GTPγS Functional Assays Research Articles

Discriminatory synergistic effect of Trp-substitutions in superagonist [(Arg/Lys) 14, (Arg/Lys) 15]nociceptin on ORL1 receptor binding and activation

ORL1 is an endogenous G protein-coupled receptor for neuropeptide nociceptin. [(R/K) 14, (R/K) 15]nociceptin is a superagonist that strongly activates the ORL1 receptor. We have previously found that substituting with Trp can reproduce the potentiation induced by Arg or Lys at position 14. In the present study, in order to ensure the effect of Trp-substitution on the activities of [(R/K) 14, (R/K) 15]nociceptin, we synthesized [W 14, (R/K) 15]nociceptin and [(R/K) 14, W 15]nociceptin. [W 14, (R/K) 15]nociceptin was found to exhibit threefold higher binding activity and 10-fold greater potency in a functional [ 35S]GTPγS functional assay as compared to wild-type nociceptin. However, when only Trp was placed in position 15, the resulting analogues, [(R/K) 14, W 15]nociceptin, showed only a moderate enhancement of binding and biological activity (2–3 fold in both). These results indicate that the placement of Trp at position 14, unlike at position 15, enhances in a synergistic fashion the interaction of nociceptin with the ORL1 receptor. The results indicate that specific interactions feasible for Arg/Lys and Trp in common must be there for aromatic residues in ORL1, thus forming a cation/π interaction or π/π hydrophobic interaction. The necessity for a favorable electrostatic interaction appears strict in position 15.

Functionalization of the 6,14-bridge of the orvinols. Part 3: Preparation and pharmacological evaluation of 18- and 19-hydroxyl substituted orvinols

The orvinols are a class of potent opioids which have been extensively studied, yet little is known about the effects of introducing substituents into the 18- and 19-positions. The etheno bridge of thevinone was hydroxylated to give both the 18- and 19-hydroxyl substituted thevinols. After 3- O-demethylation to the corresponding orvinols, binding and GTPγS functional assays indicated that hydroxyl substitution at the 18- and 19-positions differentially affects the μ opioid efficacy of orvinols.

Synthesis and pharmacological studies of new hybrid derivatives of fentanyl active at the μ-opioid receptor and I2–imidazoline binding sites

Two series of fentanyl-derived hybrid molecules bearing potent I2–imidazoline binding site (IBS) ligands (i.e., guanidine and BU224 moieties) linked with an aliphatic (m=2, 3, 4, 6, 7, 8, 9 and 12 methylene units) or aromatic spacer were prepared. Their affinities for the μ-opioid receptors and for the I2–IBS were determined through competition binding studies on human postmortem brain membranes. Whereas the BU224 hybrid molecules bound to the μ-opioid receptor and the I2–IBS in the micromolar to low micromolar range, the alkaneguanidine series exhibited remarkable affinities in the nanomolar range for both receptors. [35S]GTPγS functional assays were performed on human postmortem brain membranes with selected ligands from each series (4f and 8g) showing the highest dual affinity for the μ-opioid receptor and I2–IBS affinities. Both compounds displayed agonist properties: at the μ-opioid receptor for the alkaneguanidine derivative 4f (spacer: six methylene units) and at a G-protein coupled receptor (GPCR) which remains to be determined for 8g. The lack of analgesic properties of 4f in vivo (i.e., hot plate and writhing tests in mice), discordant with the good in vitro binding data (Ki μ=1.04±0.28nM, Ki I2=409±238nM), may possibly be due to the low intrinsic efficacy of the compound. Alternatively, a low access to the central nervous system for this kind of hybrid molecules cannot be ruled out. Two new compounds reported here (9f and 13), which were not dual acting, are worth mentioning for their outstanding binding affinities; 9f bound to the μ-opioid receptor with a picomolar affinity (Ki=0.0098±0.0033nM), whereas 13 presented an I2–IBS affinity (Ki=18±11nM) similar to the reference compound BU224.

Synthesis and Biological Evaluation of 14‐Alkoxymorphinans. Part 19

AbstractThe 14‐O‐benzylnaltrexones 3–6 were prepared from naltrexone (2) in several steps. The novel compounds were biologically evaluated in radioligand binding and in [35S]GTPγS functional assays in comparison to the reference compound naltrexone. In the binding assay, compounds 3–6 exhibited preference for κ opioid receptors, while the parent compound naltrexone shows preference for μ receptors. In the functional assay, μ antagonist potency of compounds 3–6 was in the range of naltrexone, while κ antagonist potency was considerably higher for most novel compounds in comparison to naltrexone.

Binding, pharmacological and immunological profiles of the δ-selective opioid receptor antagonist HS 378

HS 378 is a recently developed indolomorphinan with high selectivity and antagonist potency at the δ-opioid receptor. The present study was performed to characterize the opioid binding properties and pharmacological and immunological activity of HS 378 and to compare them with those of two well-known δ-opioid receptor antagonists, naltrindole (NTI) and naltriben (NTB). In vitro opioid receptor binding profiles were determined in rat brain homogenates. HS 378 showed 4.7- and 2.4- fold higher μ/δ selectivity compared to NTI and NTB, respectively. In the [ 35S]GTPγS functional assay carried out in cell lines expressing cloned human opioid receptors, HS 378 was found to be a pure δ-opioid receptor antagonist. In vitro, exposure of HS 378 resulted in an apparent dose-related suppression of concanavalin A induced rat T-lymphocyte proliferation with an IC 50 value of 0.54 μM. NTI showed also immunosuppression with an IC 50 value of 6.93 μM, whereas NTB had no effect. The IC 50 of HS 378 was 13 times lower than that of NTI and 8 times higher than that of cyclosporin A. Taken together, our findings indicate that the small molecule HS 378 has properties that may be of therapeutic value in the setting of human inflammatory diseases.

Highly Potent Nociceptin Analog Containing the Arg-Lys Triple Repeat

One of the structural characteristics of a neuropeptide nociceptin is the existence of Arg-Lys (RK) residues at positions 8–9 and 12–13; both RKs have been suggested to bind to the acidic amino acid cluster in the second extracellular loop of the seven transmembrane domain receptor ORL1. With a design strategy of attempting to obtain an analog that binds more strongly to the receptor's acidic cluster, we synthesized a series of nociceptin analogs in which the RK dipeptide unit was placed at positions 6–7, 10–11, or 14–15 adjacent to the parent RKs. Among these nociceptin analogs containing the RK triple repeat, [Arg-Lys6–7]- and [Arg-Lys10–11]nociceptins exhibited weak activities (6–9 and 60–90% of nociceptin, respectively) both in the receptor binding assay and in the [35S]GTPγS binding functional assay. In contrast, [Arg-Lys14–15]nociceptin was found to be very potent in both assays (3-fold in binding and 17-fold in GTPγS functional assay). [Arg-Lys14–15]nociceptin was the first peptide analog found to be stronger than the parent nociceptin, and structure–activity studies have suggested that the incorporated Arg-Lys14–15 interacts with either the receptor acidic amino acid cluster or the receptor aromatic amino acid residues.

(±)-4-[(N-allyl- cis-3-methyl-4-piperidinyl)phenylamino]-N,N-diethylbenzamide displays selective binding for the delta opioid receptor

Racemic 4-[(N-allyl- cis-3-methyl-4-piperidinyl)phenylamino]- N,N-diethylbenzamide ( 3a) was synthesized and found to have good affinity and selectivity for the δ receptor. these compounds can be viewed as an analog of BW373U86 and SNC-80 where an internal piperazine nitrogen has been transposed with a benzylic carbon. Functionally, 3a behaves as an agonist at the δ receptor with no measurable stimulation of either the μ or k receptor subtypes and was found to be devoid of any measurable amount of antagonist activity for any opioid receptor. A comparison of 3a to SNC-80 and DPDPE in the [ 35S]GTPγS functional assay suggests that 3a may be more like the peptide DPDPE.

N-Substituted 9beta-methyl-5-(3-hydroxyphenyl)morphans are opioid receptor pure antagonists.

The inhibition of radioligand binding and [35S]GTPgammaS functional assay data for N-methyl- and N-phenethyl-9beta-methyl-5-(3-hydroxyphenyl)morphans (5b and 5c) show that these compounds are pure antagonists at the micro, delta, and kappa opioid receptors. Since 5b and 5c have the 5-(3-hydroxyphenyl) group locked in a conformation comparable to an equatorial group of a piperidine chair conformation, this information provides very strong evidence that opioid antagonists can interact with opioid receptors in this conformation. In addition, it suggests that the trans-3, 4-dimethyl-4-(3-hydroxyphenyl)piperidine class of antagonist operates via a phenyl equatorial piperidine chair conformation. Importantly, the close relationship between the 4-(3-hydroxyphenyl)piperidines and 5-(3-hydroxyphenyl)morphan antagonists shows that the latter class of compound provides a rigid platform on which to build a novel series of opioid antagonists.

Relative efficacies of cannabinoid CB1 receptor agonists in the mouse brain

We measured (−)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-phenol (CP 55,940)-, (−)11-OH-Δ8-tetrahydrocannabinol-dimethylheptyl (HU-210)-, anandamide- and Δ9-tetrahydrocannabinol-stimulated G protein activation in mouse brain using the [35S]GTPγS functional assay. The Ki values for these drugs were determined by agonist competition binding with the cannabinoid CB1 receptor antagonist [3H] N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride ([3H]SR141716A). This information was used to calculate the efficacy for drug stimulation of G protein activity. The rank order of efficacy was CP 55,940>HU-210>anandamide>Δ9-tetrahydrocannabinol with the latter two drugs being partial agonists. Since efficacy values relate receptor occupancy to functional responses, we believe efficacy values are a better measure of drug-mediated functional responses compared with measurements of drug potency.