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1.
Anal Biochem ; 374(2): 405-10, 2008 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-18191464

RESUMEN

A membrane-based high-throughput screening (HTS) assay for active D-amino acid oxidase (DAAO) in liquid samples as well as in intact Escherichia coli cells has been developed and optimized. The detection limit of the assay was less than 1 ng per sample. The method proposed can be used for quantitative DAAO determination in the range of 0.13 to 3.60 ng enzyme per probe. The protocol was successfully tested to screen a library of E. coli clones containing mutant DAAOs active toward target substrates.


Asunto(s)
Aminoácido Oxidorreductasas/análisis , Aminoácido Oxidorreductasas/metabolismo , Aminoácidos/metabolismo , Aminoácido Oxidorreductasas/química , Aminoácido Oxidorreductasas/genética , Colodión/química , Combinación de Medicamentos , Escherichia coli/citología , Escherichia coli/enzimología , Escherichia coli/genética , Etorfina/química , Membranas Artificiales , Metotrimeprazina/química , Mutagénesis , Mutación , Reproducibilidad de los Resultados
2.
Mini Rev Med Chem ; 3(2): 137-44, 2003 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-12570846

RESUMEN

Buprenorphine is a partial agonist at the micro -opioid receptor with long duration of action and also exhibits delayed antagonist activity. Buprenorphine is finding increasing use as a treatment agent for opioid abuse, though its low efficacy is not well tolerated by all addicts. There is interest in developing a higher efficacy version of buprenorphine and in this mini-review some of the ligands recently discovered, that share with buprenorphine a profile of agonism followed by delayed antagonism, are discussed.


Asunto(s)
Analgésicos Opioides/farmacología , Buprenorfina/farmacología , Etorfina/análogos & derivados , Antagonistas de Narcóticos/farmacología , Receptores Opioides mu/antagonistas & inhibidores , Trastornos Relacionados con Sustancias/tratamiento farmacológico , Analgésicos Opioides/química , Analgésicos Opioides/uso terapéutico , Animales , Buprenorfina/efectos adversos , Buprenorfina/química , Buprenorfina/uso terapéutico , Evaluación Preclínica de Medicamentos , Etorfina/química , Etorfina/farmacología , Humanos , Hidromorfona/análogos & derivados , Hidromorfona/farmacología , Ligandos , Morfinanos/química , Morfinanos/farmacología , Antagonistas de Narcóticos/química , Antagonistas de Narcóticos/uso terapéutico , Receptores Opioides mu/agonistas , Relación Estructura-Actividad
3.
Eur J Pharmacol ; 442(1-2): 23-7, 2002 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-12020678

RESUMEN

The highly potent synthetic narcotic compound etorphine is known to cause strong analgesia, catatonia and blockade of conditioned reflexes in laboratory animals and is widely used for the immobilisation of game animals. In this study, a number of new structural analogues of etorphine, including C18-beta-structures, 3-O-methylether derivatives and saturated C7-C8 dihydro-compounds, were synthesised and examined in in vitro ligand binding experiments. Opiate receptor-mediated activation of G-proteins by these derivatives was also investigated using the [35S]GTPgammaS binding assay. The receptor binding affinity constant and G-protein stimulatory potency of the novel beta-etorphins were compared with those of the corresponding C18-alpha-derivatives. In rat brain membrane preparations, all the compounds tested displayed high affinity (K(i)'s ranging 0.4-22 nM) using [3H]naloxone in competition assays. The alpha-etorphines had somewhat higher affinity in comparison with the beta-structures. Methylether derivatives were consistently weaker than the corresponding phenolic compounds. Dihydroetorphine and beta-dihydroetorphine, which have a partially saturated ring structure, showed as good potency in the binding assays as did etorphine and beta-etorphine with C7-C8 double bonds. The etorphine derivatives were potent but naloxone-reversible activators of G-proteins in the [35S]GTPgammaS functional tests. It was also found that the C3 phenolic group is favourable for G-protein activation. On the basis of our experimental results, neither the configuration of C18 nor the saturation of the C7-C8 double bond appears to play a critical role in the biological activity of etorphines.


Asunto(s)
Etorfina/análogos & derivados , Etorfina/farmacología , Animales , Unión Competitiva/efectos de los fármacos , Encéfalo/metabolismo , Membrana Celular/metabolismo , Etorfina/química , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Naloxona/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Relación Estructura-Actividad , Radioisótopos de Azufre , Tritio
4.
Acta Pharmacol Sin ; 21(6): 536-46, 2000 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-11360688

RESUMEN

AIM: To study the mechanism of interaction of nociceptin and opioids with ORL1 receptor. METHODS: Molecular dynamics study was carried out before nociceptin was manually docked into the binding site of ORL1 receptor; DOCK4.0 program was applied to dock four stereoisomers of lofentanyl and etorphine into the binding pocket of ORL1 receptor; Binding energies were calculated, the relationship between binding energy and binding affinity was studied. RESULTS: Nociceptin fits well into the binding pocket, the N-terminal FGGF tetrapeptide is located in the inner region of the binding cavity, the nociceptin (5-7) interacts with the conservatively variable residues near the other end of binding pocket, and maybe determines selectivity of ORL1 receptor over dynorphin A, the positively charged core of nociceptin (8-13) binds predominantly with negatively charged EL-2 loop, which is thought to be able to mediate receptor activation. The shortest fully active analogue of nociceptin (1-13) is also discussed. The main difference between these two opioids and nociceptin exists in the kinds and the number of conserved and variable residues in the binding pocket and thereafter in the strength of their interaction. Prediction for binding affinities of four stereoisomers of lofentanyl has been performed based on their binding energies, the similar pharmacophore of lofentanyl and other fentanyl analogs, and the good correlation between binding energies and their experimental binding affinities (-log Ki values). CONCLUSION: Ligand docking results from this study are helpful in clarifying experimental observations of ligands interaction with opioid receptors, thus furthering biological investigations.


Asunto(s)
Fentanilo/análogos & derivados , Péptidos Opioides/química , Receptores Opioides/química , Sitios de Unión , Interacciones Farmacológicas , Etorfina/química , Fentanilo/química , Modelos Moleculares , Conformación Molecular , Péptidos Opioides/metabolismo , Receptores Opioides/metabolismo , Receptor de Nociceptina , Nociceptina
5.
Protein Eng ; 12(11): 927-42, 1999 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-10585498

RESUMEN

Refined 3D models of the transmembrane domains of the cloned delta, mu and kappa opioid receptors belonging to the superfamily of G-protein coupled receptors (GPCRs) were constructed from a multiple sequence alignment using the alpha carbon template of rhodopsin recently reported. Other key steps in the procedure were relaxation of the 3D helix bundle by unconstrained energy optimization and assessment of the stability of the structure by performing unconstrained molecular dynamics simulations of the energy optimized structure. The results were stable ligand-free models of the TM domains of the three opioid receptors. The ligand-free delta receptor was then used to develop a systematic and reliable procedure to identify and assess putative binding sites that would be suitable for similar investigation of the other two receptors and GPCRs in general. To this end, a non-selective, 'universal' antagonist, naltrexone, and agonist, etorphine, were used as probes. These ligands were first docked in all sites of the model delta opioid receptor which were sterically accessible and to which the protonated amine of the ligands could be anchored to a complementary proton-accepting residue. Using these criteria, nine ligand-receptor complexes with different binding pockets were identified and refined by energy minimization. The properties of all these possible ligand-substrate complexes were then examined for consistency with known experimental results of mutations in both opioid and other GPCRs. Using this procedure, the lowest energy agonist-receptor and antagonist-receptor complexes consistent with these experimental results were identified. These complexes were then used to probe the mechanism of receptor activation by identifying differences in receptor conformation between the agonist and the antagonist complex during unconstrained dynamics simulation. The results lent support to a possible activation mechanism of the mouse delta opioid receptor similar to that recently proposed for several other GPCRs. They also allowed the selection of candidate sites for future mutagenesis experiments.


Asunto(s)
Receptores Opioides delta/química , Receptores Opioides kappa/química , Receptores Opioides mu/química , Secuencia de Aminoácidos , Animales , Clonación Molecular , Etorfina/química , Proteínas de la Membrana/química , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Naltrexona/química , Unión Proteica , Conformación Proteica , Ingeniería de Proteínas , Estructura Secundaria de Proteína , Alineación de Secuencia
6.
Forensic Sci Int ; 101(1): 9-16, 1999 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-10376332

RESUMEN

Etorphine is a synthetic narcotic analgesic usually used in veterinary medicine. It possesses an analgesic potency up to 1000 times greater than morphine and is therefore used in low doses, primarily for tranquilising large animals. For veterinary use, etorphine is usually available in its commercial formulation as Immobilon, when in combination with acepromazine or methotrimeprazine. Due to the potency of etorphine, only very low doses are required to produce adverse or fatal effects. This paper describes a method for detecting and quantifying etorphine using HPLC with UV diode array detection (HPLC-DAD) and demonstrates the advantage of the technique for the detection of Immobilon at low doses. In a forensic case involving Immobilon, the etorphine concentrations measured in postmortem femoral vein and heart blood specimens were 14.5 and 23.5 micrograms/l, respectively. No etorphine was detected in the urine. To our knowledge this is the first time postmortem etorphine concentrations have been reported.


Asunto(s)
Analgésicos Opioides/sangre , Analgésicos Opioides/envenenamiento , Autopsia , Cromatografía Líquida de Alta Presión/métodos , Etorfina/sangre , Etorfina/envenenamiento , Metotrimeprazina/sangre , Metotrimeprazina/envenenamiento , Espectrofotometría Ultravioleta/métodos , Detección de Abuso de Sustancias/métodos , Analgésicos Opioides/química , Analgésicos Opioides/metabolismo , Combinación de Medicamentos , Sobredosis de Droga , Etorfina/química , Etorfina/metabolismo , Humanos , Masculino , Metotrimeprazina/química , Metotrimeprazina/metabolismo , Persona de Mediana Edad , Suicidio
8.
J Comput Aided Mol Des ; 5(6): 553-69, 1991 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-1667926

RESUMEN

The molecular basis underlying the divergent receptor selectivity of two cyclic opioid peptides Tyr-c[N delta-D-Orn2-Gly-Phe-Leu-] (c-ORN) and [D-Pen2,L-Cys5]-enkephalinamide (c-PEN) was investigated using a molecular modeling approach. Ring closure and conformational searching procedures were used to determine low-energy cyclic backbone conformers. Following reinsertion of amino acid side chains, the narcotic alkaloid 7 alpha-[(1R)-1-methyl-1-hydroxy-3-phenylpropyl]-6-14-endoethenotetrahy dro oripavine (PEO) was used as a flexible template for bimolecular superpositions with each of the determined peptide ring conformers using the coplanarity and cocentricity of the phenolic rings as the minimum constraint. A vector space of PEO, accounting for all possible orientations for the C21-aromatic ring of PEO served as a geometrical locus for the aromatic ring of the Phe4 residue in the opioid peptides. Although a vast number of polypeptide conformations satisfied the criteria of the opiate pharmacophore, they could be grouped into three classes differing in magnitude and sign of the torsional angle values of the tyrosyl side chain. Only class III conformers for both c-ORN and c-PEN, having tyramine dihedral angles chi 1 = 150 degrees +/- 30 degrees and chi 2 = -155 degrees +/- 20 degrees, had significant structural and conformational properties that were mutually compatible while respecting the PEO vector space. Comparison of these properties in the context of the divergent receptor selectivity of the studied opioid peptides suggests that the increased distortion of the peptide backbone in the closure region of c-PEN together with the pendant beta,beta-dimethyl group, combine to generate a steric volume which is absent in c-ORN and that may be incompatible with a restrictive topography of the mu receptor. The nature and stereochemistry of substituents adjacent to the closure region of the peptides could also modulate receptor selection by interacting with a charged (delta) or neutral (mu) subsite.


Asunto(s)
Encefalina Leucina/análogos & derivados , Encefalinas/química , Etorfina/análogos & derivados , Péptidos Cíclicos/química , Receptores Opioides/metabolismo , Gráficos por Computador , Diseño de Fármacos , Encefalina Leucina/química , Encefalina Leucina/metabolismo , Encefalinas/metabolismo , Etorfina/química , Etorfina/metabolismo , Modelos Moleculares , Estructura Molecular , Péptidos Cíclicos/metabolismo , Conformación Proteica , Receptores Opioides delta , Receptores Opioides mu , Relación Estructura-Actividad , Especificidad por Sustrato
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