Mária Szücs | Biological Research Centre of Hungarian Academy of Sciences (original) (raw)

Papers by Mária Szücs

Research paper thumbnail of Ligand-specific regulation of the endogenous mu-opioid receptor by chronic treatment with mu-opioid peptide agonists

BioMed research international, 2013

Since the discovery of the endomorphins (EM), the postulated endogenous peptide agonists of the m... more Since the discovery of the endomorphins (EM), the postulated endogenous peptide agonists of the mu-opioid receptors, several analogues have been synthesized to improve their binding and pharmacological profiles. We have shown previously that a new analogue, cis-1S,2R-aminocyclohexanecarboxylic acid(2)-endomorphin-2 (ACHC-EM2), had elevated mu-receptor affinity, selectivity, and proteolytic stability over the parent compound. In the present work, we have studied its antinociceptive effects and receptor regulatory processes. ACHC-EM2 displayed a somewhat higher (60%) acute antinociceptive response than the parent peptide, EM2 (45%), which peaked at 10 min after intracerebroventricular (icv) administration in the rat tail-flick test. Analgesic tolerance developed to the antinociceptive effect of ACHC-EM2 upon its repeated icv injection that was complete by a 10-day treatment. This was accompanied by attenuated coupling of mu-sites to G-proteins in subcellular fractions of rat brain. Al...

Research paper thumbnail of Irreversible labelling of rat brain opioid receptors by enkephalin chloromethyl ketones

Neuropeptides, 1986

Chloromethyl ketone derivatives of leucine enkephalin (LE), D-Ala2-Leu5-enkephalin (DALE) and D-A... more Chloromethyl ketone derivatives of leucine enkephalin (LE), D-Ala2-Leu5-enkephalin (DALE) and D-Ala2-D-Leu5-enkephalin (DADLE) were synthesized. They all show high affinity for rat brain opioid binding sites. Preincubation of the membrane fraction with enkephalin chloromethyl ketones causes a significant inhibition of /3H/-naloxone binding which cannot be reversed by extensive washing. It was found that the irreversible inhibition is selective for the high affinity (KD less than 1 nM) /3H/-naloxone binding site (putative mu-1 site). The irreversible blockade of opioid binding was partially protected by opiate alkaloids and opioid peptides, suggesting that non-specific labelling also occurs. Affinity of enkephalin chloromethyl ketones toward the mu sites is greater than that of the parent compounds. It was also found that the covalent inhibition of mu sites (/3H/-dihydromorphine and /3H/-DAGO binding) is more effective than that of delta sites (/3H/-DALE binding). We conclude that these chloromethyl ketone derivatives can be used as affinity labels for the opioid receptors, allowing us to study the structure of the mu receptor subtype.

Research paper thumbnail of Ligand-specific regulation of the endogenous mu-opioid receptor by chronic treatment with mu-opioid peptide agonists

BioMed research international, 2013

Since the discovery of the endomorphins (EM), the postulated endogenous peptide agonists of the m... more Since the discovery of the endomorphins (EM), the postulated endogenous peptide agonists of the mu-opioid receptors, several analogues have been synthesized to improve their binding and pharmacological profiles. We have shown previously that a new analogue, cis-1S,2R-aminocyclohexanecarboxylic acid(2)-endomorphin-2 (ACHC-EM2), had elevated mu-receptor affinity, selectivity, and proteolytic stability over the parent compound. In the present work, we have studied its antinociceptive effects and receptor regulatory processes. ACHC-EM2 displayed a somewhat higher (60%) acute antinociceptive response than the parent peptide, EM2 (45%), which peaked at 10 min after intracerebroventricular (icv) administration in the rat tail-flick test. Analgesic tolerance developed to the antinociceptive effect of ACHC-EM2 upon its repeated icv injection that was complete by a 10-day treatment. This was accompanied by attenuated coupling of mu-sites to G-proteins in subcellular fractions of rat brain. Al...

Research paper thumbnail of Ovarian sex steroid-dependent plasticity of nociceptin/orphanin FQ and opioid modulation of spinal dynorphin release

The Journal of pharmacology and experimental therapeutics, 2001

Pregnancy and its hormonal simulation via 17beta-estradiol (E(2)) and progesterone (P) are associ... more Pregnancy and its hormonal simulation via 17beta-estradiol (E(2)) and progesterone (P) are associated with spinal opioid antinociception, primarily driven by augmented dynorphin/kappa-opioid activity. This study addresses the ovarian sex steroid-activated mechanism(s) that underlie this activation using an ex vivo spinal cord preparation. In lumbar spinal cord obtained from control animals, exogenous kappa- or delta-opioid agonists (but not mu), as well as nociceptin (orphanin FQ; N/OFQ), dose dependently inhibit the stimulated release of dynorphin. Consistent with these observations, stimulated dynorphin release is enhanced following selective blockade of opioid or N/OFQ receptors, indicating that their endogenous ligands are negative modulators of dynorphin release. In lumbar spinal cord obtained from ovariectomized animals exposed to pregnancy blood levels of E(2)/P, basal and stimulated rates of dynorphin release increase approximately 2-fold. Moreover, evoked dynorphin release ...

Research paper thumbnail of Separation of kappa-opioid receptor subtype from frog brain

FEBS letters, Jan 22, 1985

Complete separation of the [3H]ethylketocyclazocine [( 3H]EKC) specific binding (kappa subtype) f... more Complete separation of the [3H]ethylketocyclazocine [( 3H]EKC) specific binding (kappa subtype) from tritiated Tyr-D-Ala2-Me-Phe4-Gly-ol5 enkephalin (DAGO) and Tyr-D-Ala2-L-Leu5-enkephalin (DALA) binding (mu-and delta-subtypes, respectively) was achieved by Sepharose-6B chromatography and sucrose density gradient centrifugation of digitonin solubilized frog brain membranes. The apparent sedimentation coefficient (s20.w) for the kappa receptor-detergent complex was 13.1 S and the corresponding Stokes radius 64 A. The isolated fractions exhibited high affinity for EKC and bremazocine, whereas mu- and delta-specific ligands were unable to compete for the [3H]EKC binding sites, indicating that the kappa subtype represents a separate molecular to compete for the [3H]EKC binding sites, indicating that the kappa subtype represents a separate molecular entity from the mu and delta receptor sites.

Research paper thumbnail of A Monoclonal Antibody Recognizing K- but Not ?- and ?-Opioid Receptors

Journal of Neurochemistry, 1991

Abstract: A monoclonal antibody (mAb), KA8, that interacts with the k-opioid receptor binding sit... more Abstract: A monoclonal antibody (mAb), KA8, that interacts with the k-opioid receptor binding site was generated. BALB/c female mice were immunized with a partially purified k-opioid receptor preparation from frog brain. Spleen cells were hybridized with SP2/0AG8 ...

Research paper thumbnail of Application of an Opioid Affinity Label for Qualitative Analysis of Mu-Receptors

Vascular Endothelium, 1991

Research paper thumbnail of Irreversible blockade of the high and low affinity (3H) naloxone binding sites by C-6 derivatives of morphinane-6-ones

Life Sciences, 1991

C-6 derivatives--hydrazones, phenylhydrazones, dinitrophenylhydrazones, oximes and semicarbazones... more C-6 derivatives--hydrazones, phenylhydrazones, dinitrophenylhydrazones, oximes and semicarbazones--of morphinane-6-ones were synthesized and their binding characteristics were studied on rat brain membranes. The dihydromorphinone and oxymorphone derivatives compete for the (3H)naloxone binding sites with high affinity, while the dihydrocodeinone and oxycodone derivatives are less potent. The affinity of the new compounds is decreased for the delta sites as compared to the parent ligands. The ligands bearing bulky substituents also bind with low affinity to the kappa sites. The modification decreased the Na(+)-index of compounds indicating their mixed agonist-antagonist character. The dihydromorphinone derivatives are all capable to block irreversibly the high affinity binding site of (3H)naloxone, whereas the dihydrocodeionone derivatives block irreversibly the low affinity site. A possible mechanism for the inhibition is suggested.

Research paper thumbnail of Irreversible blockade of the high and low affinity ( sup 3 H) naloxone binding sites by C-6 derivatives of morphinane-6-ones

Life Sciences - LIFE SCI, 1991

C-6 derivatives-hydrazones, phenylhydrazones, dinitrophenylhydrazones, oximes and semicarbazones ... more C-6 derivatives-hydrazones, phenylhydrazones, dinitrophenylhydrazones, oximes and semicarbazones - of morphinane-6-ones were synthesized and their binding characteristics were studied on rat brain membranes. The dihydromorphinone and oxymorphone derivatives compete for the (³H)naloxone binding sites with high affinity, while the dihydrocodeinone and oxycodone derivatives are less potent. The affinity of the new compounds is decreased for the delta sites as compared to the parent ligands. The ligands bearing bulky substituents also bind with low affinity to the kappa sites. The modification decreased the Na{sup +}-index of compounds indicating their mixed agonist-antagonist character. The dihydromorphinone derivatives are all capable to block irreversibly the high affinity binding site of (³H)naloxone, whereas the dihydrocodeinone derivatives block irreversibly the low affinity site. A possible mechanism for the inhibition is suggested.

Research paper thumbnail of New endomorphin analogs with mu-agonist and delta-antagonist properties

Pure and Applied Chemistry, 2000

ABSTRACT

Research paper thumbnail of Separation of κ-opioid receptor subtype from frog brain

Complete separation of the [3H]ethylketocyclazocine ([3H]EKC) specific binding (K subtype) from t... more Complete separation of the [3H]ethylketocyclazocine ([3H]EKC) specific binding (K subtype) from tritiated Tyr-D-Ala*-Me-Phe4-Gly-o15 enkephalin (DAGO) and Tyr-D-Ala*-L-Leu5-enkephalin (DALA) binding (Jand h-subtypes, respectively) was achieved by Sepharose-6B chromatography and sucrose density gradient centrifugation of digitonin solubilized frog brain membranes. The apparent sedimentation coefficient (sZo,J for the K receptor-detergent complex was 13.1 S and the corresponding Stokes radius 64 A. The isolated fractions exhibited high affinity for EKC and bremazocine, whereas p-and d-specific ligands were unable to compete for the [3H]EKC binding sites, indicating that the K subtype represents a separate molecular entity from the p and 6 receptor sites.

Research paper thumbnail of Irreversible labelling of rat brain opioid receptors by enkephalin chloromethyl ketones

Neuropeptides, 1986

ke,$one derivatives of leucine ertkephalfn (LE), D-Ala -Leu -enkephalin (DALE) and D-Ala -D-Leu -

Research paper thumbnail of Kinetics and Physical Parameters of Rat Brain Opioid Receptors Solubilized by Digitonin and CHAPS

Journal of Neurochemistry, 1986

Rat brain opioid receptors were solubilized with digitonin and a zwitterionic detergent, 3-[(3-ch... more Rat brain opioid receptors were solubilized with digitonin and a zwitterionic detergent, 3-[(3-cholamido-propy1)-dimethylammonio]-I-propanesulfonate (CHAPS). The yield of solubilization was 70-75% with digitonin and 30-35% with CHAPS. Kinetic and equilibrium studies performed from digitonin extracts resulted Abbreviations used: CHAPS, 3-[3-cholamidopropyl)-dimethyl-September 3, 1985. ammoniol-I-propanesulfonate: DADLE. o-Ala2-o-Leu5-enkeph-Address correspondence and reprint requests to Dr. J. Simon a h ; DAGO, D-Ala'-MePhe4-Gly-o15-enkephalin: DALA,

Research paper thumbnail of Solubilization and Characterization of Opioid Binding Sites from Frog (Rana esculenta) Brain

Journal of Neurochemistry, 1984

Active opioid receptors were solubilized from frog (Rana esculenta) brain membrane fractions by t... more Active opioid receptors were solubilized from frog (Rana esculenta) brain membrane fractions by the use of 1% digitonin. It was found by kinetic as well as by equilibrium measurements that both the membrane and the solubilized fractions contain two binding sites. For the membrane preparations, KD values were 0.9 and 3.6 nM, and Bmax values were 293 and 734 fmol/mg protein. For the solubilized preparations, KD values were 0.4 and 2.6 nM, an Bmax values were 35 and 266 fmol/mg protein. The stereospecificity of the binding did not change during solubilization. Both the membrane-bound and the solubilized receptors showed weak binding of enkephalin and mu-specific drugs, suggesting that they are predominantly of the kappa-type. The membrane-bound and the soluble receptors showed the same distribution of subtypes, i.e., 70% kappa, 13% mu, and 17% delta for the membrane-bound and 71% kappa, 17% mu, and 12% delta for the soluble receptors.

Research paper thumbnail of Interactions of Pathological Hallmark Proteins: TUBULIN POLYMERIZATION PROMOTING PROTEIN/p25,  -AMYLOID, AND  -SYNUCLEIN

Journal of Biological Chemistry, 2011

The disordered TPPP/p25 is a hallmark of synucleinopathies. Results: Tight binding of TPPP/p25 wi... more The disordered TPPP/p25 is a hallmark of synucleinopathies. Results: Tight binding of TPPP/p25 with ␤-amyloid results in the formation of massive aggregates both in vitro and in vivo.

Research paper thumbnail of New endomorphin analogues containing alicyclic beta-amino acids: influence on bioactive conformation and pharmacological profile

Journal of Medicinal Chemistry

Endomorphins were subjected to a number of structural modifications in a search for their bioacti... more Endomorphins were subjected to a number of structural modifications in a search for their bioactive conformations. The alicyclic beta-amino acids cis-(1 S,2 R)ACPC/ACHC, cis-(1 R,2 S)ACPC/ACHC, trans-(1 S,2 S)ACPC/ACHC, and trans-(1 R,2 R)ACPC/ACHC were introduced into endomorphins to examine the conformational effects on the bioactivity. Use of a combination of receptor binding techniques, (1)H NMR, and molecular modeling allowed the conclusion that Pro (2) substitution by these residues causes changes in structure, proteolytic stability, and pharmacological activity. It seems that the size of the alicyclic beta-amino acids does not have marked influence on the receptor binding affinities and/or selectivities. Among the new analogues, the cis-(1 S,2 R)ACPC (2) and cis-(1 S,2 R)ACHC (2)-containing derivatives displayed the highest binding potencies and efficacies in receptor binding and ligand-stimulated [ (35)S]GTPgammaS functional experiments. Molecular dynamic simulations and (1)H NMR studies of the cis-ACPC/ACHC-containing analogues revealed that many conformations are accessible, though it is most likely that these peptides bind to the mu-opioid receptor in a compact, folded structure rather than extended.

Research paper thumbnail of Characterization of rat brain opioid receptors by (Tyr-3,5-/sup 3/H)1, D-Ala2, Leu5-enkephalin binding

(Tyr-3,5-/sup 3/H)1, D-Ala2, Leu5-enkephalin ((/sup 3/H)DALA) was used for labeling the opioid re... more (Tyr-3,5-/sup 3/H)1, D-Ala2, Leu5-enkephalin ((/sup 3/H)DALA) was used for labeling the opioid receptors of rat brain plasma membranes. The labeled ligand was prepared from (Tyr-3,5-diiodo)1, D-Ala2, Leu5-enkephalin by catalytic reductive dehalogenation in the presence of Pd catalyst. The resulting (Tyr-3,5-/sup 3/H)1, D-Ala2, Leu5-enkephalin had a specific activity of 37.3 Ci/mmol. In the binding experiments steady-state level was reached at 24 degrees C within 45 min. The pseudo first order association rate constant was 0.1 min-1. The dissociation of the receptor-ligand complex was biphasic with k-1-s of 0.009 and 0.025 min-1. The existence of two binding sites was proved by equilibrium studies. The high affinity site showed a KD = 0.7 nM and Bmax = 60 fmol/mg protein; the low affinity site had a KD = 5 nM and Bmax = 160 fmol/mg protein. A series of opioid peptides inhibited (/sup 3/H)DALA binding more efficiently than morphine-like drugs suggesting that labeled ligand binds prefe...

Research paper thumbnail of A new potential affinity label for the opiate receptor

Life Sciences, 1983

D-Ala2-Leu5-enkephalin (DALA) was elongated with the methyl ester of melphalan (Mel), a nitrogen ... more D-Ala2-Leu5-enkephalin (DALA) was elongated with the methyl ester of melphalan (Mel), a nitrogen mustard on the C-terminus. The new derivative, DALA-Mel-OMe might be a potential affinity label of the opiate receptor. The compound shows high affinity in displacement experiments with an IC50 of 10 nM and 100 nM against 3H-D-Ala2-Leu5-enkephalin and 3H-naloxone, respectively. 10-100 microM of DALA-Mel-OMe causes a significant inhibition of 3H-naloxone binding, which effect can't be reversed by extensive washes. This irreversible blockade is significantly but only partially protected by high concentrations of naloxone and Leu-enkephalin. Our results suggest that DALA-Mel-OMe binds irreversibly to the opiate receptor, but nonspecific labelling also occurs.

Research paper thumbnail of 533 Intracytoplasmic sperm injection (ICSI) after testicular cancer treatment

European Urology Supplements, 2004

Research paper thumbnail of Reciprocal inhibition of G-protein signaling is induced by CB1 cannabinoid and GABAB receptor interactions in rat hippocampal membranes

Neurochemistry International, 2008

Research paper thumbnail of Ligand-specific regulation of the endogenous mu-opioid receptor by chronic treatment with mu-opioid peptide agonists

BioMed research international, 2013

Since the discovery of the endomorphins (EM), the postulated endogenous peptide agonists of the m... more Since the discovery of the endomorphins (EM), the postulated endogenous peptide agonists of the mu-opioid receptors, several analogues have been synthesized to improve their binding and pharmacological profiles. We have shown previously that a new analogue, cis-1S,2R-aminocyclohexanecarboxylic acid(2)-endomorphin-2 (ACHC-EM2), had elevated mu-receptor affinity, selectivity, and proteolytic stability over the parent compound. In the present work, we have studied its antinociceptive effects and receptor regulatory processes. ACHC-EM2 displayed a somewhat higher (60%) acute antinociceptive response than the parent peptide, EM2 (45%), which peaked at 10 min after intracerebroventricular (icv) administration in the rat tail-flick test. Analgesic tolerance developed to the antinociceptive effect of ACHC-EM2 upon its repeated icv injection that was complete by a 10-day treatment. This was accompanied by attenuated coupling of mu-sites to G-proteins in subcellular fractions of rat brain. Al...

Research paper thumbnail of Irreversible labelling of rat brain opioid receptors by enkephalin chloromethyl ketones

Neuropeptides, 1986

Chloromethyl ketone derivatives of leucine enkephalin (LE), D-Ala2-Leu5-enkephalin (DALE) and D-A... more Chloromethyl ketone derivatives of leucine enkephalin (LE), D-Ala2-Leu5-enkephalin (DALE) and D-Ala2-D-Leu5-enkephalin (DADLE) were synthesized. They all show high affinity for rat brain opioid binding sites. Preincubation of the membrane fraction with enkephalin chloromethyl ketones causes a significant inhibition of /3H/-naloxone binding which cannot be reversed by extensive washing. It was found that the irreversible inhibition is selective for the high affinity (KD less than 1 nM) /3H/-naloxone binding site (putative mu-1 site). The irreversible blockade of opioid binding was partially protected by opiate alkaloids and opioid peptides, suggesting that non-specific labelling also occurs. Affinity of enkephalin chloromethyl ketones toward the mu sites is greater than that of the parent compounds. It was also found that the covalent inhibition of mu sites (/3H/-dihydromorphine and /3H/-DAGO binding) is more effective than that of delta sites (/3H/-DALE binding). We conclude that these chloromethyl ketone derivatives can be used as affinity labels for the opioid receptors, allowing us to study the structure of the mu receptor subtype.

Research paper thumbnail of Ligand-specific regulation of the endogenous mu-opioid receptor by chronic treatment with mu-opioid peptide agonists

BioMed research international, 2013

Since the discovery of the endomorphins (EM), the postulated endogenous peptide agonists of the m... more Since the discovery of the endomorphins (EM), the postulated endogenous peptide agonists of the mu-opioid receptors, several analogues have been synthesized to improve their binding and pharmacological profiles. We have shown previously that a new analogue, cis-1S,2R-aminocyclohexanecarboxylic acid(2)-endomorphin-2 (ACHC-EM2), had elevated mu-receptor affinity, selectivity, and proteolytic stability over the parent compound. In the present work, we have studied its antinociceptive effects and receptor regulatory processes. ACHC-EM2 displayed a somewhat higher (60%) acute antinociceptive response than the parent peptide, EM2 (45%), which peaked at 10 min after intracerebroventricular (icv) administration in the rat tail-flick test. Analgesic tolerance developed to the antinociceptive effect of ACHC-EM2 upon its repeated icv injection that was complete by a 10-day treatment. This was accompanied by attenuated coupling of mu-sites to G-proteins in subcellular fractions of rat brain. Al...

Research paper thumbnail of Ovarian sex steroid-dependent plasticity of nociceptin/orphanin FQ and opioid modulation of spinal dynorphin release

The Journal of pharmacology and experimental therapeutics, 2001

Pregnancy and its hormonal simulation via 17beta-estradiol (E(2)) and progesterone (P) are associ... more Pregnancy and its hormonal simulation via 17beta-estradiol (E(2)) and progesterone (P) are associated with spinal opioid antinociception, primarily driven by augmented dynorphin/kappa-opioid activity. This study addresses the ovarian sex steroid-activated mechanism(s) that underlie this activation using an ex vivo spinal cord preparation. In lumbar spinal cord obtained from control animals, exogenous kappa- or delta-opioid agonists (but not mu), as well as nociceptin (orphanin FQ; N/OFQ), dose dependently inhibit the stimulated release of dynorphin. Consistent with these observations, stimulated dynorphin release is enhanced following selective blockade of opioid or N/OFQ receptors, indicating that their endogenous ligands are negative modulators of dynorphin release. In lumbar spinal cord obtained from ovariectomized animals exposed to pregnancy blood levels of E(2)/P, basal and stimulated rates of dynorphin release increase approximately 2-fold. Moreover, evoked dynorphin release ...

Research paper thumbnail of Separation of kappa-opioid receptor subtype from frog brain

FEBS letters, Jan 22, 1985

Complete separation of the [3H]ethylketocyclazocine [( 3H]EKC) specific binding (kappa subtype) f... more Complete separation of the [3H]ethylketocyclazocine [( 3H]EKC) specific binding (kappa subtype) from tritiated Tyr-D-Ala2-Me-Phe4-Gly-ol5 enkephalin (DAGO) and Tyr-D-Ala2-L-Leu5-enkephalin (DALA) binding (mu-and delta-subtypes, respectively) was achieved by Sepharose-6B chromatography and sucrose density gradient centrifugation of digitonin solubilized frog brain membranes. The apparent sedimentation coefficient (s20.w) for the kappa receptor-detergent complex was 13.1 S and the corresponding Stokes radius 64 A. The isolated fractions exhibited high affinity for EKC and bremazocine, whereas mu- and delta-specific ligands were unable to compete for the [3H]EKC binding sites, indicating that the kappa subtype represents a separate molecular to compete for the [3H]EKC binding sites, indicating that the kappa subtype represents a separate molecular entity from the mu and delta receptor sites.

Research paper thumbnail of A Monoclonal Antibody Recognizing K- but Not ?- and ?-Opioid Receptors

Journal of Neurochemistry, 1991

Abstract: A monoclonal antibody (mAb), KA8, that interacts with the k-opioid receptor binding sit... more Abstract: A monoclonal antibody (mAb), KA8, that interacts with the k-opioid receptor binding site was generated. BALB/c female mice were immunized with a partially purified k-opioid receptor preparation from frog brain. Spleen cells were hybridized with SP2/0AG8 ...

Research paper thumbnail of Application of an Opioid Affinity Label for Qualitative Analysis of Mu-Receptors

Vascular Endothelium, 1991

Research paper thumbnail of Irreversible blockade of the high and low affinity (3H) naloxone binding sites by C-6 derivatives of morphinane-6-ones

Life Sciences, 1991

C-6 derivatives--hydrazones, phenylhydrazones, dinitrophenylhydrazones, oximes and semicarbazones... more C-6 derivatives--hydrazones, phenylhydrazones, dinitrophenylhydrazones, oximes and semicarbazones--of morphinane-6-ones were synthesized and their binding characteristics were studied on rat brain membranes. The dihydromorphinone and oxymorphone derivatives compete for the (3H)naloxone binding sites with high affinity, while the dihydrocodeinone and oxycodone derivatives are less potent. The affinity of the new compounds is decreased for the delta sites as compared to the parent ligands. The ligands bearing bulky substituents also bind with low affinity to the kappa sites. The modification decreased the Na(+)-index of compounds indicating their mixed agonist-antagonist character. The dihydromorphinone derivatives are all capable to block irreversibly the high affinity binding site of (3H)naloxone, whereas the dihydrocodeionone derivatives block irreversibly the low affinity site. A possible mechanism for the inhibition is suggested.

Research paper thumbnail of Irreversible blockade of the high and low affinity ( sup 3 H) naloxone binding sites by C-6 derivatives of morphinane-6-ones

Life Sciences - LIFE SCI, 1991

C-6 derivatives-hydrazones, phenylhydrazones, dinitrophenylhydrazones, oximes and semicarbazones ... more C-6 derivatives-hydrazones, phenylhydrazones, dinitrophenylhydrazones, oximes and semicarbazones - of morphinane-6-ones were synthesized and their binding characteristics were studied on rat brain membranes. The dihydromorphinone and oxymorphone derivatives compete for the (³H)naloxone binding sites with high affinity, while the dihydrocodeinone and oxycodone derivatives are less potent. The affinity of the new compounds is decreased for the delta sites as compared to the parent ligands. The ligands bearing bulky substituents also bind with low affinity to the kappa sites. The modification decreased the Na{sup +}-index of compounds indicating their mixed agonist-antagonist character. The dihydromorphinone derivatives are all capable to block irreversibly the high affinity binding site of (³H)naloxone, whereas the dihydrocodeinone derivatives block irreversibly the low affinity site. A possible mechanism for the inhibition is suggested.

Research paper thumbnail of New endomorphin analogs with mu-agonist and delta-antagonist properties

Pure and Applied Chemistry, 2000

ABSTRACT

Research paper thumbnail of Separation of κ-opioid receptor subtype from frog brain

Complete separation of the [3H]ethylketocyclazocine ([3H]EKC) specific binding (K subtype) from t... more Complete separation of the [3H]ethylketocyclazocine ([3H]EKC) specific binding (K subtype) from tritiated Tyr-D-Ala*-Me-Phe4-Gly-o15 enkephalin (DAGO) and Tyr-D-Ala*-L-Leu5-enkephalin (DALA) binding (Jand h-subtypes, respectively) was achieved by Sepharose-6B chromatography and sucrose density gradient centrifugation of digitonin solubilized frog brain membranes. The apparent sedimentation coefficient (sZo,J for the K receptor-detergent complex was 13.1 S and the corresponding Stokes radius 64 A. The isolated fractions exhibited high affinity for EKC and bremazocine, whereas p-and d-specific ligands were unable to compete for the [3H]EKC binding sites, indicating that the K subtype represents a separate molecular entity from the p and 6 receptor sites.

Research paper thumbnail of Irreversible labelling of rat brain opioid receptors by enkephalin chloromethyl ketones

Neuropeptides, 1986

ke,$one derivatives of leucine ertkephalfn (LE), D-Ala -Leu -enkephalin (DALE) and D-Ala -D-Leu -

Research paper thumbnail of Kinetics and Physical Parameters of Rat Brain Opioid Receptors Solubilized by Digitonin and CHAPS

Journal of Neurochemistry, 1986

Rat brain opioid receptors were solubilized with digitonin and a zwitterionic detergent, 3-[(3-ch... more Rat brain opioid receptors were solubilized with digitonin and a zwitterionic detergent, 3-[(3-cholamido-propy1)-dimethylammonio]-I-propanesulfonate (CHAPS). The yield of solubilization was 70-75% with digitonin and 30-35% with CHAPS. Kinetic and equilibrium studies performed from digitonin extracts resulted Abbreviations used: CHAPS, 3-[3-cholamidopropyl)-dimethyl-September 3, 1985. ammoniol-I-propanesulfonate: DADLE. o-Ala2-o-Leu5-enkeph-Address correspondence and reprint requests to Dr. J. Simon a h ; DAGO, D-Ala'-MePhe4-Gly-o15-enkephalin: DALA,

Research paper thumbnail of Solubilization and Characterization of Opioid Binding Sites from Frog (Rana esculenta) Brain

Journal of Neurochemistry, 1984

Active opioid receptors were solubilized from frog (Rana esculenta) brain membrane fractions by t... more Active opioid receptors were solubilized from frog (Rana esculenta) brain membrane fractions by the use of 1% digitonin. It was found by kinetic as well as by equilibrium measurements that both the membrane and the solubilized fractions contain two binding sites. For the membrane preparations, KD values were 0.9 and 3.6 nM, and Bmax values were 293 and 734 fmol/mg protein. For the solubilized preparations, KD values were 0.4 and 2.6 nM, an Bmax values were 35 and 266 fmol/mg protein. The stereospecificity of the binding did not change during solubilization. Both the membrane-bound and the solubilized receptors showed weak binding of enkephalin and mu-specific drugs, suggesting that they are predominantly of the kappa-type. The membrane-bound and the soluble receptors showed the same distribution of subtypes, i.e., 70% kappa, 13% mu, and 17% delta for the membrane-bound and 71% kappa, 17% mu, and 12% delta for the soluble receptors.

Research paper thumbnail of Interactions of Pathological Hallmark Proteins: TUBULIN POLYMERIZATION PROMOTING PROTEIN/p25,  -AMYLOID, AND  -SYNUCLEIN

Journal of Biological Chemistry, 2011

The disordered TPPP/p25 is a hallmark of synucleinopathies. Results: Tight binding of TPPP/p25 wi... more The disordered TPPP/p25 is a hallmark of synucleinopathies. Results: Tight binding of TPPP/p25 with ␤-amyloid results in the formation of massive aggregates both in vitro and in vivo.

Research paper thumbnail of New endomorphin analogues containing alicyclic beta-amino acids: influence on bioactive conformation and pharmacological profile

Journal of Medicinal Chemistry

Endomorphins were subjected to a number of structural modifications in a search for their bioacti... more Endomorphins were subjected to a number of structural modifications in a search for their bioactive conformations. The alicyclic beta-amino acids cis-(1 S,2 R)ACPC/ACHC, cis-(1 R,2 S)ACPC/ACHC, trans-(1 S,2 S)ACPC/ACHC, and trans-(1 R,2 R)ACPC/ACHC were introduced into endomorphins to examine the conformational effects on the bioactivity. Use of a combination of receptor binding techniques, (1)H NMR, and molecular modeling allowed the conclusion that Pro (2) substitution by these residues causes changes in structure, proteolytic stability, and pharmacological activity. It seems that the size of the alicyclic beta-amino acids does not have marked influence on the receptor binding affinities and/or selectivities. Among the new analogues, the cis-(1 S,2 R)ACPC (2) and cis-(1 S,2 R)ACHC (2)-containing derivatives displayed the highest binding potencies and efficacies in receptor binding and ligand-stimulated [ (35)S]GTPgammaS functional experiments. Molecular dynamic simulations and (1)H NMR studies of the cis-ACPC/ACHC-containing analogues revealed that many conformations are accessible, though it is most likely that these peptides bind to the mu-opioid receptor in a compact, folded structure rather than extended.

Research paper thumbnail of Characterization of rat brain opioid receptors by (Tyr-3,5-/sup 3/H)1, D-Ala2, Leu5-enkephalin binding

(Tyr-3,5-/sup 3/H)1, D-Ala2, Leu5-enkephalin ((/sup 3/H)DALA) was used for labeling the opioid re... more (Tyr-3,5-/sup 3/H)1, D-Ala2, Leu5-enkephalin ((/sup 3/H)DALA) was used for labeling the opioid receptors of rat brain plasma membranes. The labeled ligand was prepared from (Tyr-3,5-diiodo)1, D-Ala2, Leu5-enkephalin by catalytic reductive dehalogenation in the presence of Pd catalyst. The resulting (Tyr-3,5-/sup 3/H)1, D-Ala2, Leu5-enkephalin had a specific activity of 37.3 Ci/mmol. In the binding experiments steady-state level was reached at 24 degrees C within 45 min. The pseudo first order association rate constant was 0.1 min-1. The dissociation of the receptor-ligand complex was biphasic with k-1-s of 0.009 and 0.025 min-1. The existence of two binding sites was proved by equilibrium studies. The high affinity site showed a KD = 0.7 nM and Bmax = 60 fmol/mg protein; the low affinity site had a KD = 5 nM and Bmax = 160 fmol/mg protein. A series of opioid peptides inhibited (/sup 3/H)DALA binding more efficiently than morphine-like drugs suggesting that labeled ligand binds prefe...

Research paper thumbnail of A new potential affinity label for the opiate receptor

Life Sciences, 1983

D-Ala2-Leu5-enkephalin (DALA) was elongated with the methyl ester of melphalan (Mel), a nitrogen ... more D-Ala2-Leu5-enkephalin (DALA) was elongated with the methyl ester of melphalan (Mel), a nitrogen mustard on the C-terminus. The new derivative, DALA-Mel-OMe might be a potential affinity label of the opiate receptor. The compound shows high affinity in displacement experiments with an IC50 of 10 nM and 100 nM against 3H-D-Ala2-Leu5-enkephalin and 3H-naloxone, respectively. 10-100 microM of DALA-Mel-OMe causes a significant inhibition of 3H-naloxone binding, which effect can't be reversed by extensive washes. This irreversible blockade is significantly but only partially protected by high concentrations of naloxone and Leu-enkephalin. Our results suggest that DALA-Mel-OMe binds irreversibly to the opiate receptor, but nonspecific labelling also occurs.

Research paper thumbnail of 533 Intracytoplasmic sperm injection (ICSI) after testicular cancer treatment

European Urology Supplements, 2004

Research paper thumbnail of Reciprocal inhibition of G-protein signaling is induced by CB1 cannabinoid and GABAB receptor interactions in rat hippocampal membranes

Neurochemistry International, 2008