Ferenc Ötvös

Binding characteristics of the novel highly selective delta agonist, [3H]Ile5,6deltorphin II

Following the description of the [3H]deltorphin II, it has been reported that the modification of deltorphin II with the substitution of Val5,6 residues by the more hydrophobic IIe5,6 residues leads to an increased affinity and selectivity. The IIe5,6 deltorphin II (Tyr-D-Ala-Phe-Gly-IIe-IIe-HH2) was tritiated by catalytic dehalogenation and labelled rat brain membrane sites with a Kd value of 0.40 nM and a Bmax of 121 fmol/mg protein. Competition binding experiments with various unlabelled subtype specific opioid receptor ligands resulted in mu/delta and kappa/delta selectivity ratios of 2400 and 18,000 respectively. Due to its high delta receptor affinity, delta selectivity and very low non-specific binding (< 20%), [3H]IIe5,6 deltorphin II, is a very useful tool for the identification and characterisation of delta opioid receptors.

Pancreatic cancer cells require an EGF receptor-mediated autocrine pathway for proliferation in serum-free conditions

In-vitro and in-vivo studies have shown that autocrine growth factors and receptors are frequently expressed in human malignancies. Few of these studies, however, provide evidence that the identified autocrine pathway is functional. In this study, a functional autocrine growth pathway in pancreatic cancer has been identified using an in-vitro cell culture system. When pancreatic cancer cells were grown without change of medium, proliferation was greater than when either medium was replaced frequently (HPAF, CAPAN-2, PANC-1 or SW1990) or cells were grown in the presence of the EGF receptor tyrosine kinase inhibitor AG1478 or the MEK inhibitor PD098059 (HPAF or CAPAN-2). Activity of extracellular-regulated kinases (ERK) 1 and 2 and c- jun and c- fos mRNA levels were significantly elevated in CAPAN-2 cells cultured continuously in serum-free medium. Collectively, the observations indicate that the EGF receptor and the ERK MAP kinase pathway mediate autocrine signals. In contrast to previous reports, the GRP and IGF-I receptors were shown not to be required for autocrine effects on pancreatic cancer cell proliferation. Autocrine stimulation of the EGF receptor can contribute to sustained mitogenic activity and proliferation of pancreatic cancer cells.

Characterization of [3H]naltrindole binding to delta opioid receptors in rat brain

[3H]Naltrindole binding characteristics were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C measured an equilibrium dissociation constant (Kd) value of 37.0 +/- 3.0 pM and a receptor density (Bmax) value of 63.4 +/- 2.0 fmol/mg protein. Association binding studies showed that equilibrium was reached within 90 min at a radioligand concentration of 30 pM. Naltrindole, as well as the ligands selective for delta (delta) opioid receptors, such as pCI-DPDPE and Deltorphin II inhibited [3H]naltrindole binding with nanomolar IC50 values. Ligands selective for mu (mu) and kappa (kappa) opioid receptors were only effective in inhibiting [3H]naltrindole binding at micromolar concentrations. From these data, we conclude that [3H]naltrindole is a high affinity, selective radioligand for delta opioid receptors.

Functionally important amino acid residues in the transient receptor potential vanilloid 1 (TRPV1) ion channel - an overview of the current mutational data

This review aims to create an overview of the currently available results of site-directed mutagenesis studies on transient receptor potential vanilloid type 1 (TRPV1) receptor. Systematization of the vast number of data on the functionally important amino acid mutations of TRPV1 may provide a clearer picture of this field, and may promote a better understanding of the relationship between the structure and function of TRPV1. The review summarizes information on 112 unique mutated sites along the TRPV1, exchanged to multiple different residues in many cases. These mutations influence the effect or binding of different agonists, antagonists, and channel blockers, alter the responsiveness to heat, acid, and voltage dependence, affect the channel pore characteristics, and influence the regulation of the receptor function by phosphorylation, glycosylation, calmodulin, PIP2, ATP, and lipid binding. The main goal of this paper is to publish the above mentioned data in a form that facilitates in silico molecular modelling of the receptor by promoting easier establishment of boundary conditions. The better understanding of the structure-function relationship of TRPV1 may promote discovery of new, promising, more effective and safe drugs for treatment of neurogenic inflammation and pain-related diseases and may offer new opportunities for therapeutic interventions.

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.

Anti-calmodulins and Tricyclic Adjuvants in Pain Therapy Block the TRPV1 Channel

Ca2+-loaded calmodulin normally inhibits multiple Ca2+-channels upon dangerous elevation of intracellular Ca2+ and protects cells from Ca2+-cytotoxicity, so blocking of calmodulin should theoretically lead to uncontrolled elevation of intracellular Ca2+. Paradoxically, classical anti-psychotic, anti-calmodulin drugs were noted here to inhibit Ca2+-uptake via the vanilloid inducible Ca2+-channel/inflamatory pain receptor 1 (TRPV1), which suggests that calmodulin inhibitors may block pore formation and Ca2+ entry. Functional assays on TRPV1 expressing cells support direct, dose-dependent inhibition of vanilloid-induced 45Ca2+-uptake at µM concentrations: calmidazolium (broad range)≥trifluoperazine (narrow range)>chlorpromazine/amitriptyline>fluphenazine>>W-7 and W-13 (only partially). Most likely a short acidic domain at the pore loop of the channel orifice functions as binding site either for Ca2+ or anti-calmodulin drugs. Camstatin, a selective peptide blocker of calmodulin, inhibits vanilloid-induced Ca2+-uptake in intact TRPV1+ cells, and suggests an extracellular site of inhibition. TRPV1+, inflammatory pain-conferring nociceptive neurons from sensory ganglia, were blocked by various anti-psychotic and anti-calmodulin drugs. Among them, calmidazolium, the most effective calmodulin agonist, blocked Ca2+-entry by a non-competitive kinetics, affecting the TRPV1 at a different site than the vanilloid binding pocket. Data suggest that various calmodulin antagonists dock to an extracellular site, not found in other Ca2+-channels. Calmodulin antagonist-evoked inhibition of TRPV1 and NMDA receptors/Ca2+-channels was validated by microiontophoresis of calmidazolium to laminectomised rat monitored with extracellular single unit recordings in vivo. These unexpected findings may explain empirically noted efficacy of clinical pain adjuvant therapy that justify efforts to develop hits into painkillers, selective to sensory Ca2+-channels but not affecting motoneurons.

Interaction of agonist peptide [3H]Tyr-d-Ala-Phe-Phe-NH2 with μ-opioid receptor in rat brain and CHO-μ/1 cell line

Opioid receptor binding properties of [3H]Tyr-D-Ala-Phe-Phe-NH2 (TAPP) were characterized in rat brain and Chinese hamster ovary (CHO) cells expressing the rat mu-receptor. In rat brain, [3H]TAPP labeled a single class of opioid sites with a dissociation constant (Kd) of 0.31 nM and maximal number of binding sites (Bmax) of 119 fmol/mg protein. In CHO-mu/1 cell membranes, the Kd and Bmax values were 0.78 nM and 1806 fmol/mg protein, respectively. Binding to rat brain was demonstrated to be pharmacologically identical to that obtained with CHO-mu/1 cell membranes and modulated by Na+ ions and guanine nucleotides. The high affinity and selectivity of [3H]TAPP together with its low non-specific binding make this radioligand a useful tool for labeling the native and cloned mu-opioid receptor.

Reversible Opening of Intercellular Junctions of Intestinal Epithelial and Brain Endothelial Cells With Tight Junction Modulator Peptides

The intercellular junctions restrict the free passage of hydrophilic compounds through the paracellular clefts. Reversible opening of the tight junctions of biological barriers is investigated as one of the ways to increase drug delivery to the systemic circulation or the central nervous system. Six peptides, ADT-6, HAV-6, C-CPE, 7-mer (FDFWITP, PN-78), AT-1002, and PN-159, acting on different integral membrane and linker junctional proteins were tested on Caco-2 intestinal epithelial cell line and a coculture model of the blood-brain barrier. All peptides tested in nontoxic concentrations showed a reversible tight junctions modulating effect and were effective to open the paracellular pathway for the marker molecules fluorescein and albumin. The change in the structure of cell-cell junctions was verified by immunostaining for occludin, claudin-4,-5, ZO-1, β-catenin, and E-cadherin. Expression levels of occludin and claudins were measured in both models. We could demonstrate a selectivity of C-CPE, ADT-6, and HAV-6 peptides for epithelial cells and 7-mer and AT-1002 peptides for brain endothelial cells. PN-159 was the most effective modulator of junctional permeability in both models possibly acting via claudin-1 and -5. Our results indicate that these peptides can be effectively and selectively used as potential pharmaceutical excipients to improve drug delivery across biological barriers.

Binding characteristics of [3H]14-methoxymetopon, a high affinity mu-opioid receptor agonist.

The highly potent µ‐opioid receptor agonist 14‐methoxymetopon (4,5α‐epoxy‐3‐hydroxy‐14β‐methoxy‐5β,17‐dimethylmorphinan‐6‐one) was prepared in tritium labelled form by a catalytic dehalogenation method resulting in a specific radioactivity of 15.9 Ci/mmol. Opioid binding characteristics of [3H]14‐methoxymetopon were determined using radioligand binding assay in rat brain membranes. [3H]14‐Methoxymetopon specifically labelled a single class of opioid sites with affinity in low subnanomolar range (Ki = 0.43 nm) and maximal number of binding sites of 314 fmol/mg protein. Binding of [3H]14‐methoxymetopon was inhibited by ligands selective for the µ‐opioid receptor with high potency, while selective κ‐opioids and δ‐opioids were weaker inhibitors. 14‐Methoxymetopon increased guanosine‐5′‐O‐(3‐[35S]thio)‐triphosphate ([35S]GTPγS) binding with an EC50 of 70.9 nm, thus, providing evidence for the agonist character of this ligand. The increase of [35S]GTPγS binding was inhibited by naloxone and selective µ‐opioid antagonists, indicating a µ‐opioid receptor‐mediated action. [3H]14‐Methoxymetopon is one of the few nonpeptide µ‐opioid receptor agonists available in radiolabelled form up to now. Due to its high affinity and selectivity, high stability and extremely low nonspecific binding (<10%), this radioligand would be an important and useful tool in probing µ‐opioid receptor mechanisms, as well as to promote a further understanding of the opioid system at the cellular and molecular level.

Inhibition of forebrain μ-opioid receptor signaling by low concentrations of rimonabant does not require cannabinoid receptors and directly involves μ-opioid receptors.

Increasing number of publications shows that cannabinoid receptor 1 (CB(1)) specific compounds might act in a CB(1) independent manner, including rimonabant, a potent CB(1) receptor antagonist. Opioids, cannabinoids and their receptors are well known for their overlapping pharmacological properties. We have previously reported a prominent decrease in μ-opioid receptor (MOR) activity when animals were acutely treated with the putative endocannabinoid noladin ether (NE). In this study, we clarified whether the decreased MOR activation caused by NE could be reversed by rimonabant in CB(1) receptor deficient mice. In functional [(35)S]GTPγS binding assays, we have elucidated that 0.1mg/kg of intraperitoneal (i.p.) rimonabant treatment prior to that of NE treatment caused further attenuation on the maximal stimulation of Tyr-d-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO), which is a highly specific MOR agonist. Similar inhibitory effects were observed when rimonabant was injected i.p. alone and when it was directly applied to forebrain membranes. These findings are cannabinoid receptor independent as rimonabant caused inhibition in both CB(1) single knockout and CB(1)/CB(2) double knockout mice. In radioligand competition binding assays we highlighted that rimonabant fails to displace effectively [(3)H]DAMGO from MOR in low concentrations and is highly unspecific on the receptor at high concentrations in CB(1) knockout forebrain and in their wild-type controls. Surprisingly, docking computational studies showed a favorable binding position of rimonabant to the inactive conformational state of MOR, indicating that rimonabant might behave as an antagonist at MOR. These findings were confirmed by radioligand competition binding assays in Chinese hamster ovary cells stably transfected with MOR, where a higher affinity binding site was measured in the displacement of the tritiated opioid receptor antagonist naloxone. However, based on our in vivo data we suggest that other, yet unidentified mechanisms are additionally involved in the observed effects.