RÉSUMÉ
The dermorphin-derived tetrapeptide H-Dmt-d-Arg-Phe-Lys-NH(2) (Dmt = 2',6'-dimethyltyrosine) ([Dmt(1)]DALDA) is a highly potent and selective mu-opioid agonist capable of crossing the blood-brain barrier and producing a potent, centrally mediated analgesic effect when given systemically. For the purpose of biodistribution studies by fluorescence techniques, [Dmt(1)]DALDA analogues containing various fluorescent labels [dansyl, anthraniloyl (atn), fluorescein, or 6-dimethylamino-2'-naphthoyl] in several different locations of the peptide were synthesized and characterized in vitro in the guinea-pig ileum and mouse vas deferens assays, and in mu-, delta- and kappa-opioid receptor-binding assays. The analogues showed various degrees of mu receptor-binding selectivity, but all of them were less mu-selective than the [Dmt(1)]DALDA parent peptide. Most analogues retained potent, full mu-agonist activity, except for one with fluorescein attached at the C-terminus (3a) (partial mu-agonist) and one containing beta-(6'-dimethylamino-2'-naphthoyl)alanine (aladan) in place of Phe(3) (4) (mu- and kappa-antagonist). The obtained data indicate that the receptor-binding affinity, receptor selectivity and intrinsic efficacy of the prepared analogues vary very significantly, depending on the type of fluorescent label used and on its location in the peptide. The results suggest that the biological activity profile of fluorescence-labeled peptide analogues should always be carefully determined prior to their use in biodistribution studies or other studies. One of the analogues containing the atn group (2a) proved highly useful in a study of cellular uptake and intracellular distribution by confocal laser scanning microscopy.
Sujet(s)
Colorants fluorescents , Oligopeptides/composition chimique , Récepteur mu/composition chimique , Récepteur mu/métabolisme , Tyrosine/analogues et dérivés , 2-Naphtylamine/analogues et dérivés , Alanine/analogues et dérivés , Animaux , Composés dansylés , Fluorescéines , Souris , Oligopeptides/métabolisme , Tyrosine/composition chimique , Tyrosine/métabolisme , ortho-AminobenzoatesRÉSUMÉ
Recent studies showed that dermorphin and enkephalin analogues containing two methyl groups at the 2',6'-positions of the Tyr(1) aromatic ring and lacking an N-terminal amino group were moderately potent delta and mu opioid antagonists. These results indicate that a positively charged N-terminal amino group may be essential for signal transduction but not for receptor binding and suggested that its deletion in agonist opioid peptides containing an N-terminal 2',6'-dimethyltyrosine (Dmt) residue may represent a general way to convert them into antagonists. In an attempt to develop dynorphin A (Dyn A)-derived kappa opioid antagonists, we prepared analogues of [Dmt(1)]Dyn A(1-11)-NH2 (1), in which the N-terminal amino group was either omitted or replaced with a methyl group. This was achieved by replacement of Tyr(1) with 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp]. Compounds were tested in the guinea pig ileum and mouse vas deferens bioassays and in rat and guinea pig brain membrane receptor binding assays. All analogues turned out to be potent kappa antagonists against Dyn A(1-13) and the non-peptide agonist U50,488 and showed only weak mu and delta antagonist activity. The most potent and most selective kappa antagonist of the series was [(2S)-Mdp(1)]Dyn A(1-11)-NH2 (5, dynantin), which showed subnanomolar kappa antagonist potency against Dyn A(1-13) and very high kappa selectivity both in terms of its K(e) values determined against kappa, mu, and delta agonists and in terms of its ratios of kappa, mu, and delta receptor binding affinity constants. Dynantin is the first potent and selective Dyn A-derived kappa antagonist known and may complement the non-peptide kappa antagonists norbinaltorphimine and GNTI as a pharmacological tool in opioid research.
Sujet(s)
Dynorphines/synthèse chimique , Fragments peptidiques/synthèse chimique , Récepteur kappa/antagonistes et inhibiteurs , Animaux , Fixation compétitive , Encéphale/métabolisme , Dynorphines/composition chimique , Dynorphines/pharmacologie , Cochons d'Inde , Iléum/effets des médicaments et des substances chimiques , Iléum/physiologie , Techniques in vitro , Mâle , Souris , Contraction musculaire/effets des médicaments et des substances chimiques , Muscles lisses/effets des médicaments et des substances chimiques , Muscles lisses/physiologie , Fragments peptidiques/composition chimique , Fragments peptidiques/pharmacologie , Dosage par compétition , Rats , Récepteur kappa/métabolisme , Relation structure-activité , Conduit déférent/effets des médicaments et des substances chimiques , Conduit déférent/physiologieRÉSUMÉ
The tetrapeptide DALDA (H-Tyr-D-Arg-Phe-Lys-NH2) is a polar and selective mu agonist showing poor penetration of the placental and blood-brain barriers. In an effort to enhance the potency of DALDA, analogues containing 2',6'-dimethyltyrosine (Dmt), N,2',6'-trimethyltyrosine (Tmt), 2'-methyltyrosine (Mmt) or 2'-hydroxy,6'-methyltyrosine (Hmt) in place of Tyr1, or Orn or alpha,gamma-diaminobutyric acid (A2bu) in place of Lys4, were synthesized. All compounds displayed high mu receptor selectivity in the rat and guinea pig brain membrane binding assays and most of them were more potent mu agonists than DALDA in the mu receptor-representative guinea pig ileum assay, with [Dmt1]DALDA showing the highest potency. Because of its extraordinary mu agonist potency, high mu selectivity, polar character (charge of 3 + ) and metabolic stability, [Dmt1]DALDA has potential for use in obstetrical or peripheral analgesia.
Sujet(s)
Analgésiques/synthèse chimique , Analgésiques/pharmacologie , Oligopeptides/synthèse chimique , Oligopeptides/pharmacologie , Analgésiques/composition chimique , Analgésiques/métabolisme , Animaux , Encéphale/effets des médicaments et des substances chimiques , Encéphale/métabolisme , Cochons d'Inde , Iléum/effets des médicaments et des substances chimiques , Iléum/métabolisme , Techniques in vitro , Oligopeptides/composition chimique , Oligopeptides/métabolisme , Rats , Récepteur mu/agonistes , Récepteur mu/métabolismeRÉSUMÉ
Recently we reported using minilibraries to replace Lys(9) [somatostatin (SRIF) numbering] of the potent somatostatin agonist L-363,301 (c[-Pro-Phe-D-Trp-Lys-Thr-Phe-]) to generate the potent neurokinin receptor (NK-1) antagonist c[-Pro-Phe-D-Trp-p-F-Phe-Thr-Phe-]. This novel cyclic hexapeptide did not bind the SRIF receptor. Thus, a single mutation converted L-363,301, a SRIF agonist with potency ca. 2-8 times the potency of SRIF in laboratory animals,(24) into a selective NK-1 receptor antagonist with an IC(50) of 2 nM in vitro. During the screening of the same libraries for ligands of the delta-opioid receptor, we identified four compounds (1-4) which represent a new class of delta-opioid antagonists, some of which were also NK-1 receptor antagonists. The most potent delta-opioid antagonist, c[-Pro-1-Nal-D-Trp-Tyr-Thr-Phe-] (2), showed a K(e) value of 128 nM in the mouse vas deferens assay and a delta-receptor binding affinity constant of 152 nM in the rat brain membrane binding assay. These results are of interest because they represent a novel class of delta-opioid antagonists and, like two previously reported delta-opioid antagonists, they lack a positive charge. To examine further the requirement for a positive charge in the delta-opioid ligands, we prepared two analogues of the beta-casomorphin-derived mixed mu-agonist/delta-antagonist, H-Dmt-c[-D-Orn-2-Nal-D-Pro-Gly-] (7), in which we eliminated the positive charge either through formylation of the primary amino group (5) or by the deletion of this N-terminal amino group (6). These latter compounds proved to be delta-opioid antagonists with K(e) values in the 16-120 nM range, as well as fairly potent mu-opioid antagonists (K(e) approximately 200 nM). These six compounds provide the most convincing evidence to date that there is no requirement for a positive charge in mu- and delta-opioid receptor antagonists. In addition, cyclic hexapeptide 4 lacks a phenolic hydroxyl group. Taken together, these data suggest that the prevailing assumptions about delta- and mu-opioid receptor binding need revision and that the receptors for these opioid ligands have much in common with the NK-1 and somatostatin receptors.
Sujet(s)
Antagonistes narcotiques/synthèse chimique , Oligopeptides/synthèse chimique , Peptides cycliques/synthèse chimique , Récepteur mu/antagonistes et inhibiteurs , Animaux , Fixation compétitive , Encéphale/métabolisme , Cochons d'Inde , Iléum/effets des médicaments et des substances chimiques , Techniques in vitro , Ligands , Mâle , Souris , Contraction musculaire/effets des médicaments et des substances chimiques , Muscles lisses/effets des médicaments et des substances chimiques , Antagonistes narcotiques/composition chimique , Antagonistes narcotiques/pharmacologie , Antagonistes du récepteur de la neurokinine-1 , Oligopeptides/composition chimique , Oligopeptides/métabolisme , Oligopeptides/pharmacologie , Peptides cycliques/composition chimique , Peptides cycliques/métabolisme , Peptides cycliques/pharmacologie , Dosage par compétition , Rats , Récepteur de la neurokinine 1/métabolisme , Récepteur mu/métabolisme , Relation structure-activité , Conduit déférent/effets des médicaments et des substances chimiquesRÉSUMÉ
The development of novel delta opioid antagonists and delta opioid agonists structurally derived from the prototype delta antagonist TIPP (H-Tyr-Tic-Phe-Phe-OH), is reviewed. Both delta antagonists and delta agonists with extraordinary potency and unprecedented delta receptor selectivity were discovered. Some of them are already widely used as pharmacological tools and are also of interest as potential therapeutic agents for use in analgesia. The results of the performed structure-activity studies revealed that the delta antagonist versus delta agonist behavior of this class of compounds depended on very subtle structural differences in diverse locations of the molecule. These observations can be best explained with a receptor model involving a number of different inactive and active receptor conformations.
Sujet(s)
Antagonistes narcotiques/composition chimique , Antagonistes narcotiques/pharmacologie , Oligopeptides/composition chimique , Oligopeptides/pharmacologie , Peptides opioïdes/composition chimique , Peptides opioïdes/pharmacologie , Récepteur delta/agonistes , Récepteur delta/antagonistes et inhibiteurs , Tétrahydroisoquinoléines , Animaux , Dipeptides/composition chimique , Dipeptides/pharmacologie , Humains , Techniques in vitro , Relation structure-activitéRÉSUMÉ
The discovery of the prototype delta opioid antagonists TIPP (H-Tyr-Tic-Phe-Phe-OH) and TIP (H-Tyr-Tic-Phe-OH) in 1992 was followed by extensive structure-activity relationship studies, leading to the development of analogues that are of interest as pharmacological tools or as potential therapeutic agents. Stable TIPP-derived delta opioid antagonists with subnanomolar delta receptor binding affinity and extraordinary delta receptor selectivity include TIPP[Psi] (H-Tyr-TicPsi[CH(2)NH]Phe-Phe-OH] and TICP[Psi] (H-Tyr-TicPsi[CH(2)NH]Cha-Phe-OH); Cha: cyclohexylalanine), which are widely used in opioid research. Theoretical conformational analyses in conjunction with the pharmacological characterization of conformationally constrained TIPP analogues led to a definitive model of the receptor-bound conformation of H-Tyr-Tic-(Phe-Phe)-OH-related delta opioid antagonists, which is characterized by all-trans peptide bonds. Further structure-activity studies revealed that the delta antagonist vs delta agonist behavior of TIP(P)-derived compounds depended on very subtle structural differences in diverse locations of the molecule and suggested a delta receptor model involving a number of different inactive receptor conformations. A further outcome of these studies was the identification of a new class of potent and very selective dipeptide delta agonists of the general formula H-Tyr-Tic-NH-X (X = arylalkyl), which are of interest for drug development because of their low molecular weight and lipophilic character. Most interestingly, TIPP analogues containing a C-terminal carboxamide group displayed a mixed mu agonist/delta antagonist profile, and thus were expected to be analgesics with a low propensity to produce tolerance and physical dependence. This turned out to be the case with the TIPP-derived mu agonist/delta antagonist DIPP-NH(2)[Psi] (H-Dmt-TicPsi[CH(2)NH]Phe-Phe-NH(2)); Dmt: 2',6'- dimethyltyrosine).
Sujet(s)
Antagonistes narcotiques/synthèse chimique , Oligopeptides/pharmacologie , Peptides opioïdes/synthèse chimique , Tétrahydroisoquinoléines , Modèles moléculaires , Antagonistes narcotiques/pharmacologie , Peptides opioïdes/pharmacologie , Conformation des protéines , Récepteurs aux opioïdes/agonistesRÉSUMÉ
Cholecystokinin is a gastrointestinal and neuropeptide which has been implicated in a wide range of physiological and behavioral processes. We have developed a sensitive and specific assay system to measure the various forms of cholecystokinin (CCK) in human plasma. This 3-step system involves i) extraction of CCK fragments from plasma using reverse phase chromatography; ii) separation of peptides by high performance liquid chromatography; and iii) detection and quantification of peptides with a double-antibody radioimmunoassay, using an antibody raised against cholecystokinin tetrapeptide (CCK-4) coupled to thyroglobulin and 125I Bolton-Hunter CCK-4 as tracer. The antibody detects CCK-4, sulfated CCK-8 (CCK-8S) and nonsulfated CCK-8 (CCK-8ns) with equal affinity. The lower limit of detection is 2.7 fmol, with an ED50 of 10.6 +/- 2.2 fmol. Mean CCK-like immunoreactivity (CCK-LI) in the plasma of 12 healthy subjects was determined to be 12.9 +/- 2.1 pM CCK-4 equivalents. Concentrations of each individual peptide in plasma were determined to be 1.0 +/- 0.2 pM, 3.4 +/- 0.8 pM and 1.9 +/- 0.4 pM for CCK-4, CCK-8s and CCK-8ns respectively.