Synthesis and binding affinities of methylvesamicol analogs for the acetylcholine transporter and sigma receptor

We synthesized methylvesamicol analogs 13-16 and investigated the binding characteristics of 2-[4-phenylpiperidino]cyclohexanol (vesamicol) and methylvesamicol analogs 13-16, with a methyl group introduced into the 4-phenylpiperidine moiety, to sigma receptors (sigma-1, sigma-2) and to vesicular acetylcholine transporters (VAChT) in membranes of the rat brain and liver. In competitive inhibition studies, (-)-o-methylvesamicol [(-)-OMV] (13) (Ki=6.7 nM), as well as (-)-vesamicol (Ki=4.4 nM), had a high affinity for VAChT. (+)-p-Methylvesamicol [(+)-PMV] (16) (Ki=3.0 nM), as well as SA4503 (Ki=4.4 nM), reported as a sigma-1 mapping agent for positron emission tomography (PET), had a high affinity for the sigma-1 receptor. The binding affinity of (+)-PMV (16) for the sigma-1 receptor (Ki=3.0 nM) was about 13 times higher than that for the sigma-2 (sigma-2) receptor (Ki=40.7 nM). (+)-PMV (16) (Ki=199 nM) had a much lower affinity for VAChT than SA4503 (Ki=50.2 nM) and haloperidol (Ki=41.4 nM). These results showed that the binding characteristics of (-)-OMV (13) to VAChT were similar to those of (-)-vesamicol and that (+)-PMV (16) bound to the sigma-1 receptor with high affinity. In conclusion, (-)-OMV (13) and (+)-PMV (16), which had a suitable structure, with a methyl group for labeling with 11C, may become not only a new VAChT ligand and a new type of sigma receptor ligand, respectively, but may also become a new target compound of VAChT and the sigma-1 receptor radioligand for PET, respectively.     

Characterization of radioiodinated (-)-ortho-iodovesamicol binding in rat brain preparations

We investigated the binding characteristics of optical isomers of three iodovesamicol analogs to vesicular acetylcholine transporters (VAChT) and to sigma receptors (sigma-1, sigma-2) in rat brains. In competitive inhibition studies, (-)-enantiomers [(-)-ortho-iodovesamicol ((-)-oIV), (-)-meta-iodovesamicol ((-)-mIV), (-)-vesamicol] displayed a higher affinity for VAChT than (+)-enantiomer [(+)-oIV, (+)-mIV, (+)-vesamicol]. (-)-oIV and (-)-mIV showed the same high affinity for VAChT as (-)-vesamicol. For sigma receptors(sigma-1, sigma-2), (-)-oIV (Ki = 62.2 nM (to sigma-1) and 554 nM(to sigma-2)) showed a lower affinity than (-)-mIV (Ki = 4.5 nM (to sigma-1) and 42.9 nM (to sigma-2)). Furthermore, in a saturation binding study, (-)-[125I]-oIV exhibited a Kd of 17.4 +/- 5.1 nM with a maximum number of binding sites, Bmax, of 559 +/- 51 fmol/ mg of protein. These results showed that (-)-oIV binds to vesicular acetylcholine transporters (VAChT) more selectively than (-)-mIV, previously reported as a VAChT mapping agent, and may be suitable for VAChT imaging studies.     

In Vivo Differences between Two Optical Isomers of Radioiodinated o-iodo-trans-decalinvesamicol for Use as a Radioligand for the Vesicular Acetylcholine Transporter

Purpose

To develop a superior VAChT imaging probe for SPECT, radiolabeled (-)-OIDV and (+)-OIDV were isolated and investigated for differences in their binding affinity and selectivity to VAChT, as well as their in vivo activities.

Procedures

Radioiodinated o-iodo-trans-decalinvesamicol ([¹²⁵I]OIDV) has a high binding affinity for vesicular acetylcholine transporter (VAChT) both in vitro and in vivo. Racemic [¹²⁵I]OIDV was separated into its two optical isomers (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV by HPLC. To investigate VAChT binding affinity (Ki) of two OIDV isomers, in vitro binding assays were performed. In vivo biodistribution study of each [¹²⁵I]OIDV isomer in blood, brain regions and major organs of rats was performed at 2,30 and 60 min post-injection. In vivo blocking study were performed to reveal the binding selectivity of two [¹²⁵I]OIDV isomers to VAChT in vivo. Ex vivo autoradiography were performed to reveal the regional brain distribution of two [¹²⁵I]OIDV isomers and (-)-[¹²³I]OIDV for SPECT at 60 min postinjection.

Results

VAChT binding affinity (Ki) of (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV was 22.1 nM and 79.0 nM, respectively. At 2 min post-injection, accumulation of (-)-[¹²⁵I]OIDV was the same as that of (+)-[¹²⁵I]OIDV. However, (+)-[¹²⁵I]OIDV clearance from the brain was faster than (-)-[¹²⁵I]OIDV. At 30 min post-injection, accumulation of (-)-[¹²⁵I]OIDV (0.62 ± 0.10%ID/g) was higher than (+)-[¹²⁵I]OIDV (0.46 ± 0.07%ID/g) in the cortex. Inhibition of OIDV binding showed that (-)-[¹²⁵I]OIDV was selectively accumulated in regions known to express VAChT in the rat brain, and ex vivo autoradiography further confirmed these results showing similar accumulation of (-)-[¹²⁵I]OIDV in these regions. Furthermore, (-)-[¹²³I]OIDV for SPECT showed the same regional brain distribution as (-)-[¹²⁵I]OIDV.

Conclusion

These results suggest that radioiodinated (-)-OIDV may be a potentially useful tool for studying presynaptic cholinergic neurons in the brain.     

Purification and characterization of the guinea pig sigma-1 receptor functionally expressed in Escherichia coli

Sigma receptors once considered as a class of opioid receptors are now regarded as unique orphan receptors, distinguished by the ability to bind various pharmacological agents such as the progesterone (steroid), haloperidol (anti-psychotic), and drugs of abuse such as cocaine and methamphetamine. The sigma-1 receptor is a 223 amino acid protein, proposed to have two transmembrane segments. We have developed a scheme for the purification of the guinea pig sigma-1 receptor following overexpression in Escherichia coli as a maltose binding protein (MBP) fusion and extraction with Triton X-100. Affinity chromatography using an amylose column and Ni2+ affinity column was used to purify the sigma-1 receptor. The sigma-1 receptor purified by this method is a 26 kDa polypeptide as assessed by SDS-PAGE, binds sigma ligands with high affinity and can be specifically photoaffinity labeled with the sigma-1 receptor photoprobe, [125I]-iodoazidococaine. Ligand binding using [3H]-(+)-pentazocine indicated that approximately half of the purified protein in Triton X-100 bound to radioligand. The MBP-sigma-1 receptor and the sigma-1 receptor in 0.5% triton were maximally stable for approximately two weeks at -20 degrees C in buffer containing 30% glycerol.     

A sigma-1 receptor selective analogue of BD1008. A potential substitute for (+)-opioids in sigma receptor binding assays

A simple, achiral monoamine sigma-1 (sigma1) receptor selective ligand (sigma2Ki/sigma1Ki>2000) is described, which could replace the chiral (+)-pentazocine or dextrallorphan as a sigma1 masking agent in sigma2 binding assays.     

Synthesis and evaluation of optically pure [3H]-(+)-pentazocine, a highly potent and selective radioligand for sigma receptors

Tritium-labeled (+)-pentazocine ([3H]-1b) of specific activity 26.6 Ci/mmol was synthesized in 3 steps starting with (+)-normetazocine (2) of defined optical purity. [3H]-1b has been characterized as a highly selective ligand for labeling of sigma receptors. Competition data revealed that [3H]-1b could be displaced from guinea pig brain membrane preparations with a number of commonly used sigma receptor ligands. [3H]-1b exhibited saturable, enantioselective binding with a Kd of 5.13 +/- 0.97 nM and a Bmax of 1146 +/- 122 fmol/mg protein. Phencyclidine (PCP) displaced [3H]-1b with low affinity while MK-801 was inactive, thus indicating insignificant activity at the PCP-binding site; apomorphine failed to displace [3H]-1b indicating lack of dopamine receptor cross-reactivity. Since the affinity of [3H]-1b is about 6 times that of the two commonly employed sigma ligands ((+)-3-[3H]PPP and [3H]DTG) and since it is more selective for sigma receptors than the benzomorphan [3H]SKF-10,047, it represents the first example of a highly selective benzomorphan based sigma receptor ligand. [3H]-1b should prove useful for further study of the structure and function of sigma receptors.     

Characterization of [3H]desmethylimipramine binding in bovine adrenal medulla: interactions with sigma- and (or) phencyclidine-receptor ligands.

High-affinity binding sites (apparent KD 2.87 nM) for [3H]desmethylimipramine ([3H]DMI), have been demonstrated and characterized in membrane preparations of bovine adrenal medulla. The binding of [3H]DMI improved upon pretreatment of the membrane with KCl and was saturable, sodium dependent, and potently inhibited by nisoxetine and imipramine. [3H]DMI binding was also inhibited by various phencyclidine (PCP)- and (or) sigma-receptor ligands, with the following order of potency: haloperidol > rimcazole > (-)-butaclamol > dextromethorphan > MK-801 > (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP) > PCP > N-(2-thienyl)cyclohexyl-3,4-piperidine (TCP) > (+)-SKF-10047 > (-)-SKF-10047. The inhibition produced by sigma ligands was not attributed to stimulation of either sigma 1- or sigma 2-receptors, owing to inactivity of the selective sigma-receptor ligands (+)-pentazocine and 1,3-di(2-tolyl)guanidine (DTG). The inhibition of [3H]DMI binding by sigma- and PCP-receptor ligands was not attributed to PCP1- or PCP2-receptor stimulation, owing to the decreased potency (100-fold) of these ligands in [3H]DMI assays compared with the affinity for brain PCP1 sites, and the ineffectiveness of the PCP2-ligand N-(1-(2-benzo(b)thiophenyl)cyclohexyl)piperidine (BTCP). Scatchard analysis of the inhibition by the sigma-ligands haloperidol and (+)-3-PPP, as well as the PCP1 receptor ligand MK-801, demonstrated noncompetitive interaction with the site bound by [3H]DMI. These studies indicate that bovine adrenomedullary membranes possess a specific receptor for the noradrenaline uptake inhibitor [3H]DMI, which is sensitive to allosteric modulation produced by PCP and sigma-ligands.     

Chiral, nonracemic (piperazin-2-yl)methanol derivatives with sigma-receptor affinity

Starting with the proteinogenic amino acid (S)-serine a series of chiral nonracemic (piperazin-2-yl)methanols 3 with various N-4 substituents is described. The key step in the synthesis of 3 is the reaction of the chloroacetamide 5 with various primary amines to yield the diastereomeric bicyclic piperazinediones cis-6 and trans-6. The scope and limitation of this transformation is thoroughly investigated. The alpha1- and sigma2-receptor affinities of the piperazines 3 are determined in receptor binding studies with guinea pig brain and rat liver membrane preparations using [3H]-labeled (+)-pentazocine and ditolylguanidine, respectively. It was found, that an additional phenyl residue in the N-4 substituent is favorable to high sigma1-receptor affinity. In this series the p-methoxybenzyl substituted piperazine 3d reveals the highest sigma1-receptor affinity (Ki=12.4 nM) with selectivity toward sigma2-, NMDA-, kappa-opioid, and mu-opioid receptors.     

Effects of streptozotocin-induced diabetes on neuronal sigma receptors in the rat brain

To study the effect of diabetes mellitus on the density of sigma receptors, in vitro binding experiments were conducted in whole brain homogenate membranes of 5-week and 10-week control rats and streptozotocin (STZ)-induced diabetic rats. sigma-1 Receptors were labelled with [3H](+)-pentazocine while sigma-2 receptors were labelled with [3H] 1,3-di-o-tolylguanidine (DTG) in the presence of 0.5 microM (+)-pentazocine to mask sigma-1 sites. Non-specific binding was determined in the presence of 20 microM haloperidol. Scatchard analysis revealed a 27% (p<0.01) decreased in sigma-1 receptor density and a 33% (p<0.01) decreased in sigma-2 receptor density in whole brain of 10-week STZ-diabetic rats. No statistically significant difference was found in the sigma receptor content of 5-week STZ-diabetic rats. These results provide evidence that neuronal sigma receptors are reduced in 10-week STZ-diabetic rats and suggest that changes in sigma receptors may play a role in diabetes related abnormalities. Further evaluation is required to determine whether changes observed in the brain are homogeneous for either or both sigma receptor subtypes as well as potential links between other CNS receptor changes previously observed in STZ-induced diabetic rats.     

Interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with nicotinic acetylcholine receptor subtypes expressed in cell lines and rat cortex

The interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with different nicotinic acetylcholine receptor (nAChR) subtypes was studied in cell lines and rat cortex. MPA showed an affinity (Ki = 1.21 nM) which was higher than anatoxin-a > (−)-nicotine > (+)-[R]nornicotine > (−)-[S]nornicotine > and (+)-nicotine, but lower than cytisine (Ki = 0.46 nM) in competing for (−)-[³H]nicotine binding in M10 cells, which stably express the recombinant ₄β₂ nAChR subtype. A one-binding site model was observed in all competing experiments between (−)-[³H]nicotine binding and each of the agonists studied in M10 cells. MPA showed a 13-fold higher affinity for (−)-[³H]nicotine binding sites compared to the [³H]epibatidine binding sites in rat cortical membranes. In human neuroblastoma SH-SY5Y cells, which predominantly express the ₃ nAChR subunit mRNA, MPA displaced [³H]epibatidine binding from a single population of the binding sites with an affinity in the same nM range as that observed MPA in displacing [³H]epibatidine binding in rat cortical membranes. Chronic treatment of M10 cells with MPA significantly up-regulated the number of (−)-[³H]nicotine binding sites in a concentration dependent manner. Thus MPA appears to have higher affinity to 4-subunit containing receptor subtype than ₃-subunit containing receptor subtype of nAChRs. Furthermore MPA binds to ₄β₂ receptor subtype with higher affinity than (−)-nicotine and behaves, opposite to cytisine, as a full agonist in up-regulating the number of nAChRs. © 1998 Elsevier Science Ltd. All rights reserved.     

Characterization of a new radioiodinated probe for the alpha2C adrenoceptor in the mouse brain

[125I]17alpha-hydroxy-20alpha-yohimban-16beta-(N-4-p6 hydroxyphenethyl)carboxamide or [125I]rauwolscine-OHPC, a new radioiodinated probe derived from rauwolscine was synthesized and its binding characteristics investigated on sections of the mouse caudate putamen. [125I]rauwolscine-OHPC binding was saturable and revealed interaction with a single class of binding sites (KD= 0.171 nM, Bmax = 3082 pCi/mg of tissue). The kinetically derived affinity was in close agreement with the affinity evaluated by saturation experiments: k(-1)/k(+1)(0.0403 min(-1)/114 10(6) M(-1) min(-1))=0.35 nM. Competition studies revealed interaction with one single class of binding sites for each of the twelve compounds tested. The rank of potency suggested an interaction with alpha2 adrenoceptors (atipamezole > or = RX 821002 > yohimbine > (-)epinephrine). Moreover, the good affinity of [125I] rauwolscine-OHPC binding sites for spiroxatrine, yohimbine, WB 4101, the relatively good affinity for prazosin (Ki =37.4 nM) and the affinity ratio prazosin/oxymetazoline (37.4/43.4=0.86) were consistent with an alpha2C selective labelling of [125I]rauwolscine-OHPC. The distribution of [125I]rauwolscine-OHPC binding sites in mouse brain was characterized by autoradiography. The density of binding sites was high in the islands of Calleja, accumbens nucleus, caudate putamen and olfactory tubercles, moderate in the hippocampus, amygdala and anterodorsal nucleus of the thalamus. These findings demonstrated that [125I]rauwolscine-OHPC is a useful radioiodinated probe to label alpha2C adrenoceptors in mouse brain.     

125I] 3-quinuclidinyl 4-iodobenzilate: a high affinity, high specific activity radioligand for the M1 and M2-acetylcholine receptors

We have prepared a radioiodinated ligand which binds with high affinity to the muscarinic acetylcholine receptor (m-AChR). A derivative of 3-quinuclidinyl benzilate, [125I] labeled (R) 1-aza-bicyclo(2.2.2)oct-3-yl (R,S)-alpha-hydroxy-alpha-(4-[125I]iodophenyl)phenyl acetate (4- IQNB ) exhibits an affinity for the m-AChR from corpus striatum higher than that of (R) [3H] QNB. Additionally, [125I] 4- IQNB exhibits receptor selectivity for the M1 receptor since the affinity for the receptor from dog and rat heart is lower than that using dog or rat corpus striatum.     

Syntheses and evaluation of a homologous series of aza-vesamicol as improved radioiodine-labeled probes for sigma-1 receptor imaging

Sigma-1 receptor imaging probes for determining the expression levels are desirable for diagnoses of various diseases and companion diagnoses of therapeutic agents targeting the sigma-1 receptor. In this study, we aimed to develop probes with higher affinity for the sigma-1 receptor. For this purpose, we synthesized and evaluated compounds, namely, vesamicol derivatives, in which alkyl chains of varying chain length were introduced between a piperazine ring and a benzene ring. The binding affinity of the vesamicol derivatives for the sigma-1 receptor tended to increase depending on the length of the alkyl chain between the benzene ring and the piperazine ring. The sigma-1 receptor of 2-(4-(3-phenylpropyl)piperazin-1-yl)cyclohexan-1-ol (5) (K_i?=?5.8?nM) exhibited the highest binding affinity; therefore, we introduced radioiodine into the benzene ring in 5. The radioiodine labeled probe [¹²⁵I]2-(4-(3-(4-iodophenyl)propyl)piperazin-1-yl)cyclohexan-1-ol ([¹²⁵I]10) showed high accumulation in the sigma-1 receptor expressing DU-145 cells both in vitro and in vivo. Co-injection of [¹²⁵I]10 with an excess level of a sigma receptor ligand, haloperidol, resulted in a significant decrease in the tumor accumulation in vitro and in vivo, indicating sigma receptor-mediated tumor uptake. These results provide useful information for developing sigma-1 receptor imaging probes.     

Evaluation of the eutomer of 4-{3-(4-chlorophenyl)-3-hydroxypyrrolidin-1-yl}-1-(4-fluorophenyl)butan-1-one, {(+)-SYA 09}, a pyrrolidine analog of haloperidol

Enantiomeric separation of the racemic 4-{3-(4-chlorophenyl)-3-hydroxypyrrolidin-1-yl}-1-(4-fluorophenyl)butan-1-one, a pyrrolidine analog of haloperidol, {(+/-)-SYA 09}, and subsequent binding studies revealed that most of the binding affinity at dopamine and serotonin receptors resides in the (+)-isomer {(+)-SYA 09} or the eutomer. Further pharmacological evaluation of the eutomer revealed that it has a higher affinity for the dopamine D4 (DAD4) receptor subtype (Ki = 3.6 nM) than for the DAD2 subtype (Ki = 51.1 nM) with a ratio of 14.2 (D2Ki/D4Ki ratio = 14.2). In an animal model of antipsychotic efficacy, the (+)-SYA 09 was efficacious with an ED50 value of 1.6 mg/kg, i.p., and at twice this value, (+)-SYA 09 did not induce significant catalepsy in rats.     

SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of [3H]dextromethorphan and sigma ligands to guinea pig brain.

The DM1/sigma 1 site binds dextromethorphan (DM) and sigma receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of [3H]dextromethorphan, [3H]3-(-3-Hydroxyphenyl)-N-(1-propyl)piperidine and (+)-[3H]1,3-Di-o-tolyl-guanidine ([3H]DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drug has identical nM Ki values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM1/sigma 1 site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed Ki values of 9-13 and 3-4 microM respectively against the three labeled ligands. These results, the broad specificity of the DM1/sigma 1 binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor. These findings may have important implications for the understanding of the therapeutic, side effects and toxicity of several neurotropic drugs.     

In vitro and in vivo pharmacological profile of 4-(4-fluorobenzylidene)-1-[2-[5-(4-fluorophenyl)-1H-pyrazol-4-yl] ethyl] piperidine (NRA0161)

Atypical antipsychotic properties of 4-(4-fluorobenzylidene)-1-[2-[5-(4-fluorophenyl)-1H-pyrazol-4-yl]ethyl] piperidine (NRA0161) were investigated by in vitro receptor affinities, in vivo receptor occupancies and findings were compared with those of risperidone and haloperidol in rodent behavioral studies. In in vitro receptor binding studies, NRA0161 has a high affinity for human cloned dopamine D(4) and 5-HT(2A) receptor with Ki values of 1.00 and 2.52 nM, respectively. NRA0161 had a relatively high affinity for the alpha(1) adrenoceptor (Ki; 10.44 nM) and a low affinity for the dopamine D(2) receptor (Ki; 95.80 nM). In in vivo receptor binding studies, NRA0161 highly occupied the 5-HT(2A) receptor in rat frontal cortex. In contrast, NRA0161 did not occupy the striatal D(2) receptor. In behavioral studies, NRA0161, risperidone and haloperidol antagonized the locomotor hyperactivity in mice, as induced by methamphetamine (MAP). At a higher dosage, NRA0161, risperidone and haloperidol dose-dependently antagonized the MAP-induced stereotyped behavior in mice and NRA0161 dose-dependently and significantly induced catalepsy in rats. The ED(50) value in inhibiting the MAP-induced locomotor hyperactivity was 30 times lower than that inhibiting the MAP-induced stereotyped behavior and 50 times lower than that which induced catalepsy.These findings suggest that NRA0161 may have atypical antipsychotic activities yet without producing extrapyramidal side effects.     

Probing the steroid binding domain-like I (SBDLI) of the sigma-1 receptor binding site using N-substituted photoaffinity labels

Radioiodinated photoactivatable photoprobes can provide valuable insights regarding protein structure. Previous work in our laboratory showed that the cocaine derivative and photoprobe 3-[ (125)I]iodo-4-azidococaine ([ (125)I]IACoc) binds to the sigma-1 receptor with 2-3 orders of magnitude higher affinity than cocaine [Kahoun, J. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1393-1397]. Using this photoprobe, we demonstrated the insertion site for [ (125)I]IACoc to be Asp188 [Chen, Y. (2007) Biochemistry 46, 3532-3542], which resides in the proposed steroid binding domain-like II (SBDLII) region (amino acids 176-194) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. An additional photoprobe based on the sigma-1 receptor ligand fenpropimorph, 1- N-(2-3-[ (125)I]iodophenyl)propane ([ (125)I]IAF), was found to label a peptide in both the SBDLII and steroid binding domain-like I (SBDLI) (amino acids 91-109) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. In this report, we describe two novel strategically positioned carrier-free, radioiodinated photoaffinity labels specifically designed to probe the putative "nitrogen interacting region" of sigma-1 receptor ligands. These two novel photoprobes are (-)-methyl 3-(benzoyloxy)-8-2-(4-azido-3-[ (125)I]iodobenzene)-1-ethyl-8-azabicyclo[3.2.1]octane-2-carboxylate ([ (125)I]-N-IACoc) and N-propyl- N-(4-azido-3-iodophenylethyl)-3-(4-fluorophenyl)propylamine ([ (125)I]IAC44). In addition to reporting their binding affinities to the sigma-1 and sigma-2 receptors, we show that both photoaffinity labels specifically and covalently derivatized the pure guinea pig sigma-1 receptor (26.1 kDa) [Ramachandran, S. (2007) Protein Expression Purif. 51, 283-292]. Cleavage of the photolabeled sigma-1 receptor using Endo Lys C and cyanogen bromide (CNBr) revealed that the [ (125)I]-N-IACoc label was located primarily in the N-terminus and SBDLI-containing peptides of the sigma-1 receptor, while [ (125)I]IAC44 was found in peptide fragments consistent with labeling of both SBDLI and SBDLII.     

A multireceptorial binding reinvestigation on an extended class of sigma ligands: N-[omega-(indan-1-yl and tetralin-1-yl)alkyl] derivatives of 3,3-dimethylpiperidine reveal high affinities towards sigma1 and EBP sites

New 1-[omega-(2,3-dihydro-1H-inden-1-yl)- and (2,3-dihydro-5-methoxy-1H-inden-1-yl)alkyl]- and 1-[omega-(1,2,3,4-tetrahydronaphthalen-1-yl)- and (6-methoxy- or 6-fluoro-1,2,3,4-tetrahydronaphthalen-1-yl)alkyl] derivatives of 3,3-dimethylpiperidine were synthesized, as homologous compounds of an existing series of sigma ligands, in order to carry out sigma receptor subtypes structure-affinity relationships. The new compounds and some of their related analogues, already reported, were tested in new multireceptorial radioligand binding assays. As reference compounds, the known sigma(1) ligands SA 4503, BD 1008 and NE 100 were also prepared and tested. All reported compounds showed high sigma(1) affinity assayed by (+)-[(3)H]-pentazocine on guinea-pig brain (apparent K(i)=1.75-72.2 nM) and moderate or low sigma(2) affinity by [(3)H]-DTG on rat liver, in contrast with previous results. One tertiary amine function spaced by a five-membered chain from a phenyl group is the structural feature shared by the most active compounds 26 and 43 and some reference sigma(1) ligands. The reported sigma(1) ligands, including reference compounds, also demonstrated a high affinity towards EBP (Delta(8)-Delta(7) sterol isomerase) site (apparent K(i)=0.48-14.8 nM) and some of them (37 and 44) were good ligands at L-type Ca(++) channel. 1-[4-(2,3-Dihydro-1H-inden-1-yl)butyl]-3,3-dimethylpiperidine (26) was the best mixed sigma(1) and EBP ligand (apparent K(i)=1.75 and 1.54 nM, respectively) with a good selectivity versus sigma(2) receptor (138- and 157-fold, respectively).     

MS-377, a selective sigma receptor ligand, indirectly blocks the action of PCP in the N-methyl-D-aspartate receptor ion-channel complex in primary cultured rat neuronal cells

MS-377 ((R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate) is a antipsychotic agent that binds to sigma-1 receptor. MS-377 showed anti-dopaminergic and anti-serotonergic activities and antagonistic action against phencyclidine (PCP)-induced behaviors in an animal model. These anti-psychotic activities of MS-377 are attributable to association with sigma-1 receptor. However, the mechanism by which the sigma-1 receptor ligands exact those numerous effects remains to be elucidated. In the present study, we evaluated the effect of MS-377 on N-methyl-D-aspartate (NMDA) receptor ion-channel complex in primary cultured rat neuronal cells. First, we examined the effect of MS-377 on NMDA-induced Ca2+ influx with fura-2/ AM loaded cells. MS-377 showed no effects on the basal Ca2+ concentration and NMDA-induced Ca2+ influx by itself PCP and SKF-10047 reduced the NMDA-induced increase in intracellular Ca2+ concentration. Pre-incubation of 1 microM MS-377 was found to significantly block the reduction by PCP or SKF-10047 of the NMDA-induced Ca2+ influx. Second, the effect of MS-377 on [3H]MK-801 intact cell binding was examined. PCP, haloperidol and (+)-pentazocine inhibited [3H]MK-801 binding, although MS-377 showed no effect by itself Pre-treatment of MS-377 markedly reversed the inhibition of [3H]MK-801 binding by PCP in a dose-dependent manner. These effects of MS-377 may depend on its affinity for the sigma-1 receptor, because MS-377 is a selective sigma-1 receptor ligand without any affinity for NMDA receptor ion-channel complex. These observations suggest that the MS-377 indirectly modulated the NMDA receptor ion-channel complex, and the anti-psychotic activities of MS-377, in part, are attributable to such on action via sigma-1 receptor.     

In vitro and in vivo binding of (E)- and (Z)-N-(iodoallyl)spiperone to dopamine D2 and serotonin 5-HT2 neuroreceptors

Apparent affinities (Ki) of (E)- and (Z)-N-(iodoallyl)spiperone [E)- and (Z)-NIASP) for dopamine D2 and serotonin 5-HT2 receptors were determined in competition binding assays. (Z)-NIASP (Ki 0.35 nM, D2; Ki 1.75 nM, 5-HT2) proved slightly more potent and selective for D2 sites in vitro than (E)-NIASP (Ki 0.72 nM, D2; Ki 1.14 nM, 5-HT2). In vivo, radioiodinated (E)- and (Z)-[125I]-NIASP showed regional distributions in mouse brain which are consonant with prolonged binding to dopamine D2 receptors accompanied by a minor serotonergic component of shorter duration. Stereoselective, dose-dependent blockade of (E)-[125I]-NIASP uptake was found for drugs binding to dopamine D2 sites, while drugs selective for serotonin 5-HT2, alpha 1-adrenergic and dopamine D1 receptors did not inhibit radioligand binding 2 hr postinjection. Specific binding in striatal tissue was essentially irreversible over the time course of the study, and (E)-[125I]-NIASP gave a striatal to cerebellar tissue radioactivity concentration of 16.9 to 1 at 6 hr postinjection. Thus, (E)-[125I]-NIASP binds with high selectivity and specificity to dopamine D2 sites in vivo.