1/2=91 min. The Kd calculated from k−1/k+1 was 0.084 nM. The low have sub-nanomolar affinity and N1000 fold selectivity for the σ1 dissociation rate permits the use of filtration for the separation of free from subtype relative to σ2 (Binding of SN56 to non-σ bound radioligand (while the association rate binding sites was tested with a battery of receptors and transporters supports 90 min incubations for attaining steady-state binding.
including, adrenergic α1, adrenergic α2, adrenergic β1, adrenergic β2,histamine H1, histamine H2, mu opioid, delta opioid, kappa opioid,dopamine D 3.3. Saturation binding 1, dopamine D2, serotonin 5HT2a, serotonin 5HT3, GABAA, dopamine transporter, and serotonin transporters. Of the binding sitestested, only α shows the results of the saturation binding study of [3H]-SN56.
and H1 showed affinities greater than 1 μM The binding affinity of [3H]-SN56 was K iα2 = 205 nM, and KiH1 = 311 nM respectively) d = 0.069 ± 0.007 nM which however, the affinity of SN56 for the σ represents a 70 fold higher affinity than reported for [3H](+)- 1 receptor is approximately 350 times higher than its affinity for either of these receptors, indicating a pentazocine (Kd =4.8 nM) Receptor density favorable selectivity profile for the development of a radioprobe for (Bmax) as determined by saturation binding with [3H]-SN56 was 340± use in radioligand binding studies.
In the present study, [3H]-SN56 exhibited N95% specific binding to σ1 in rat brain membranes at concentrations up to 10 times the Kd.
3.4. Competition binding However, non-specific binding of [3H]-SN56 to the glass fiber filtersused to separate bound from free radioligand was 25–35% at 10 times Tabulated values of Kis determined in this study using [3H]-SN56 the Kd concentration, resulting in a final specific binding signal of versus values reported by using [3H](+)- 65–75% of total observed binding. [3H]-SN56 exhibited saturable and pentazocine are shown in . In a comparison of binding reversible binding to a single high affinity site in rat membranes with profiles of the sites labeled by [3H]-SN56 versus [3H](+)-pentazocine a binding profile similar to that observed for [3H](+)-pentazocine.
is shown in a correlation plot of Ki values obtained experimentally The Bmax observed for [3H]-SN56 (340 ± 10 fmol/mg) was consistent versus values reported by For the group of with the range of values reported in the literature for rat brain labeled hallmark σ ligands tested, there was a significant correlation between with [3H]-BHDP, [3H]-SA4503, or [3H](+)-pentazocine ( the affinities obtained using the novel versus conventional radioli- ). In addition, the Ki values of classical gand. Of particular note is the higher affinity of (+)-pentazocine as σ compounds in competition binding assays against [3H]-SN56 were compared to (−)-pentazocine in the assays, a stereoselectivity consistent with those reported in the literature against the well pattern that is consistent with binding to σ1 receptors.
established σ1 radioligand [3H](+)-pentazocine ().
The association and dissociation kinetics of [3H]-SN56 were also shown to be amenable for filtration assays.
reported a Ki of 0.56 nM for SN56 versus [3H](+)- 2 membranes prepared from guinea pig brain. Similarly, we obtained a Ki of 0.38 nM versus [3H](+)-pentazocine in P2 Summary of data from competition binding experiments.
H]-SN56 Bound (fmole)
Fig. 3. Total and non-specific binding of [3H]-SN56 to rat brain membranes. Samples contained 0.7 nM [3H]-SN56, in a total volume of 0.5 ml. Data points represent the meanof three independent determinations of duplicate samples at each concentration.
a Bowen et al., 1993.
J.A. Fishback et al. / European Journal of Pharmacology 653 (2011) 1–7 amounts from 5–50 μg in volumes from 50–1750 μl, and conditionswhere the ratio of receptor concentration ([receptor]) to Kd were from0.42– 147. As the volume was decreased or the amount of membrane was increased in these studies, the calculated Kd and Bmax increased.
H]-SN563 1
When no corrections were made for radioligand depletion, with [receptor]/Kd = 0.42 (the lowest ratio tested, corresponding to 5 μg in 1750 μl) the calculated Kd was approximately 2.3 times the "true"value, and with [receptor]/Kd = 1.25 the calculated Kd was approxi- mately 2.8 times the true value. When the Kds were recalculated,taking into account radioligand depletion, the resulting values were 2.1 and 3.9 times the true value, respectively.
log K (nM) [3H]-(+)-Pentazocine
The conditions chosen for our saturation studies utilized a [receptor]/Kd of 1. No corrections were made for radioligand Fig. 5. Comparison of Ki values determined experimentally with [3H]-SN56 versus depletion because it was not possible to accurately assess what values determined with [3H](+)-pentazocine by r2 = 0.89. 1)1haloperidol, 2) (+)-pentazocine, 3) (−)-pentazocine, 4) DTG.
portion of non-specific binding was due to binding to membraneversus binding to filter. Based on Carter's work, we might expect our membranes prepared from rat brain (data not shown). These values are results to overestimate the Kd of [3H]-SN56 by 2 to 3 fold.
5 fold higher than the affinity determined with saturation and kinetic Carter's studies for competition binding did not model the studies of [3H]-SN56 ( 0.07 nM and 0.08 nM respectively). We suspect approach we utilized, with [receptor] = Kd[3H]-SN56 and concentra- that depletion of the non-labeled ligand results in an erroneously high tion [3H]-SN56 = 10 times Kd[3H]-SN56, so useful comparisons to their value for the Ki of SN56 as determined by competition binding.
data are not possible. However, as stated previously, the Ki values we Systematic errors resulting from the use of high receptor derived for the σ ligands tested correlated well with values reported concentrations may also contribute to errors in both the determina- in the literature.
tion of Kd from saturation and kinetic experiments, and in the No significant difference in binding affinities was observed determination of Kis of un-labeled compounds. However, these errors following labeling with [3H](+)-pentazocine versus [3H]-SN56 for are minimal, quantifiable, and in practice when [3H]-SN56 is used for the ligands tested. A larger pool of compounds will need to be competition binding assays of σ1 ligands, our preliminary results screened to confirm that this relationship is maintained. However, suggest they have no impact on our Ki determinations as compared to compounds that show significant differences in affinity following historical data for the compounds tested.
labeling with the two different radioligands may provide valuable While most researchers are familiar with radioligand depletion due insight into differences in the binding of σ ligands from different to excessive receptor concentration, depletion of the un-labeled ligand chemical classes.
occurs when the affinity of the un-labeled compound greatly exceeds [3H]-SN56 appears to provide similar binding characteristics the affinity of the labeled compound compared to [3H](+)-pentazocine with some noteworthy advan- ). used computer modeling to tages. Synthetically, [3H]-SN56 can be produced more easily and in derive estimates of the error in the determination of Ki of ligands significantly higher yields than [3H](+)-pentazocine. The chemical exhibiting higher affinities than the radioligands utilized in their stability of [3H]-SN56 is also expected to be greater than that of [3H] measurement; the authors projected that for an un-labeled ligand (+)-pentazocine, which degrades over time, resulting in increased with a true affinity 100 times greater than the radioligand (as in our background levels.
case), 10% radioligand depletion would result in an experimentally A number of radiolabeled σ1 ligands have been reported in the determined Kd 6 times higher than the true value. Thus, the 5 fold literature but none have been widely accepted as a replacement for difference between the affinity of SN56, as determined by competition [3H](+)-pentazocine in competition binding studies. The two best binding, and the affinity of [3H]-SN56 determined with saturation and candidates, SA4503 () and BHDP ( kinetic studies may be explained by this phenomena.
), have been studied in rat brain membranes in tritiated and un- Practical considerations dictated that we use relatively high receptor labeled forms and exhibit approximately 100 fold higher affinity for concentrations; this introduces systematic error that is quantifiable and σ1 versus σ2 with no significant binding to other common receptors or within acceptable limits. When possible, experimental conditions for transporters However, binding experiments should be chosen so that the receptor concentra- differences in relative expression of σ1 versus σ2 in disease states, tion is less than 10% of the Kd of the radioligand to minimize radioligand tissues, or cell lines may compromise the accurate analysis of σ1 depletion However, with a radioligand with sub- binding with these radioligands since their σ1/σ2 selectivity ratios just nanomolar affinity this would require multi-milliliter sample volumes.
meet the 100 fold difference generally accepted as the minimum Our experiments required 100 μg of tissue to obtain adequate signal for difference required for discriminating receptor subtypes. Because precise detection. We chose 0.5 ml sample volumes because we intend [3H]-SN56 displays a N1000 fold higher affinity for σ1 versus σ2, to adapt this method to a higher throughput 96-well method where changes in σ expression would not impact measurements performed sample volumes are more limited than in test tube based binding with this radioligand as much as with the other proposed alternatives.
determinations. To ensure that N90% of added radioligand was "free" In vitro binding studies with receptor specific radioligands have (unbound) in competition binding experiments run under these historically been important in the discovery and characterization of conditions, we utilized high concentrations of [3H]-SN56. While these receptors, and continue to play a central role in the process of drug conditions are not ideal, they are tolerated if required for detection and discovery. Application of this technology to the σ receptor has the error in values obtained with the method are known and within an resulted in: 1) identification of σ receptors as a unique receptor type acceptable range ( ), 2) confirmation that σ receptors have at least examined the effects of assay miniaturization two subtypes (and 3) using the human muscarinic M3 receptor expressed in CHO cells. This determination of its anatomical distribution ( cell line expressed receptor at 5 pmol/mg, and the novel radioligand Radioligand binding tested, [3H]-NMS (1-[N-methyl-3H] scopolamine methyl chloride) studies continue to play a primary role in selecting σ ligands for in vivo had an affinity of 0.42 pM. In saturation binding studies, receptor testing because there are no widely accepted in vitro functional assays concentrations were varied over a wide range, with membrane for σ1 activation.
J.A. Fishback et al. / European Journal of Pharmacology 653 (2011) 1–7 In addition to its service in the initial characterization of the σ Carter, C.M., Leighton-Davies, J.R., Charlton, S.J., 2007. Miniaturized receptor binding assays: complications arising from ligand depletion. J. Biomol. Screen. 12, 255–266.
receptor, radioligand binding data has contributed to the elucidation Chang, K.J., Jacobs, S., Cuatrecasas, P., 1975. Quantitative aspects of hormone–receptor of 1) structural elements that define high affinity σ1 ligands, and 2) interactions of high affinity. Effect of receptor concentration and measurement of specific amino acids of the σ dissociation constants of labeled and unlabeled hormones. Biochim. Biophys. Acta 1 protein that are critical for selective, 406, 294–303.
high affinity binding of known ligands ( Cobos, E.J., Entrena, J.M., Nieto, F.R., Cendan, C.M., Del Pozo, E., 2008. Pharmacology and therapeutic potential of sigma1 receptor ligands. Curr. Neuropharmacol. 6, 344–366.
Studies correlating structure with binding affinity are ongoing; de Costa, B.R., Bowen, W.D., Hellewell, S.B., Walker, J.M., Thurkauf, A., Jacobson, A.E., numerous examples of such labors have been reported in the Rice, K.C., 1989. Synthesis and evaluation of optically pure [3H]–(+)–pentazo-cine, a highly potent and selective radioligand for sigma receptors. FEBS Lett. 251, literature, with the majority of effort focused on identifying subtype specific ligands ( de Costa, B., Radesca, L., Dominguez, C., Di Paolo, L., Bowen, W.D., 1992. Synthesis and receptor binding properties of fluoro- and iodo-substituted high affinity sigmareceptor ligands: identification of potential PET and SPECT sigma receptor imaging σ1 Radioligands have also demonstrated utility in radio-imaging agents. J. Med. Chem. 35, 2221–2230.
studies; several examples of the successful use of the σ1 radioligand Glennon, R.A., 2005. Pharmacophore identification for sigma-1 (σ1) receptor binding: [11C-SA4503] as a positron emission tomography (PET) imaging agent application of the "deconstruction−reconstruction−elaboration" approach. MiniRev. Med. Chem. 5, 927–940.
have been reported in recent years ( Goldstein, A., Barrett, R.W., 1987. Ligand dissociation constants from competition binding Displacement of [11C]-SA4503 by assays: errors associated with ligand depletion. Mol. Pharmacol. 31, 603–609.
Guitart, X., Codony, X., Monroy, X., 2004. Sigma receptors: biology and therapeutic 1 active antidepressant fluvoxamine confirmed that fluvoxamine potential. Psychopharmacology (Berl.) 174, 301–319.
occupies σ1 receptors in healthy human male volunteers ( Hanner, M., Moebius, F.F., Flandorfer, A., Knaus, H.G., Striessnig, J., Kempner, E., Other studies showed reduced σ1 receptor density in Glossmann, H., 1996. Purification, molecular cloning, and expression of the defined brain regions of Alzheimer's and Parkinson's patients mammalian sigma1-binding site. Proc. Natl Acad. Sci. USA 93, 8072–8077.
Hashimoto, K., Ishiwata, K., 2006. Sigma receptor ligands: possible application as (). Also, supporting a role for σ1 in cancer, therapeutic drugs and as radiopharmaceuticals. Curr. Pharm. Des. 12, 3857–3876.
reported that steroid hormones that are Hellewell, S.B., Bowen, W.D., 1990. A sigma-like binding site in rat pheochromocytoma (PC12) cells: decreased affinity for (+)-benzomorphans and lower molecular ligands displace [11C]-SA4503 from tumors in rats (). Thus, these studies suggest σ weight suggest a different sigma receptor form from that of guinea pig brain. Brain Res. 527, 244–253.
may hold significant diagnostic value for psychiatric diseases and Hellewell, S.B., Bruce, A., Feinstein, G., Orringer, J., Williams, W., Bowen, W.D., 1994. Rat liver and kidney contain high densities of σ1 and σ2 receptors: characterization by Future advances in the field of σ receptor therapeutic development ligand binding and photoaffinity labeling. Eur. J. Pharmacol. 268, 9–18.
Holl, R., Schepmann, D., Frohlich, R., Grunert, R., Bednarski, P.J., Wunsch, B., 2009.
will require greater knowledge of the nature of the interaction of the σ Dancing of the second aromatic residue around the 6, 8-diazabicyclo[3.2.2]nonane receptor with its ligands and protein binding partners. Additional framework: influence on sigma receptor affinity and cytotoxicity. J. Med. Chem. 52, tools needed to further this knowledge include new subtype specific Ishikawa, M., Ishiwata, K., Ishii, K., Kimura, Y., Sakata, M., Naganawa, M., Oda, K., agonist and antagonist ligands, radioligands, and other affinity labels Miyatake, R., Fujisaki, M., Shimizu, E., Shirayama, Y., Iyo, M., Hashimoto, K., 2007.
and probes. The development of other technologies, such as high High occupancy of sigma-1 receptors in the human brain after single oral throughput methods for the determination of binding affinities, and in administration of fluvoxamine: a positron emission tomography study using[11C]SA4503. Biol. Psychiatry 62, 878–883.
vitro functional assays, will also hasten efforts to design and identify Ishiwata, K., Kobayashi, T., Kawamura, K., Matsuno, K., 2003. Age-related changes of the new selective σ ligands with potential therapeutic value.
binding of [3H]SA4503 to sigma1 receptors in the rat brain. Ann. Nucl. Med. 17, In conclusion, the results of our studies suggest that [3H]-SN56 Jansen, K.L., Dragunow, M., Faull, R.L., Leslie, R.A., 1991a. Autoradiographic visualisation possesses high affinity and selectivity for the σ1 receptor, and appears of [3H]DTG binding to sigma receptors, [3H]TCP binding sites, and L-[3H]glutamate to be a viable alternative for [3H](+)-pentazocine in radioligand binding to NMDA receptors in human cerebellum. Neurosci. Lett. 125, 143–146.
binding assays. Further, because [3H]-SN56 has a N70 fold higher Jansen, K.L., Faull, R.L., Dragunow, M., Leslie, R.A., 1991b. Autoradiographic distribution affinity for the σ of sigma receptors in human neocortex, hippocampus, basal ganglia, cerebellum, 1 receptor than [3H](+)-pentazocine, competition pineal and pituitary glands. Brain Res. 559, 172–177.
binding studies require less radioligand and membrane, resulting in Kekuda, R., Prasad, P.D., Fei, Y.J., Leibach, F.H., Ganapathy, V., 1996. Cloning and significant efficiencies in resources when performing the assays. Thus, functional expression of the human type 1 sigma receptor (hSigmaR1). Biochem.
[3H]-SN56 represents another valuable tool for the study of the σ Biophys. Res. Commun. 229, 553–558.
Klouz, A., Tillement, J.P., Boussard, M.F., Wierzbicki, M., Berezowski, V., Cecchelli, R., receptor and the development of σ1 based therapeutics.
Labidalle, S., Onteniente, B., Morin, D., 2003. [3H]BHDP as a novel and selectiveligand for sigma1 receptors in liver mitochondria and brain synaptosomes of therat. FEBS Lett. 553, 157–162.
Martin, W.R., Eades, C.G., Thompson, J.A., Huppler, R.E., Gilbert, P.E., 1976. The effects of morphine- and nalorphine-like drugs in the nondependent and morphine- This study was supported by grants from the National Institute on dependent chronic spinal dog. J. Pharmacol. Exp. Ther. 197, 517–532.
Matsumoto, R.R., Bowen, W.D., Tom, M.A., Vo, V.N., Truong, D.D., De Costa, B.R., 1995.
Drug Abuse (DA023205 and DA013978).
Characterization of two novel σ receptor ligands: antidystonic effects in ratssuggest σ receptor antagonism. Eur. J. Pharmacol. 280, 301–310.
Matsumoto, R.R., Bowen, W.D., Walker, J.M., Patrick, S.L., Zambon, A.C., Vo, V.N., Truong, D.D., De Costa, B.R., Rice, K.C., 1996. Dissociation of the motor effects of (+)-pentazocine from binding to σ1 sites. Eur. J. Pharmacol. 301, 31–40.
Ablordeppey, S.Y., Fischer, J.B., Glennon, R.A., 2000. Is a nitrogen atom an important Matsuno, K., Nakazawa, M., Okamoto, K., Kawashima, Y., Mita, S., 1996. Binding pharmacophoric element in sigma ligand binding? Bioorg. Med. Chem. 8, properties of SA4503, a novel and selective σ1 receptor agonist. Eur. J. Pharmacol.
306, 271–279.
Ablordeppey, S.Y., Fischer, J.B., Law, H., Glennon, R.A., 2002. Probing the proposed Maurice, T., Su, T.P., 2009. The pharmacology of sigma-1 receptors. Pharmacol. Ther.
phenyl-A region of the sigma-1 receptor. Bioorg. Med. Chem. 10, 2759–2765.
124, 195–206.
Bowen, W.D., Hellewell, S.B., McGarry, K.A., 1989. Evidence for a multi-site model of the Mei, J., Pasternak, G.W., 2001. Molecular cloning and pharmacological characterization rat brain σ receptor. Eur. J. Pharmacol. 163, 309–318.
of the rat sigma1 receptor. Biochem. Pharmacol. 62, 349–355.
Bowen, W.D., DeCosta, B.R., Hellewell, S.B., Walker, J.M., Rice, K.C., 1993. [3H]-(+)- Mesangeau, C., Narayanan, S., Green, A.M., Shaikh, J., Kaushal, N., Viard, E., Xu, Y.T., Pentazocine: a potent and highly selective benzomorphan-based probe for sigma-1 Fishback, J.A., Poupaert, J.H., Matsumoto, R.R., McCurdy, C.R., 2008. Conversion of a receptors. Mol. Pharmacol. 1, 117–126.
highly selective sigma-1 receptor-ligand to sigma-2 receptor preferring ligands Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram with anticocaine activity. J. Med. Chem. 51, 1482–1486.
quantities of protein utilizing the principle of protein−dye binding. Anal. Biochem.
Pan, Y.X., Mei, J., Xu, J., Wan, B.L., Zuckerman, A., Pasternak, G.W., 1998. Cloning and 72, 248–254.
characterization of a mouse sigma1 receptor. J. Neurochem. 70, 2279–2285.
Bylund, D.B., Toews, M.L., 1993. Radioligand binding methods: practical guides and tips.
Rybczynska, A.A., Elsinga, P.H., Sijbesma, J.W., Ishiwata, K., de Jong, J.R., de Vries, E.F., Am. J. Physiol. 265, 421–429.
Dierckx, R.A., van Waarde, A., 2009. Steroid hormones affect binding of the sigma Bylund, D.B., Deupree, J.D., Toews, M.L., 2004. Radioligand-binding methods for ligand 11C-SA4503 in tumour cells and tumour-bearing rats. Eur. J. Nucl. Med. Mol.
membrane preparations and intact cells. Meth. Mol. Biol. 259, 1–28.
Imaging 36, 1167–1175.
J.A. Fishback et al. / European Journal of Pharmacology 653 (2011) 1–7 Seth, P., Leibach, F.H., Ganapathy, V., 1997. Cloning and structural analysis of the cDNA activity of 2(3H)-benzoxazolone and 2(3H)-benzothiazolone derivatives. J. Med.
and the gene encoding the murine type 1 sigma receptor. Biochem. Biophys. Res.
Chem. 41, 1138–1145.
Commun. 241, 535–540.
Walker, J.M., Bowen, W.D., Goldstein, S.R., Roberts, A.H., Patrick, S.L., Hohmann, A.G., Seth, P., Fei, Y.J., Li, H.W., Huang, W., Leibach, F.H., Ganapathy, V., 1998. Cloning and DeCosta, B., 1992. Autoradiographic distribution of [3 H](+)-pentazocine and [3 H] functional characterization of a sigma receptor from rat brain. J. Neurochem. 70, 1, 3-di-o-tolylguanidine (DTG) binding sites in guinea pig brain: a comparative study. Brain Res. 581, 33–38.
Seth, P., Ganapathy, M.E., Conway, S.J., Bridges, C.D., Smith, S.B., Casellas, P., Ganapathy, Yamamoto, H., Miura, R., Yamamoto, T., Shinohara, K., Watanabe, M., Okuyama, S., V., 2001. Expression pattern of the type 1 sigma receptor in the brain and identity of Nakazato, A., Nukada, T., 1999. Amino acid residues in the transmembrane domain critical anionic amino acid residues in the ligand-binding domain of the receptor.
of the type 1 sigma receptor critical for ligand binding. FEBS Lett. 445, 19–22.
Biochim. Biophys. Acta 1540, 59–67.
Yous, S., Poupaert, J.H., Lesieur, I., Depreux, P., Lesieur, D., 1994. AlCl3-DMF reagent in Su, T.P., 1982. Evidence for sigma opioid receptor: binding of [3H]SKF-10047 to etorphine- the Friedel-Crafts reaction. Application to the acylation reaction of 2(3H)- inaccessible sites in guinea-pig brain. J. Pharmacol. Exp. Ther. 223, 284–290.
benzothiazolones. J. Org. Chem. 59, 1574–1576.
Tam, S.W., 1983. Naloxone-inaccessible sigma receptor in rat central nervous system.
Yous, S., Wallez, V., Belloir, M., Caignard, D., McCurdy, C.R., 2005. Novel 2(3H)- Proc. Natl Acad. Sci. USA 80, 6703–6707.
benzothiazolones as highly potent and selective sigma-1 receptor ligands. Med.
Toyohara, J., Sakata, M., Ishiwata, K., 2009. Imaging of sigma1 receptors in the human Chem. Res. 14, 158–168.
brain using PET and [11C]SA4503. Cent. Nerv. Syst. Agents Med. Chem. 9, 190–196.
Zampieri, D., Grazia Mamolo, M., Laurini, E., Zanette, C., Florio, C., Collina, S., Rossi, D., Ucar, H., Van derpoorten, K., Cacciaguerra, S., Spampinato, S., Stables, J.P., Depovere, P., Azzolina, O., Vio, L., 2009. Substituted benzo[d]oxazol-2(3H)-one derivatives with Isa, M., Masereel, B., Delarge, J., Poupaert, J.H., 1998. Synthesis and anticonvulsant preference for the sigma1 binding site. Eur. J. Med. Chem. 44, 124–130.

Source: http://www.ucl.eu/cps/ucl/doc/ir-ldri/images/Fishback2011.pdf