Guinea Pig Brain Membranes Research Articles

The C-terminal pentapeptide acein analogue (JMV3315) stimulates dopamine release in the brain

We have previously reported the synthesis and biological activity of a newly identified peptide of sequence H–Pro–Pro–Thr–Thr–Thr–Lys–Phe–Ala–Ala–OH called acein that is able to stimulate dopamine release in the brain of rodents in vivo and ex vivo by interacting with angiotensin converting enzyme (ACE). In the present piece of work, we studied the structure–activity relationships of acein using displacement experiments of the labelled ligand [125I]Tyr–Pro–Pro–Thr–Thr–Thr–Lys–Phe–Ala–Ala–OH on guinea pig brain membranes, known to have high-affinity acein binding sites. We determined that the C-terminal pentapeptide H–Thr–Lys–Phe–Ala–Ala–OH is the minimal structure able to interact with high affinity (Ki (inhibitory constant) 13 ± 2 nM) with acein binding sites. Among the analogues of the pentapeptide that were synthesized, the pentapeptide H–Thr–Lys–Tyr–Ala–Ala–OH showed the highest affinity (Ki 3.7 ± 1.0 nM). Accordingly, this pentapeptide was able to stimulate dopamine release from striatal slices taken from the sensorimotor territory of rats.

Allosteric modulation of sigma receptors by BH3 mimetics ABT-737, ABT-263 (Navitoclax) and ABT-199 (Venetoclax)

ABT-737, ABT-263 (Navitoclax) and ABT-199 (Venetoclax) are under intensive preclinical and clinical investigation as treatments for hematologic and other malignancies. These small molecules mimic pro-death B-cell lymphoma-2 (Bcl-2) Homology 3 (BH3) domain-only proteins. They also bear a structural resemblance to certain sigma (σ) receptor ligands. Moreover, the Bcl-2 and σ receptor protein families are both located primarily at the endoplasmic reticulum, mediate cell death and survival through protein–protein interactions, and physically associate. Accordingly, we examined the ability of the ABT series of BH3 mimetics to interact with σ receptors using radioligand-binding techniques. Negative allosteric modulation of [3H](+)-pentazocine, an agonist, binding to σ1 receptors in guinea pig brain membranes was observed for ABT-737, ABT-263 and ABT-199. Findings included reduction of specific binding to distinct plateaus in concentration-dependent fashion, significant slowing of radioligand dissociation kinetics, and decreases in radioligand affinity with no or modest changes in maximal receptor densities. Using a ternary complex model, dissociation constants (KX) for modulator binding to the σ1 receptor ranged from 1 to 2.5 μM, while negative cooperativity factors (α), representing the changes in affinity of ligand and modulator when bound as a ternary complex with the receptor, ranged from 0.15 to 0.42. These observations were extended and reinforced by studies using intact small cell (NCI-H69) and non-small cell (NCI-H23) lung cancer cells, and by using an antagonist σ1 receptor radioligand, E-N-1-(3′-[125I]iodoallyl)-N′-4-(3″,4″-dimethoxyphenethyl)piperazine, in mouse brain membranes. By contrast, exploratory studies indicate marked enhancement of the σ2 receptor binding of [3H]1,3-di-(o-tolyl)guanidine/(+)-pentazocine in NCI-H23 cells and guinea pig brain membranes. These findings raise intriguing questions regarding mechanism and potential functional outcomes.

Radioiodination and preclinical evaluation of 4-benzyl-1-(3-[125I]-iodobenzylsulfonyl)piperidine as a breast tumor imaging tracer in mouse

4-Benzyl-1-(3-iodobenzylsulfonyl)piperidine, 4-B-IBSP, has shown high-binding affinity to both sigma (σ) receptors in our previous work. In current study, radiolabeling and preclinical evaluation of 4-benzyl-1-(3-[125I]-iodobenzylsulfonyl)piperidine, 4-B-[125I]IBSP, in human ductal breast carcinoma (T47D) cells and in breast adenocarcinoma-bearing BALB/c mice are described. Radioiodination of this new σ ligand was performed by a palladium-catalyzed stannylation approach followed by oxidative iododestannylation reaction using Iodo-Gen. Competition-binding assays for binding of 4-B-[125I]IBSP to guinea pig brain membranes and toT47D cells were performed with known σ ligands. The selectivity and binding characteristics (B max and K d) were analyzed. In vitro stability and in vivo blood metabolism studies werealso evaluated. Moreover, biodistribution studies were performed in normal and into the tumor-bearing mice at interval time points post-injection (p.i.). Both in vitro and in vivo blockade experiments were done in the presence of the σ receptors blocking agents. Radioiodinated ligand was obtained in high yield and high specific activity. The σ inhibition constants (K i, nM) for 4-(3-iodobenzyl)-1-(benzylsulfonyl)piperazine (4-IBBSPz), (+)-pentazocine, haloperidol, DTG, and 4-B-IBSP were 1.37 ± 0.19, 3.90 ± 0.77, 2.69 ± 0.33, 30.62 ± 2.01, and 0.61 ± 0.05, respectively. 4-B-[125I]IBSP bound to σ receptor sites preferably to very high-affinity binding sites on T47D cells. The radioligand showed acceptable in vitro and in vivo stabilities in the blood pool. However, in vivo biodistribution studies in normal Swiss albino mice revealed fast clearance of 4-B-[125I]IBSP from blood and the other normal organs. Biodistribution experiments of 4-B-[125I]IBSP in breast adenocarcinoma tumor-bearing BALB/c mice showed a relatively high tumor uptake at 30min p.i. (4.13 ± 0.95) that reaches to 1.57 ± 0.24 even after 240min p.i. A pre-injection of 4-B-IBSP and haloperidol with 4-B-[125I]IBSP resulted in 36-57% decrease in activity in the tumor, liver, and brain at 60min p.i. The high affinity of 4-B-[125I]IBSP to σ receptor-binding sites, its relatively high uptake, and preferential retention in the tumor as well as an increasing trend observed in the tumor to blood and in the tumor to muscle ratios suggests that an iodine-123 labeled counterpart, 4-B-[123I]IBSP, would be a promising σ radioligand for pursuing further studies to assess its potential for breast tumors imaging with SPECT.

Scouting new sigma receptor ligands: Synthesis, pharmacological evaluation and molecular modeling of 1,3-dioxolane-based structures and derivatives

Herein we report the synthesis and biological activity of new sigma receptor (σR) ligands obtained by combining different substituted five-membered heterocyclic rings with appropriate σR pharmacophoric amines. Radioligand binding assay, performed on guinea pig brain membranes, identified 25b (1-(1,4-dioxaspiro[4.5]decan-2-ylmethyl)-4-benzylpiperazine) as the most interesting compound of the series, displaying high affinity and selectivity for σ1R (pKiσ1 = 9.13; σ1/σ2 = 47). The ability of 25b to modulate the analgesic effect of the κ agonist (−)-U-50,488H and μ agonist morphine was evaluated in vivo by radiant heat tail-flick test. It exhibited anti-opioid effects on both κ and μ receptor-mediated analgesia, suggesting an agonistic behavior at σ1R. Docking studies were performed on the theoretical σ1R homology model. The present work represents a new starting point for the design of more potent and selective σ1R ligands.

Synthesis and pharmacological evaluation of [(3)H]HS665, a novel, highly selective radioligand for the kappa opioid receptor.

Herein we report the radiolabeling and pharmacological investigation of a novel radioligand, the N-cyclobutylmethyl substituted diphenethylamine [(3)H]HS665, designed to bind selectively to the kappa opioid peptide (KOP) receptor, a target of therapeutic interest for the treatment of a variety of human disorders (i.e., pain, affective disorders, drug addiction, and psychotic disorders). HS665 was prepared in tritium-labeled form by a dehalotritiated method resulting in a specific activity of 30.65 Ci/mmol. Radioligand binding studies were performed to establish binding properties of [(3)H]HS665 to the recombinant human KOP receptor in membranes from Chinese hamster ovary cells stably expressing human KOP receptors (CHOhKOP) and to the native neuronal KOP receptor in guinea pig brain membranes. Binding of [(3)H]HS665 was specific and saturable in both tissue preparations. A single population of high affinity binding sites was labeled by [(3)H]HS665 in membranes from CHOhKOP cells and guinea pig brain with similar equilibrium dissociation constants, Kd, 0.45 and 0.64 nM, respectively. Average receptor density of [(3)H]HS665 recognition sites were 5564 and 154 fmol/mg protein in CHOhKOP cells and guinea pig brain, respectively. This study shows that the new radioligand distinguishes and labels KOP receptors specifically in neuronal and cellular systems expressing KOP receptors, making this molecule a valuable tool in probing structural and functional mechanisms governing ligand-KOP receptor interactions in both a recombinant and native in vitro setting.

Relationship between cerebral sigma-1 receptor occupancy and attenuation of cocaine's motor stimulatory effects in mice by PD144418.

Psychostimulant effects of cocaine are mediated partly by agonist actions at sigma-1 (σ1) receptors. Selective σ1 receptor antagonists attenuate these effects and provide a potential avenue for pharmacotherapy. However, the selective and high affinity σ1 antagonist PD144418 (1,2,3,6-tetrahydro-5-[3-(4-methylphenyl)-5-isoxazolyl]-1-propylpyridine) has been reported not to inhibit cocaine-induced hyperactivity. To address this apparent paradox, we evaluated aspects of PD144418 binding in vitro, investigated σ1 receptor and dopamine transporter (DAT) occupancy in vivo, and re-examined effects on locomotor activity. PD144418 displayed high affinity for σ1 sites (Ki 0.46 nM) and 3596-fold selectivity over σ2 sites (Ki 1654 nM) in guinea pig brain membranes. No appreciable affinity was noted for serotonin and norepinephrine transporters (Ki >100 μM), and the DAT interaction was weak (Ki 9.0 μM). In vivo, PD144418 bound to central and peripheral σ1 sites in mouse, with an ED50 of 0.22 μmol/kg in whole brain. No DAT occupancy by PD144418 (10.0 μmol/kg) or possible metabolites were observed. At doses that did not affect basal locomotor activity, PD144418 (1, 3.16, and 10 μmol/kg) attenuated cocaine-induced hyperactivity in a dose-dependent manner in mice. There was good correlation (r(2) = 0.88) of hyperactivity reduction with increasing cerebral σ1 receptor occupancy. The behavioral ED50 of 0.79 μmol/kg corresponded to 80% occupancy. Significant σ1 receptor occupancy and the ability to mitigate cocaine's motor stimulatory effects were observed for 16 hours after a single 10.0 μmol/kg dose of PD144418.

Synthesis and biological evaluation of a novel sigma-1 receptor antagonist based on 3,4-dihydro-2(1H)-quinolinone scaffold as a potential analgesic

The synthesis and sigma-1 receptor (σ1R) antagonist activity of a new series of 3,4-dihydro-2(1H)-quinolinone derivatives are reported. The new compounds were evaluated in vitro in sigma-1 and sigma-2 receptor-binding assays in guinea pig brain membranes. The structure–activity relationship led us to the promising derivative 7-(3-(piperidin-1-yl)propoxy)-1-(4-fluorobenzyl)-3,4-dihydro-2(1H)-quinolinone (35). The compounds with highest affinity and greatest selectivity were further profiled, and compound 35 had a high binding constant for sigma-1 receptor (Kiσ1 = 1.22 nM) and high sigma-1/2 selectivity (1066-fold). Thus, compound 35, which proved to be an antagonist of sigma-1 receptor, emerged as the most interesting candidate. In addition, compound 35 exerted dose-dependent anti-nociceptive effects in the formalin test. These characteristics suggested that the potent and selective compound 35 could be a potent candidate for pain treatment.

Characterization of Ligand Binding to the σ1Receptor in a Human Tumor Cell Line (RPMI 8226) and Establishment of a Competitive Receptor Binding Assay

The standard assay for the determination of σ(1) receptor affinities of novel compounds is a competitive binding assay using [(3)H]-(+)-pentazocine as radioligand and membrane preparations from guinea pig brain. Herein, a novel competitive binding assay was developed employing the hematopoietic cell line of human multiple myeloma (RPMI 8226), which expresses a large amount of the human σ(1) receptor. Membrane fragments of RPMI 8226 cells were prepared and characterized. A Western blot analysis confirmed the high density of σ(1) receptors in this cell line. Assay conditions were carefully optimized leading to an incubation period of 120 min, an incubation temperature of 37°C, and receptor material for each well was prepared from 300,000 cells. It was shown that a large excess (10 μM) of (+)-pentazocine, haloperidol, and di-o-tolylguanidine provided the same results during determination of the nonspecific binding. Saturation experiments with the radioligand [(3)H]-(+)-pentazocine led to a K(d)-value of 36±0.3 nM and a B(max)-value of 477±7 fmol/mg protein. These data resulted in approximately 122,000 σ(1) binding sites per cell. The assay was validated by using six known σ(1) ligands and eight σ(1) ligands prepared in our lab. The K(i)-values determined with RPMI 8226-derived receptor material are in good accordance with the K(i)-values obtained with guinea pig brain membrane preparations. Compared with guinea pig brain preparations, the RPMI 8226-derived receptor material represents a better standardized receptor material with a high density of human σ(1) receptors.

Development of novel N-methyl and N-allyl-substituted oxazolomorphinans and their interaction with opioid receptors

Methods The chemistry involved the design and synthesis of two sets of oxazolomorphinans having the new heteroring anellated to the A-ring of the morphinan backbone. Binding affinities of the newly synthesized compounds at opioid receptors were determined by in vitro competition binding assays using rat brain (μ, δ) and guinea pig brain ( ) membranes and employing [H]DAMGO (μ), [H] [Ile]deltorphin II (δ) and [H]U-69,593 ( ) as specific opioid radioligands. The in vitro pharmacological activities were established using [S]GTPgS functional assays in membranes from Chinese hamster ovary (CHO) cells expressing human opioid receptors.

Opioid receptor selectivity of peptide models of β-endorphin*

Two peptides, designed to contain structural models of the proposed hydrophilic linker domain (residues 6-12) and amphiphilic alpha-helical domain (residues 13-29) in beta-endorphin, have been tested for their abilities to mimic the opioid receptor selectivity profile of the natural hormone. In competitive binding assays employing guinea-pig brain membranes, both peptides displayed a much higher affinity for mu- and delta-opioid receptors than for kappa opioid receptors. Relative to beta-endorphin, the peptide models were 2-3 times more potent in the mu and kappa receptor binding assays, and about equipotent in the delta receptor binding assay. In guinea-pig ileum assays, one peptide was equipotent to beta-endorphin and the other was twice as potent. Like beta-endorphin, their actions on this tissue were highly sensitive to naloxone antagonism, indicating that they were mediated by mu receptors and not kappa receptors. In view of the design of the two peptide models, and their minimal homology to the natural hormone, these results provide additional evidence in support to our proposal for the functional conformation of beta-endorphin.

σ1 and σ2 receptor binding affinity and selectivity of SA4503 and fluoroethyl SA4503

SA4503, a potent sigma(1) receptor agonist, is reported as having 103-fold higher affinity for sigma(1) (IC(50) = 17.4 nM) than sigma(2) (IC(50) = 1,784 nM) sites in guinea pig brain membranes. Modest structural changes appear to have major effects on sigma(1)/sigma(2) selectivity. The fluoroethyl analog, FE-SA4503, is described as having high primary affinity for sigma(2) sites (IC(50) = 2.11 nM) and a weaker interaction with sigma(1) sites (IC(50) = 6.48 nM). SA4503 and FE-SA4503 have been radiolabeled for PET studies, and both bind selectively to sigma(1) receptors in animal and human brain in vivo. We prepared SA4503 and FE-SA4503 as reference compounds for radioligand development efforts. In our hands, SA4503 is 14-fold selective for sigma(1) (K(i) = 4.6 nM) over sigma(2) (K(i) = 63.1 nM) sites in guinea pig brain homogenates. Further, FE-SA4503 exhibits the same 14-fold selectivity for sigma(1) (K(i) = 8.0 nM) over sigma(2) (K(i) = 113.2 nM) receptors. The main differences from previously reported values stem from sigma(2) affinity determinations. This protocol, displacement of [(3)H]DTG binding to sigma(2) sites using (+)-pentazocine (200 nM) to mask sigma(1) sites, was validated by the proper rank order of sigma(2) inhibitory potencies shown by a panel of additional ligands: ifenprodil > haloperidol > DTG >> (+)-pentazocine. Robust Pearson correlation (r = 1.0, P = 0.002; slope = 0.97) was observed for our pK(i) values against those from a prior study by others. The findings have bearing on structure-activity relationships for this active series, and on conclusions that might be drawn from experiments relying upon defined sigma(1)/sigma(2) binding selectivity.

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 σ1- and σ2-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 σ1-receptor affinity. In this series the p-methoxybenzyl substituted piperazine 3d reveals the highest σ1-receptor affinity (Ki=12.4 nM) with selectivity toward σ2-, NMDA-, κ-opioid, and μ-opioid receptors.

Chiral, nonracemic (piperazin-2-yl)methanol derivatives with σ-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 σ 1- and σ 2-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 σ 1-receptor affinity. In this series the p-methoxybenzyl substituted piperazine 3d reveals the highest σ 1-receptor affinity ( K i=12.4 nM) with selectivity toward σ 2-, NMDA-, κ-opioid, and μ-opioid receptors.

A study of bile acids as opioid receptor ligands in rat brain membranes

There is evidence to suggest that the pruritus that results from liver disease is mediated, at least in part, by opioid receptor-ligand interactions; a central component has been proposed. Opiate drugs with agonist activity at opioid receptors induce naloxone-reversible pruritus. Bile acids accumulate in tissues in liver disease. We studied the ability of bile acids to displace specific opioid ligands from opioid receptors in rat and guinea pig brain membrane preparation in binding assays. None of the bile acids studied displaced significantly the opioid ligands from their receptors suggesting that bile acids in vitro are not opioid receptor ligands. The results of this study do not support a role of these bile acids as direct pruritogens by an opioid receptor-mediated mechanism.

Identification and localisation of the sodium channel alpha subunits carrying the Ttx-sensitive sodium current (INa) in the enteric nervous system of the guinea PIG intestine

The sodium channel protein consists of an alpha subuint in association with auxiliary beta subunits. The 5 neuronal alpha subnnit isoforms that carry the TTX-sensittve current are differentially expressed according to the neuronal types and have different functional properties, which result in distinct conductances in specific cells. Our work was designed in order to identify which alpha subunits carry the TTX-sensitive Na current in the enteric nervous system. Experiments were performed on fixed whole mount preparations of the myenteric and submucous plexuses of guinea pig (GP) small and large intestine using polydonal antibodies (ABs) raised against rat and/or human epitopes of the following alpha subunits : Navl.1, Nay1.2, Navl.3, Navl.6 and Nay1.7. Positive controls on non enteric nervous system were obtained for all ABs except that against Nay1.7. AB selectivity was verified using Western blots performed on rat and GP brain membrane preparations. Only NaV1.2 and Navl.3 alpha subunits were expressed in enteric neurons. NaV1.2 was expressed in the majority of the enteric neurons and immunoreactivity was located in the soma. Interestingly, it was highly expressed in a single axon hillock of the enteric sensory neurons. Some of the myenteric NaV1.2 immunoreactive non-sensory neurons are likely to be motor neurons that innervate the longitudinal muscle. Navl.3-fike immunoreactivity was observed only in descending filamentous neurons It was located in the soma, the long ramifying dendrites and the axon. Numerous smooth nerve fibers were also labeled. These fibres are extrinsic in origin since they enter the gut wall in large bundles running from the mesentery. Using Western blots performed on adult and new born rat and GP brain membrane preparations, the Navl.2 AB, raised against epitopes located in the I-I1 intracellular loop (M1 IL), stained a band at 230 kDa. Using rat brain membrane preparations, the Navl.3 AB, also raised against an epitope located in 1-II 1L, stained a band at 130 kDa. A band of similar low molecular weight was also stained using a Pan AB raised against the loop llI-IV. The expression of both the 230 and 130 kDa protein was age-dependent as previously described for Navl.2 and Navl 3 (Felts eta;. 1997). Our data strongly suggests that in enteric sensory neurons and in some motor neurons of the GP the TTX-sensitive INa is exclusively car'ned by the Navl.2 alpha subunit. This work is supported by GSK and CNRS.

Synthesis of (+)-cis-N-(4-isothiocyanatobenzyl)-N-normetazocine, an isothiocyanate derivative of N-benzylnormetazocine as acylant agent for the sigma(1) receptor.

(+)-cis-N-(4-Isothiocyanatobenzyl)-N-normetazocine (BNIT) (+)-(4) was designed and synthesized as a derivative of the potent and selective sigma(1) receptor ligand (+)-cis-N-benzyl-N-normetazocine for irreversibly blocking sigma(1) binding sites. Pretreatment of guinea pig brain membranes with BNIT (0.1, 1, and 5 microM) caused a concentration-dependent loss of binding of the selective sigma(1) ligand [(3)H]-(+)-pentazocine. Binding experiments with [(3)H]-1,3-di(2-tolyl)guanidine ([(3)H]-DTG), a ligand of sigma(1) and sigma(2) receptors, showed that pretreatment with BNIT blocked only the sigma(1) component of [(3)H]-DTG binding.

Novel Spiropiperidines as Highly Potent and Subtype Selective σ-Receptor Ligands. Part 1

A series of spiro[[2]benzopyran-1,4'-piperidines] and spiro[[2]benzofuran-1,4'-piperidines] of general structure 10 is prepared, and the affinity for sigma(1)- and sigma(2)-receptors is investigated by means of radioligand binding assays. The synthesis of the spiropiperidines 14a and 23 proceeds from bromine/lithium exchange of the bromoacetals 11 and 21, addition to piperidin-4-one 12a, and subsequent cyclization. Systematic variations of the substituent R at the nitrogen atom, the group X in position 3, and the ring size of the oxygen heterocycle are performed. The sigma(1)- and sigma(2)-receptor affinities are determined with guinea pig brain and rat liver membrane preparations using [(3)H]-labeled (+)-pentazocine and ditolylguanidine, respectively. Test results show that a benzyl residue at the piperidine nitrogen atom and a methoxy group in position 3 are advantageous for high sigma(1)-receptor affinity. In this series the 1'-benzyl-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,4'-piperidine] (14a) and the 1'-benzyl-3-methoxy-3H-spiro[[2]benzofuran-1,4'-piperidine] (23) are among the most potent sigma(1)-ligands interacting in the low nanomolar range with sigma(1)-receptors (14a, K(i) = 1.29 nM; 23, K(i) = 1.14 nM). Variation of the nitrogen substituent R from benzyl to H, alkyl, phenyl, or omega-phenylalkyl and the group X from methoxy to hydroxy, carbonyl, or alkyloxy led to reduced sigma(1)-receptor affinity. In addition to their high sigma(1)-receptor affinity, the spiropiperidines 14a and 23 display excellent selectivity toward sigma(2)-receptors (sigma(1)/sigma(2) = 2708 and 1130) and several other receptor and reuptake systems. Introduction of a polar hydroxy group in position 3 and elongation of the distance between the piperidine nitrogen atom and the phenyl moiety result in ligands with considerable sigma(2)-receptor affinity and therefore diminished sigma(1)/sigma(2)-receptor selectivity. The hemiacetalic 1'-(3-phenylpropyl)-3,4-dihydrospiro[[2]benzopyran-1,4'-piperidin]-3-ol (15e) represents the most active sigma(2)-receptor ligand in this series with a K(i) value of 83.1 nM.

2',6'-Dimethyltyrosine]dynorphin A(1-11)-NH2 analogues lacking an N-terminal amino group: potent and selective kappa opioid antagonists.

Recent studies showed that dermorphin and enkephalin analogues containing two methyl groups at the 2',6'-positions of the Tyr(1) aromatic ring and lacking an N-terminal amino group were moderately potent delta and mu opioid antagonists. These results indicate that a positively charged N-terminal amino group may be essential for signal transduction but not for receptor binding and suggested that its deletion in agonist opioid peptides containing an N-terminal 2',6'-dimethyltyrosine (Dmt) residue may represent a general way to convert them into antagonists. In an attempt to develop dynorphin A (Dyn A)-derived kappa opioid antagonists, we prepared analogues of [Dmt(1)]Dyn A(1-11)-NH2 (1), in which the N-terminal amino group was either omitted or replaced with a methyl group. This was achieved by replacement of Tyr(1) with 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp]. Compounds were tested in the guinea pig ileum and mouse vas deferens bioassays and in rat and guinea pig brain membrane receptor binding assays. All analogues turned out to be potent kappa antagonists against Dyn A(1-13) and the non-peptide agonist U50,488 and showed only weak mu and delta antagonist activity. The most potent and most selective kappa antagonist of the series was [(2S)-Mdp(1)]Dyn A(1-11)-NH2 (5, dynantin), which showed subnanomolar kappa antagonist potency against Dyn A(1-13) and very high kappa selectivity both in terms of its K(e) values determined against kappa, mu, and delta agonists and in terms of its ratios of kappa, mu, and delta receptor binding affinity constants. Dynantin is the first potent and selective Dyn A-derived kappa antagonist known and may complement the non-peptide kappa antagonists norbinaltorphimine and GNTI as a pharmacological tool in opioid research.

A Novel γ-Secretase Assay Based on Detection of the Putative C-terminal Fragment-γ of Amyloid β Protein Precursor

Alzheimer's disease is characterized by the deposits of the 4-kDa amyloid beta peptide (A beta). The A beta protein precursor (APP) is cleaved by beta-secretase to generate a C-terminal fragment, CTF beta, which in turn is cleaved by gamma-secretase to generate A beta. Alternative cleavage of the APP by alpha-secretase at A beta 16/17 generates the C-terminal fragment, CTFalpha. In addition to A beta, endoproteolytic cleavage of CTF alpha and CTF beta by gamma-secretase should yield a C-terminal fragment of 57-59 residues (CTF gamma). However, CTF gamma has not yet been reported in either brain or cell lysates, presumably due to its instability in vivo. We detected the in vitro generation of A beta as well as an approximately 6-kDa fragment from guinea pig brain membranes. We have provided biochemical and pharmacological evidence that this 6-kDa fragment is the elusive CTF gamma, and we describe an in vitro assay for gamma-secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTF gamma fragment. Three compounds previously identified as gamma-secretase inhibitors, pepstatin-A, MG132, and a substrate-based difluoroketone (t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2, 2-difluoropentanoyl-Val-Ile-OMe), reduced the yield of CTF gamma, providing additional evidence that the fragment arises from gamma-secretase cleavage. Consistent with reports that presenilins are the elusive gamma-secretases, subcellular fractionation studies showed that presenilin-1, CTF alpha, and CTF beta are enriched in the CTF gamma-generating fractions. The in vitro gamma-secretase assay described here will be useful for the detailed characterization of the enzyme and to screen for gamma-secretase inhibitors.

Synthesis and in vitro opioid activity profiles of DALDA analogues

The tetrapeptide DALDA (H-Tyr- d-Arg-Phe-Lys-NH 2) is a polar and selective μ agonist showing poor penetration of the placental and blood–brain barriers. In an effort to enhance the potency of DALDA, analogues containing 2′,6′-dimethyltyrosine (Dmt), N,2′,6′-trimethyltyrosine (Tmt), 2′-methyltyrosine (Mmt) or 2′-hydroxy,6′-methyltyrosine (Hmt) in place of Tyr 1, or Orn or α,γ-diaminobutyric acid (A 2bu) in place of Lys 4, were synthesized. All compounds displayed high μ receptor selectivity in the rat and guinea pig brain membrane binding assays and most of them were more potent μ agonists than DALDA in the μ receptor-representative guinea pig ileum assay, with [Dmt 1]DALDA showing the highest potency. Because of its extraordinary μ agonist potency, high μ selectivity, polar character (charge of 3+) and metabolic stability, [Dmt 1]DALDA has potential for use in obstetrical or peripheral analgesia.