Biomedical subjects

F Porreca

Publications and source records attributed to F Porreca.

At least 91 records · Page 5Linked to original sources

PubMed1997

Extraordinary potency of a novel delta opioid receptor agonist is due in part to increased efficacy.

A new cyclic opioid peptide of sequence Tyr-D-Pen-Gly-Phe-Cys-Phe (HBP2) was examined in the mouse isolated vas deferens (MVD) bioassay. Studies with receptor-selective opioid antagonists showed the peptide to be highly selective for delta opioid receptors. HBP2 and the standard delta agonist DPDPE were simultaneously compared using the technique of partial irreversible receptor blockade; data were analyzed using the operational model of pharmacologic agonism. HBP2 was approximately 160 times as potent as DPDPE; estimation of the affinity and efficacy of the two peptides revealed that the potency increase was due to a 5.3-fold increase in efficacy, as well as a 37-fold increase affinity. This contrasts with our previous findings with other cyclic enkephalin analogs, in which increased affinity was achieved without a change in apparent efficacy. Analysis of concentration-response curve shape suggested in addition the possibility of heterogeneity in transduction mechanisms for MVD delta receptors.

Animals↗

PubMed1997

Opioid peptides: simultaneous delta agonism and mu antagonism in somatostatin analogues.

Four isomers of the Somatostatin analogue H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) were made with beta-MePhe in position 1 and assayed for opioid binding in rat brain, biological activity in MVD and GPI bioassays, and antinociception in mouse warm-water tail flick assays. The analogues displayed varying potencies and biological activities including: simultaneous delta receptor agonism/mu receptor antagonism, mu receptor antagonism, and delta receptor agonism. These analogues demonstrated that the N-terminal residue is important for receptor potency/selectivity and signal transduction. These analogues my represent leads to therapeutic agents that yield analgesia via delta agonist effects, yet lack side effects associated with mu activity.

Analgesics↗

PubMed1997

Spinally administered dynorphin A produces long-lasting allodynia: involvement of NMDA but not opioid receptors.

The endogenous opioid peptide dynorphin A has non-opioid effects that can damage the spinal cord when given in high doses. Dynorphin has been shown to increase the receptive field size of spinal cord neurons and facilitate C-fiber-evoked reflexes. Furthermore, endogenous dynorphin levels increase following damage to the spinal cord, injury to peripheral nerves, or inflammation. In this study, sensory processing was characterized following a single, intrathecal injection of dynorphin A (1-17) in mice. A single intrathecal injection of dynorphin A (1-17) (3 nmol, i.t.) induced mechanical allodynia (hind paw, von Frey filaments) lasting 70 days, tactile allodynia (paint brush applied to flank) lasting 14 days, and cold allodynia (acetone applied to the dorsal hind paw) lasting 7 days. Similarly, dynorphin A (2-17) (3 nmol, i.t.), a non-opioid peptide, induced cold and tactile allodynia analogous to that induced by dynorphin A (1-17), indicating the importance of non-opioid receptors. Pretreatment with the NMDA antagonists, MK-801 and LY235959, but not the opioid antagonist, naloxone, blocked the induction of allodynia. Post-treatment with MK-801 only transiently blocked the dynorphin-induced allodynia, suggesting the NMDA receptors may be involved in the maintenance of allodynia as well as its induction. We have induced a long-lasting state of allodynia and hyperalgesia by a single intrathecal injection of dynorphin A (1-17) in mice. The allodynia induced by dynorphin required NMDA receptors rather than opioid receptors. This result is consistent with results in rats and with signs of clinically observed neuropathic pain. This effect of exogenously administered dynorphin raises the possibility that increased levels of endogenous dynorphins associated with spinal cord injuries may participate in the genesis and maintenance of neuropathic pain.

Animals↗

PubMed1997

Differential activities of intrathecal MK-801 or morphine to alter responses to thermal and mechanical stimuli in normal or nerve-injured rats.

Nerve ligation injury in rats results in reduced nociceptive and non-nociceptive thresholds, similar to some aspects of clinical conditions of neuropathic pain. Since underlying mechanisms of hyperalgesia and allodynia may differ, the present study investigated the pharmacology of morphine and MK-801 in rats subjected to a tight ligation of the L5 and L6 nerve roots or to a sham-operation procedure. Response to acute nociception was measured by (a) withdrawal of a hindpaw from a radiant heat source, (b) withdrawal of the tail from a radiant heat source or (c) the latency to a rapid flick of the tail following immersion in water at different noxious temperatures. Mechanical thresholds were determined by measuring response threshold to probing the hindpaw with von Frey filaments. Nerve ligation produced a significant, stable and long-lasting decrease in threshold to mechanical stimulation (i.e., tactile allodynia) when compared to sham-operated controls. Standardization of the diameter of the filaments (to that of the largest filament) did not alter the response threshold in nerve-injured animals. Nerve ligation produced decreased response latency of the ipsilateral paw (i.e., hyperalgesia) when compared to that of sham-operated rats. Tail-flick latencies to thermal stimuli induced by water at constant temperatures (48 degrees, 52 degrees or 55 degrees C) or by radiant heat were not significantly different between nerve-injured and sham-operated groups. At doses which were not behaviorally toxic, MK-801 had no effect on tactile allodynia. At these doses, MK-801 blocked decreased paw withdrawal latency to radiant heat in nerve-injured rats, but did not significantly elevate the response threshold of sham-operated rats. Systemic (i.p.) or intracerebroventricular (i.c.v.) doses of morphine previously shown to be antiallodynic in nerve-ligated rats did not affect the response to probing with von Frey filaments in sham-operated controls. Intrathecal (i.t.) morphine did not change paw withdrawal thresholds elicited by von Frey filaments of either nerve-ligated rats (as previously reported) or of sham-operated rats at doses maximally effective against thermal stimuli applied to the tail or foot. Spinal morphine produced dose-dependent antinociception in both nerve-injured and sham-operated groups in the foot-flick test but was less potent in the nerve-injured group. Presuppression of hyperalgesia of the foot with i.t. MK-801 in nerve-injured animals did not alter the potency of i.t. morphine. I.t. morphine was also active in the tail-flick tests with decreased potency in nerve-injured animals and, at some stimulus intensities, with a decreased efficacy as well. These data emphasize the distinction between the inactivity of morphine to suppress mechanical withdrawal thresholds (as elicited by von Frey filaments) and the activity of this compound to block the response to an acute thermal nociceptive stimulus in sham-operated or nerve-injured rats. It appears that nerve ligation injury produces a thermal allodynia/hyperalgesia which is likely dependent upon opioid-sensitive small-diameter primary afferent fibers and a mechanical allodynia which may be largely independent of small-fiber input.

Analgesics, Opioid↗

PubMed1997

Salivary epidermal growth factor plays a role in protection of ileal mucosal integrity.

The role of salivary epidermal growth factor (EGF) in the maintenance of ileal mucosal integrity was studied by evaluating the effects of sialoadenectomy on luminal EGF levels, ileal tissue resistance (Rt), and unidirectional flux of [51Cr]EDTA. Mice in groups 1 (SLX) and 2 (SLX + EGF) were subjected to sialoadenectomy, while mice in groups 3 (Sham) and 4 (Sham + EGF) underwent a sham procedure. All animals received normal diet and water, except that EGF (100 ng/ml) was added to water for SLX + EGF and Sham + EGF mice. At seven days after surgery, luminal EGF levels in gastrointestinal segments and ileal Rt were significantly reduced by sialoadenectomy, which was prevented by EGF supplementation. Unidirectional flux of [51Cr]EDTA was 6- to 22-fold greater in the ileum of sialoadenectomized mice, which was prevented by EGF administration. Results suggest that salivary EGF may be the major source of intestinal EGF, and it may play a role in maintenance of ileal mucosal integrity.

Animals↗

PubMed1997

Synergistic antinociceptive interactions of morphine and clonidine in rats with nerve-ligation injury.

BACKGROUND: Ligation injury of the L5/L6 nerve roots in rats produces behavioral signs representative of clinical conditions of neuropathic pain, including tactile allodynia and thermal and mechanical hyperalgesia. In this model, intrathecal morphine shows no antiallodynic activity, as well as decreased antinociceptive potency and efficacy. This study was designed to explore the antinociceptive activity of intrathecal clonidine alone or in combination with intrathecal morphine (1:3 fixed ratio) in nerve-injured rats. The aims, with this study, were to use nerve-injured animals to determine: (1) whether the antinociceptive potency and efficacy of intrathecal clonidine was altered, and (2) whether the combination of intrathecal morphine and clonidine would act synergistically to produce antinociception. METHODS: Unilateral nerve injury was produced by ligation of the L5 and L6 spinal roots of male Sprague-Dawley rats. Sham-operated rats underwent a similar surgical procedure but without nerve ligation. Morphine and clonidine were given intrathecally through implanted catheters alone or in a 1:3 fixed ratio. Nociceptive responses were measured by recording tail withdrawal latency from a 55 degrees C water bath, and data were calculated as % maximal possible effect (%MPE). RESULTS: Morphine produced a dose-dependent antinociceptive effect in both sham-operated and nerve-injured rats. The doses calculated to produce a 50 %MPE (i.e., A50) (+/-95% confidence intervals [CI]) were 15 +/- 4.9 micrograms and 30 +/- 18 micrograms, respectively. Though morphine was able to produce a maximal response (100%) in sham-operated rats, the maximal response achieved in nerve-injured animals was only 69 +/- 21.9 %MPE. Clonidine produced a dose-dependent effect, with an A50 (+/-95% CI) of 120 +/- 24 micrograms in sham-operated rats. In nerve-ligated rats, clonidine produced a maximal effect that reached a plateau of 55 +/- 10.9 %MPE and 49 +/- 10.2 %MPE at 100 and 200 micrograms, respectively, preventing the calculation of an A50. In sham-operated rats, a morphine-clonidine mixture produced maximal efficacy, with an A50 (+/-95% CI) of 15 +/- 9.2 micrograms (total dose), significantly less than the theoretical additive A50 of 44 +/- 10 micrograms. In L5/L6 nerve-ligated rats, the morphine-clonidine combination produced maximal efficacy, with an A50 (+/-95% CI) of 11 +/- 5.4 micrograms (total dose), which was significantly less than the theoretical additive A50 of 118 +/- 73 micrograms, indicating a synergistic antinociceptive interaction. The intrathecal injection of [D-Ala2, NMePhe4, Gly-ol]enkephalin (DAMGO) produced A50 values of 0.23 microgram (range, 0.09-0.6) and 0.97 microgram (range, 0.34-2.7) in sham-operated and ligated rats, respectively. Phentolamine (4 mg/kg, intraperitoneally) produced no antinociceptive effect alone and attenuated, rather than enhanced, the effect of morphine in both groups of rats. CONCLUSIONS: These data show that: (1) clonidine, like morphine, loses antinociceptive potency and efficacy after nerve ligation injury, and (2) strongly suggest that a spinal combination of morphine and clonidine synergize under conditions of nerve injury to elicit a significant antinociceptive action when either drug alone may be lacking in efficacy. It is unlikely that the synergy of morphine with clonidine is due to an attenuation of spinal sympathetic outflow by clonidine, because the sympatholytic agent phentolamine produced an opposing effect on morphine antinociception. The data suggest that combinations of morphine and clonidine may prove useful in controlling pain in patients with neuropathic conditions.

Adrenergic alpha-Agonists↗

PubMed1997

Synthesis and biological properties of beta-MePhe3 analogues of deltorphin I and dermenkephalin: influence of biased chi 1 Phe3 residues on peptide recognition for delta-opioid receptors.

Using the method of conformational constraint, we have designed and synthesized analogues of deltorphin I and dermenkephalin containing each of the four stereoisomers (2S,3S; 2S,3R; 2R,3S; 2R,3R) of the unusual amino acid beta-methylphenylalanine in position three. The potency and selectivity of these analogues were evaluated by radioreceptor binding assays in the rat brain using [3H]CTOP (mu-ligand) and[3H]p-C1Phe4]DPDPE (delta-ligand), and by bioassay using the mouse vas deferens (delta-receptor assay) and guinea pig ileum (mu-receptor assay) assays. The substitution of a beta-MePhe for Phe3 in deltorphin I and dermenkephalin has a large and variable effect on the bioactivities of the synthesized analogues. The synthesized analogues are somewhat less potent than the native peptides. Both [(2S,3R)-beta-MePhe3]deltorphin and [(2S,3R)-beta-MePhe3] dermenkephalin are more selective, however, and interact essentially specifically with the receptor in the binding assays and bioassays. The bioassay data in vitro of the synthesized analogues of deltorphin I and dermenkephalin follow the same general trends as the receptor binding data. These results demonstrate that topographical modifications of the side-chain conformation of critical structural moieties in a ligand can significantly modulate both the potency and receptor selectivity for ligands that have multiple sites of biological activity, and they illustrate that this approach has general application to peptide and peptidomimetic ligand design.

Aminobutyrates↗

PubMed1997

Para-substituted phenylalanine-4 analogues of [L-Ala3]DPDPE: highly selective delta opioid receptor ligands.

Several para-substituted Phe4 analogues of the delta 1-selective antagonist [L-Ala3]DPDPE (DPADPE) were prepared and evaluated for their brain-binding and in vitro pharmacological effects. Unlike the p-haloPhe4 analogues of DPDPE and the deltorphins, similar analogues of DPADPE with electron-withdrawing groups substituted at the para-position of the Phe4 aromatic ring did not all have increased potency and selectivity for delta opioid receptors, but all retained high potency and selectivity for delta opioid receptors greater than DPDPE.

Animals↗

PubMed1997

Antisense targeting of delta opioid receptors in NG 108-15 cells: direct correlation between oligodeoxynucleotide uptake and receptor density.

Antisense oligodeoxynucleotides (ODN) have been used to inhibit the function of a number of structurally defined neurotransmitter receptors in vivo by transiently disrupting their expression in the CNS. However, issues concerning the cellular and molecular mechanisms of these ODN often raise questions about the specificity of such ODN-mediated "knock-down" of target proteins. This study sought to extend our in vivo "knock-down" of the delta opioid receptor (DOR) by targeting this receptor in the NG 108-15 cells with an antisense ODN for the DOR and by using a polyclonal antibody raised against this receptor to determine the efficiency and selectivity of the antisense ODN in inhibiting expression of the DOR. By fluorescence tagging the ODN and immunofluorescence labeling the DOR, we monitored the uptake efficiency of the ODN and the DOR density in individual cells that had been treated with the antisense ODN or with a mismatch control. Quantitative fluorescence image analysis showed that the uptake of ODN by NG 108-15 cells was time- and concentration-dependent and that it was not uniform within a population. Treatment with the antisense ODN elicited an inverse correlation between DOR immunoreactivity and the ODN fluorescence in individual cells. No correlation was found in cells treated with the mismatch control. These findings suggest that the antisense ODN-mediated "knock-down" of the DOR is governed by the sequence specificity of the ODN and the efficiency of its uptake by the target cells in a time- and concentration-dependent manner. These data provide further evidence in support of the selectivity of antisense ODN targeting and the utility of these molecules as an effective tool in neuropharmacological studies.

Animals↗

PubMed1996

Selective blockade of peripheral delta opioid agonist induced antinociception by intrathecal administration of delta receptor antisense oligodeoxynucleotide.

Previous studies have shown that intrathecal (i.t.) administration of antisense, but not mismatch, oligodeoxynucleotides (ODNs) to the cloned delta opioid receptor (DOR) can inhibit the antinociceptive actions of i.t. delta (delta), but not mu (mu) or kappa (kappa), opioid agonists. As a major portion of spinal opioid receptors are localized on the central terminals of the small afferent fibers, we hypothesized that the effects of antisense ODNs given i.t. might be the result of actions at the level of the cell body in the dorsal root ganglion (DRG). This possibility was investigated by assessing the antinociceptive actions of an i.t. or intrapaw (ipaw) administered mu (morphine), delta ([D-Ala2, Glu4]deltorphin) or kappa (CI977) opioid agonist in rats treated with i.t. saline or antisense or mismatch ODNs to the DOR (12.5 micrograms, twice-daily for 3 days). The opioid agonists produced significant antinociception in the 5% formalin-flinch test following either i.t. or ipaw administration. DOR antisense ODN treatment blocked the antinociceptive actions of both i.t. or ipaw [D-Ala2, Glu4]deltorphin without affecting the antinociceptive actions of i.t. or ipaw morphine or CI977. Radioligand binding studies with [3H]naltrindole (NTI), a delta selective antagonist, indicated an approximate 50% decrease in delta opioid receptors in the lumbar spinal cord following i.t. DOR antisense, but not mismatch, ODN treatment. DOR antisense or mismatch ODN treatment did not affect nu or kappa radioligand binding in lumbar spinal cord. These data suggest the possibility that peripheral proteins can be targeted with i.t. antisense ODNs providing significant opportunities for the exploration of the physiological and pathological significance of these substances.

Analgesics, Opioid↗

PubMed1996

Tramadol, M1 metabolite and enantiomer affinities for cloned human opioid receptors expressed in transfected HN9.10 neuroblastoma cells.

Tramadol hydrochloride is a centrally acting synthetic analgesic in widespread clinical use. Despite different degrees of opioid-like characteristics in preclinical tests, it is characterized by lack of full naloxone reversibility or naloxone-precipitated withdrawal in humans. To investigate this apparent discrepancy, the present study measured the affinity of tramadol (and its enantiomers) and an active O-desmethyl metabolite (M1) (and its enantiomers) to cloned human opioid receptors of the mu, delta and kappa type stably expressed in HN9.10 neuroblastoma cells. At mu sites, the Ki values for tramadol, its (+) and (-) enantiomers, M1, and its (+) and (-) enantiomers were 17000, 15700, 28800, 3190, 153 and 9680 nM, respectively, compared to 7.1 nM for morphine. These results are consistent with the suggestion of a non-opioid contribution to the clinical profile of tramadol.

Brain Neoplasms↗

PubMed1996

Antiallodynic effects of a CCKB antagonist in rats with nerve ligation injury: role of endogenous enkephalins.

Cholecystokinin (CCK) may act as an endogenous anti-opioid and blockade of CCK receptors can enhance the potency and efficacy of morphine. This effect is blocked by opioid delta (delta) receptor antagonists, suggesting a tonic inhibitory action of CCK to diminish the release and/or availability of endogenous enkephalins. The present studies have further evaluated this possibility by studying the antiallodynic actions of a CCKB antagonist (L365,260) alone, or in the presence of thiorphan (a neutral endopeptidase inhibitor) in a model of peripheral neuropathy. Animals subjected to nerve injury, but not sham controls, exhibited long lasting, stable mechanical allodynia. Intrathecal (i.t.) administration of L365,260 or thiorphan alone did not alter allodynia. However, co-administration of these compounds produced a significant antiallodynic action which was antagonized by receptor selective doses of naltrindole, an opioid delta receptor antagonist. In addition, antisera to [Leu5]enkephalin, but not to [Met5]enkephalin, also blocked the antiallodynic action of thiorphan plus L365,260. These data suggest that blockade of CCKB receptors may enhance the actions or availability of endogenous [Leu5]enkephalin or a like substance which can elicit a significant antiallodynic action via opioid delta receptors when its degradation is by inhibited by thiorphan. The data suggest that delta opioids are involved in regulation of some aspects of nerve-injury induced pain.

Animals↗

PubMed1996

Immunofluorescence analysis of antisense oligodeoxynucleotide-mediated 'knock-down' of the mouse delta opioid receptor in vitro and in vivo.

We have previously used antisense oligodeoxynucleotides (ODN) to the cloned delta opioid receptor (DOR) to inhibit the antinociceptive response to spinally administered delta opioid receptor selective agonists in mice. Here we have examined the effect of DOR antisense ODN treatment on the level of DOR expressed in NG 108-15 cells and the spinal cord, through immuno-fluorescence microscopy, to determine the efficiency and selectivity of the antisense ODN-mediated "knock-down' of the DOR in these tissues. Antisense ODN, but not mismatch control, treatment resulted in a significant reduction in DOR immunoreactivity (-ir) in NG 108-15 cells and spinal cord. Thus, the inhibition of antinociceptive response to intrathecal delta selective agonists by DOR antisense ODN correlates with the loss of DOR-ir in the superficial layers of the dorsal horn of the spinal cord.

Animals↗

PubMed1996

Effects of modifications of residues in position 3 of dynorphin A(1-11)-NH2 on kappa receptor selectivity and potency.

Tyrosine1 and phenylalanine4 in dynorphin A (Dyn A) have been reported to be important residues for opioid agonist activity and for potency at kappa receptors. The glycine residues in the 2 and 3 positions of dynorphin A may affect the relative orientation of the aromatic rings in positions 1 and 4, but their flexibility precludes careful analysis. To examine these effects on dynorphin A, we previously have synthesized the linear analogues [D-Ala3]Dyn A(1-11)-NH2 (2) and [Ala3]Dyn A(1-11)-NH2 (3) and reported their biological activities. Analogues 2 and 3 displayed affinities for the central kappa opioid receptor (IC50 = 0.76 and 1.1 nM, respectively) similar to that of Dyn A(1-11)-NH2 (1) (IC50 = 0.58 nM) and greatly enhanced selectivities for kappa vs mu and kappa vs delta receptors (IC50 ratios of 350 and 1300 for 2, and 190 and 660 for 3, respectively). These results suggest that the structure and lipophilicity of the amino acid present in position 3 of Dyn A(1-11)-NH2 as well as the conformational changes they induce in the message sequence of dynorphin have important effects on potency and selectivity for kappa opioid receptors. To further investigate structure-activity relationships involving the residue at the 3 position of Dyn A(1-11)-NH2, a series of Dyn A analogues with aromatic, charged, and aliphatic side chain substitutions at the 3 position was designed, synthesized, and evaluated for their affinities for kappa, mu, and delta opioid receptors. It was found that analogues with lipophilic amino acids at the 3 position of Dyn A(1-11)-NH2 generally displayed higher affinity but similar selectivities for the kappa receptor than analogues with charged residues at the same position. It is suggested that the structural, configurational, and steric/lipophilic effects of amino acids at position 3 of Dyn A(1-11)-NH2 may play an important role in potency and selectivity for the kappa receptor.

Amino Acid Sequence↗

PubMed1996

Epidermal growth factor protects mouse ileal mucosa from Triton X-100-induced injury.

Luminal exposure of mouse ileal mucosal sheets in vitro to 0.3% Triton X-100 for 10 min resulted in a 45.3 +/- 4.4% decrease in tissue resistance (Rt). Luminal pretreatment of tissue with epidermal growth factor (EGF, 30 nM) reduced the Triton X-100-induced decrease in Rt to 10.2 +/- 3.5%. This effect of luminal EGF was concentration-related with an A50 value of 7.4 nM. EGF applied to the basal buffer also produced a similar protective effect, but it was 5.3-fold less potent than luminal EGF. Morphological analysis indicated the formation of denudations at villi tips by Triton X-100 which was prevented by EGF pretreatment. These results suggest that EGF may play a role in the protection of ileal mucosal integrity.

Animals↗

PubMed1996

Probes for narcotic receptor-mediated phenomena. 21. Novel derivatives of 3-(1,2,3,4,5,11-hexahydro-3-methyl-2,6-methano-6H-azocino[4,5-b]indol- 6-yl)-phenols with improved delta opioid receptor selectivity.

Derivatives of racemic and optically pure levorotatory 3-(1,2,3,4,5,11-hexahydro-3-methyl-2,6-methano-6H-azocino[4,5-b]in dol-6-yl)phenols containing methoxy substituents in the C10', C9', and C8' positions (compounds 9-11, respectively) were synthesized and characterized by spectroscopic and X-ray methods. The binding affinities for the mu, delta and kappa 1 opioid receptors and activity in the guinea pig ileum (GPI) and mouse vas deferens (MVD) functional bioassays were determined for these compounds. A methoxy substituent in the C8' position decreases the binding affinity for both the mu and delta receptors, while a C10' methoxy substituent has little effect on either binding affinity. Interestingly, a methoxy group at the C9' position in the levorotatory series provides compound (-)-10 which exhibits both enhanced in vitro affinity and selectivity for the delta opioid receptor relative to the unsubstituted derivative (-)-8 and is the most selective (mu/delta IC50 ratio 17.9, kappa 1/delta IC50 ratio 314) and highest affinity (IC50 3.7 nM) delta receptor ligand for this novel class of compounds. The results of the GPI and MVD bioassays are more dramatic and indicate that (-)-10 is an agonist for the delta receptor (IC50 49.0 nM) with substantial selectivity for the delta versus the mu receptor borne out by a GPI/MVD IC50 ratio of > 612.

Analgesics↗

PubMed1996

Design, synthesis, and biological activities of cyclic lactam peptide analogues of dynorphine A(1-11)-NH2.

We previously have reported four possible binding conformation of dynorphin A (Dyn A) for the central kappa opioid receptors, induced by the address sequence, using a molecular mechanics energy minimization approach. The lowest energy conformation was found to exhibit an alpha-helical conformation in the cyclized address sequence. It was suggested that an alpha-helical conformation in the cyclized address sequence or a helical conformation induced by the conformational characteristics of the message sequence may be important for binding potency and kappa opioid receptor selectivity. Side chain to side chain lactam bridges between the i and i + 4 positions have been shown to stabilize alpha-helical conformation. Thus, a series of cyclic lactam analogues of dynorphin A(1-11)-NH2 have been designed, synthesized and evaluated by the guinea pig brain (GPB) binding assay and guinea pig ileum (GPI) bioassay to evaluate the conformational analysis prediction and, further, to investigate the conformational requirements for high potency and selectivity for kappa opioid receptors. Positions 2-6, 3-7, and 5-9 were chosen as the sites for incorporating cyclic conformational constraints. Cyclization between D-Asp(2) and Lys(6) in c[D-Asp(2),Lys(6)]Dyn A(1-11)-NH2 led to an analogue with pronounced potency and selectivity enhancement for the mu opioid receptor, whereas cyclization between D-Asp(3) and Lys(7) in c[D-Asp(3),Lys(7)]Dyn A(1-11)-NH2 led to a potent ligand (IC(50) 4.9 nM) with kappa receptor selectivity. The other analogues in the series proved to be less selective. The biological results led to the suggestion that the binding conformation for the kappa receptor may have structural requirements that are distinct from those of mu and delta receptors. Interestingly, analogues with a D-Asp at position 2, 3, or 9 were found to be more potent for the kappa receptor than analogues with an L-Asp at the same positions. It is suggested that the incorporation of D-Asp into position 2, 3, or 9 of Dyn A(1-11)-NH2 may have stereochemical and conformational effects on the nearby amino acids which can help discriminate the preference between kappa, mu, and delta receptors.

Amino Acid Sequence↗

PubMed1996

Conformational restriction of Tyr and Phe side chains in opioid peptides: information about preferred and bioactive side-chain topology.

The side chain of Tyr and Phe was fixed into the gauche(-) or gauche(+) conformation by using the Tic Htc structures, and into the trans conformation by using an aminobenzazepine-type (Aba) structure. When incorporated into dermorphin or deltorphin II, the Tic and Htc analogues all showed a large decrease in both mu and delta affinities and activities. Fixation of Phe(3) in the trans rotamer resulted in a large increase in delta affinity in the dermorphin analogue, whereas in the [Aba(3)-Gly(4)] deltorphin II analogue, good delta affinity is maintained despite the removal of the Glu side chain. Whereas several authors propose a gauche(-) preferred conformation for the Phe(3) side chain, these results suggest a trans conformation at the delta receptor. The use of these conformationally constrained residues for evaluating the preferred solution conformation in the flexible N-terminal tripeptide Tyr-D-Ala-Phe is illustrated. The (1)H-nmr parameters--chemical shift, temperature dependence, and nuclear Overhauser effects to the D-Ala(2) methyl protons in the different analogues--provide direct evidence to confirm the proposed sandwich conformation in the native peptides.

Amino Acid Sequence↗