Krisztina Monory

Specific activation of the μ opioid receptor (MOR) by endomorphin 1 and endomorphin 2

The recently discovered endomorphin 1 (Tyr‐Pro‐Trp‐Phe‐NH2) and endomorphin 2 (Tyr‐Pro‐Phe‐Phe‐NH2) were investigated with respect to their direct receptor‐binding properties, and to their ability to activate G proteins and to inhibit adenylyl cyclase in both cellular and animal models. Both tetrapeptides activated G proteins and inhibited adenylyl cyclase activity in membrane preparations from cells stably expressing the μ opioid receptor, an effect reversed by the μ receptor antagonist CTAP (d‐Phe‐Cys‐Tyr‐d‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2), but they had no influence on cells stably expressing the δ opioid receptor. To further establish the selectivity of these peptides for the μ opioid receptor, brain preparations of mice lacking the μ opioid receptor gene were used to study their binding and signalling properties. Endomorphin 2, tritiated by a dehalotritiation method resulting in a specific radioactivity of 1.98u2003TBq/mmol (53.4u2003Ci/mmol), labelled the brain membranes of wild‐type mice with a Kd value of 1.77u2003nm and a Bmax of 63.33u2003fmol/mg protein. In membranes of mice lacking the μreceptor gene, no binding was observed, and both endomorphins failed to stimulate [35S]guanosine‐5′‐O‐(3‐thio)triphosphate ([35S]GTPγS) binding and to inhibit adenylyl cyclase. These data show that endomorphins are capable of activating G proteins and inhibiting adenylyl cyclase activity, and all these effects are mediated by the μ opioid receptors.

Opioid binding profiles of new hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxymorphinans

Several hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxycodeinones and 14-alkoxydihydrocodeinones were synthesised [1] and characterised in in vitro radioligand binding assays in rat brain membrane preparations. The tested compounds show the highest affinity for the mu opioid binding sites and most of them have agonist character. Subtype analysis of the binding shows mu2 specificity. However, some of these ligands are able to block partially (40-60%) the high affinity (putative mu1) opioid binding sites while all of them act as reversible ligands at the low affinity (putative mu2) sites.

μ-Opioid receptor specific antagonist cyprodime : characterization by in vitro radioligand and [35S]GTPγS binding assays

The use of compounds with high selectivity for each opioid receptor (μ, δ and κ) is crucial for understanding the mechanisms of opioid actions. Until recently non-peptide μ-opioid receptor selective antagonists were not available. However, N-cyclopropylmethyl-4,14-dimethoxy-morphinan-6-one (cyprodime) has shown a very high selectivity for μ-opioid receptor in in vivo bioassays. This compound also exhibited a higher affinity for μ-opioid receptor than for δ- and κ-opioid receptors in binding assays in brain membranes, although the degree of selectivity was lower than in in vitro bioassays. Cyprodime has recently been radiolabelled with tritium resulting in high specific radioactivity (36.1 Ci/mmol). We found in in vitro binding experiments that this radioligand bound with high affinity (Kd 3.8±0.18 nM) to membranes of rat brain affording a Bmax of 87.1±4.83 fmol/mg. Competition studies using μ, δ and κ tritiated specific ligands confirmed the selective labelling of cyprodime to a μ-opioid receptor population. The μ-opioid receptor selective agonist [d-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO) was readily displaced by cyprodime (Ki values in the low nanomolar range) while the competition for δ- ([d-Pen2,d-Pen5]enkephalin (DPDPE)) and κ- (5α,7α,8β-(−)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]-benzene-acetamide (U69,593)) opioid receptor selective compounds was several orders of magnitude less. We also found that cyprodime inhibits morphine-stimulated [35S]GTPγS binding. The EC50 value of morphine increased about 500-fold in the presence of 10 μM cyprodime. These findings clearly indicate that cyprodime is a useful selective antagonist for μ-opioid receptor characterization.

Synthesis and biological evaluation of 14-alkoxymorphinans. 14.1 14-Ethoxy-5-methyl substituted indolomorphinans with δ opioid receptor selectivity

Abstract The 5-methyl and 14-ethoxy substituted analogues (compounds 2 – 4) of the δ opioid receptor antagonist naltrindole showed similar selectivity when compared with the reference drug. Compound 2 was a δ receptor antagonist in the mouse vas deferens preparation (MVD) exhibiting considerably higher selectivity ratios than naltrindole, while compound 4 was found to be a full and potent δ receptor agonist in the MVD.

Novel Delta-Opioid-Receptor-Selective Ligands in the 14-Alkoxy-Substituted Indolo- and Benzofuromorphinan Series

Several new indolo- and benzofuromorphinans substituted at the positions 5 and 14 were prepared and tested in vitro by means of opioid-receptor binding and functional ([35S]GTPγS binding) assays. All compounds 1u2009–u200911 displayed high affinity for δ opioid-binding sites (Tableu20051). Compound 4 proved to be an agonist, and all other compounds were antagonists. The presence of a Me group at position 5 induced no change in δ affinity (see 1vs.3), but decreased the μ and κ affinities. An EtO group at position 14 conferred a very high affinity and also high selectivity to δ opioid receptors (see 2 and 10). Chain elongation of the 14-alkoxy group resulted in compounds with reduced δ affinity and selectivity (see 4 and 11 and also 5u2009–u20099). The results of the present study indicate that the 5- and 14-positions of indolo- and benzofuromorphinans represent critical sites that could be a trigger to develop new compounds with increased δ affinity and/or selectivity.

Effects of cannabinoids in Krox-24 targeted mice.

Krox-24 is an immediate early gene encoding a zinc-finger transcription factor implicated in several adaptive responses, and its induction by cannabinoids has been reported. We used mice targeted in the Krox-24 gene to specifically dissect the role of this protein in the acute and chronic central actions of cannabinoids. We report here on the ability of cannabinoids to activate G-proteins and to inhibit adenylyl cyclase, and to elicit behavioral responses in wild-type and mutant mice. The behavioral parameters and the biochemical correlates of abstinence after Δ9-THC withdrawal were evaluated. We show that Krox-24 is not involved in the acute analgesic effects of Δ9-THC and in the SR precipitated Δ9-THC withdrawal syndrome.

Characterization of N,N(Me)2-Dmt-Tic-OH, a delta selective opioid dipeptide antagonist

N,N(Me)2-Dimethyl-tyrosine-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid-OH (N,N(Me)2-Dmt-Tic-OH) is a very selective δ opioid dipeptide with elevated antagonist activity. We have radiolabelled this compound by catalytic tritiation of the N,N(Me)2-Dmt(3,5′−12)-Tic-OH precursor. The ligand labelled rat brain membranes with a Kd value of 0.42 nM and a Bmax of 63.12 fmol/mg protein. The new tritiated ligand showed high affinity for the δ opioid receptor whereas its binding at μ and κ opioid receptors was weak. N,N(Me)2-Dmt-Tic-OH was able to inhibit the agonist-stimulated binding of the non-hydrolysable GTP analogue [35S]GTPγS, thus attenuating the activation of G proteins via opioid receptors. This simple opioid dipeptide in both normal and labelled form may serve as a useful tool to study δ opioid receptors in vitro and in vivo.