An in vitro polysome display system for identifying ligands from very large peptide libraries.

We have used an in vitro protein synthesis system to construct a very large library of peptides displayed on polysomes. A pool of DNA sequences encoding 10(12) random decapeptides was incubated in an Escherichia coli S30 coupled transcription/translation system. Polysomes were isolated and screened by affinity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin B. The mRNA from the enriched pool of polysomes was recovered, copied into cDNA, and amplified by the polymerase chain reaction (PCR) to produce template for the next round of in vitro synthesis and selection. A portion of the amplified template from each round was cloned into a filamentous phagemid vector to determine the specificity of peptide binding by phage ELISA and to sequence the DNA. After four rounds of affinity selection, the majority of clones encoded peptides that bound specifically to the antibody and contained a consensus sequence that is similar to the known epitope for the antibody. Synthetic peptides corresponding to several of these sequences have binding affinities ranging from 7 to 140 nM. The in vitro system described here has the potential to screen peptide libraries that are three to six orders of magnitude larger than current biological peptide display systems.

Aquaretic effect of the stable dynorphin-A analog E2078 in the human.

Dynorphin-A is an endogenously released hormone that is now recognized as a kappa-opioid receptor agonist. Because kappa-agonists have been reported to induce water diuresis through negative modulation of antidiuretic hormone release and/or action, in the present study we investigated the aquaretic effects and safety of E2078, a metabolically stable dynorphin-A analog, in 21 healthy subjects after single (1.0, 2.0, 3.0, 5.0, 7.5 and 10.0 mg, i.m.) and repeated (5.0 mg t.i.d. for 4 and 1/3 days) administration. E2078 dose-dependently increased the 0 to 4-hr urine volume, which plateaued between 1032.4 +/- 130.1 and 1286.2 +/- 113.0 ml/4 hr (mean +/- S.E.M.) at doses of between 5.0 and 10.0 mg. The drug decreased urine osmolality (Uosm) markedly, by 17 to 27%, and the free-water clearance (CH2O) became positive, changing from -1.8 +/- 0.2 (placebo) to 0.8 +/- 0.3-2.8 +/- 0.4 ml/min after a single E2078 administration (P < .01). In the repeated-dose study, the first dose of E2078 increased the 0 to 5-hr urine output (1256 +/- 164.9 ml/5 h), lowered Uosm (151.8 +/- 13.3 mOsm/kg) and brought about a positive CH2O (1.9 +/- 0.2 ml/min). These values were similar to those seen after the single dose, but after the subsequent doses these aquaretic effects were attenuated, although the ranges of these same parameters were still significantly greater (P < .01-0.05) (441.4 +/- 102.4-585.3 +/- 131.9 ml/5 hr, 322.8 +/- 21.9-378.2 +/- 47.7 mOsm/kg and -0.2 +/- 0.2-(-)0.6 +/- 0.2 ml/min, respectively) than the day -1 base line (164.1 +/- 41.3 ml/5 hr, 992.0 +/- 80.6 mOsm/kg and -1.2 +/- 0.2 ml/min, respectively). Urinary excretion of electrolytes (Na, K and Cl) was not altered during either study period. A single E2078 administration reduced plasma antidiuretic hormone dose-dependently. On repeated dosing, the plasma concentration had rebounded to approximately 3 pg/ml by the time of the first dose on days 3 and 5, which lowered it again. The present results suggest that E2078 is a safe and effective aquaretic and could be a useful therapeutic tool for patients with water-retaining diseases.

Basic peptides can be imported into yeast mitochondria by two distinct targeting pathways. Involvement of the peptide-sensitive channel of the outer membrane.

The interaction of several basic peptides with yeast mitochondria has been analyzed. The peptides were selected for their ability to block a cationic channel of the outer membrane, the peptide-sensitive channel. These peptides possess common characteristics, such as a net positive charge superior to 2 and the capacity to form amphiphilic structures. They can be divided into two classes as follows: peptides of class I derived from mitochondrial signal peptides, such as the presequence of cytochrome c oxidase subunit IV, e.g. pCyt OX IV (1-12) Y; and peptides of class II unrelated to the mitochondria, such as dynorphin B (1-13). Class I peptides inhibited the translocation of a chimeric protein, cytochrome b2-DHFR, into the mitochondrial matrix, whereas peptides of class II failed to inhibit this import. Experiments with iodinated pCyt OX IV (1-12) Y and dynorphin B (1-13) showed, however, that both types of peptides were imported into yeast mitochondria in vitro and subsequently degraded. At 30 degrees C, two import mechanisms could be distinguished; the mitochondrial presequences (class I) were translocated into the matrix in a temperature- and potential-sensitive manner, probably along the general import pathway, while class II dynorphin B (1-13) was imported into the intermembrane space by a process that was neither temperature- nor potential-sensitive. At 0 degree C, both peptides were imported in a class II manner. The class II characteristics suggested the existence of a direct pathway into the intermembrane space, which may be associated with the peptide-sensitive channel. This hypothesis is substantiated by the competition for the import into the mitochondria between peptides of the two classes. The import of pCyt OX (1-12) Y was inhibited at 30 degrees C only by peptides of class I, IV whereas, at 0 degree C, this import was also inhibited by peptides of class II. Import of peptides of the latter class was inhibited by peptides of the two classes both at 0 degree C and 30 degrees C.

Dynorphin B is present in sensory and parasympathetic nerves innervating pial arteries.

Dynorphin B (dyn B) in trigeminal ganglion cells and in perivascular nerve fibers in pial arteries was demonstrated in rat, guinea-pig, and monkey by immunohistochemistry. The pathway from the trigeminal ganglion, which runs via the nasociliary nerve and ethmoidal foramen to the pial arteries, was shown in rat by retrograde tracer technique and nerve section. In the guinea-pig the peptide was demonstrated to coexist with substance P and calcitonin gene-related peptide in neurons of the trigeminal ganglion and pial nerve fibers, i.e., it was present in cerebrovascular sensory nerves with primarily nociceptive function. Another finding in guinea-pig was a coexistence of dyn B with vasoactive intestinal polypeptide in the pial nerve fibers and neurons of the sphenopalatine ganglion, indicating a presence also in parasympathetic nerves to the cerebral vessels. No vasomotor effect of dyn B could be detected in isolated segments of rat pial arteries, which rules out a direct postsynaptic effect on vascular tone. The peptide did not display a prejunctional modulatory action on the adrenergic nerves present in the vessels. The function of dyn B in the cerebrovascular nerves is discussed.

Facilitation of self-stimulation of ventral tegmentum by microinjection of opioid receptor subtype agonists.

Intracranial self-stimulation (ICSS) evoked from the ventral tegmental area-substantia nigra (VTA-SN) and lateral hypothalamus-medial forebrain bundle (LH-MFB) was assessed following microinjections of mu (Tyr-D-Ala2-N-Me-Phe4-Gly5ol: DAGO), delta-(D-Ala2, D-Met5)-enkephalin: DADME) or kappa (Dynorphin-B or Rimorphin) opioid receptor subtype agonists or saline into either VTA-SN or LH-MFB. The current intensity was fixed at an optimum level to obtain 60-70% of the maximum asymptotic response rate. DAGO (5 micrograms/0.5 microliters), DADME (2 micrograms/0.5 microliters) or Dynorphin B (0.5 microgram/0.5 microliters) injected into VTA-SN facilitated the self-stimulation rates of VTA-SN by 27%, 32%, and 59%, respectively. These microinjections did not alter the self-stimulation of LH-MFB when effects of these injections were still persisting in VTA-SN. Similar doses of these opioid receptor agonists injected into LH-MFB had no significant effect on the self-stimulation rates of either LH-MFB or VTA-SN. The facilitatory effects of DADME were completely abolished by naloxone (30 mg/kg IP). Taken together, these results suggest that all three opioid receptor subtypes of ventral tegmentum and not of lateral hypothalamus are involved in the electrically evoked self-stimulation of VTA-SN.

Structural requirements of bioactive peptides for interaction with endopeptidase 22.19.

A series of biologically active peptides and related compounds (opioid peptides, neurotensin, and bradykinin) were used as substrates or competitive inhibitors to study the structural requirements for peptide interaction with endopeptidase 22.19. The kinetics of hydrolysis of these peptides indicated that, in contrast to other proteases, the substrate specificity of endopeptidase 22.19 is not determined by the amino acids flanking the sensitive bonds of the substrates. The competition between bioactive peptide analogues and the quenched fluorescence substrate of endopeptidase 22.19 indicated that their length and their flexibility may be the dominant factors to explain their binding specificities. These peculiar features of endopeptidase 22.19 may be of importance to understand the physiological processes of conversion and inactivation of biologically active peptides.

Voltage-dependent effects of opioid peptides on hippocampal CA3 pyramidal neurons in vitro.

Opioid peptides, and especially the dynorphins, have been localized to several circuits in the CA3 hippocampal region, yet electrophysiological studies often find mixed effects of opiates on the excitability of CA3 neurons. Reasoning that these mixed effects might involve voltage-dependent actions, we tested the effect of several opiates on CA3 pyramidal neurons using single-electrode voltage-clamp recording in a slice preparation of rat hippocampus. In most CA3 neurons, the voltage-dependent K+ current known as the M-current (IM) was uniquely sensitive to the opioid peptides, with the direction of response dependent upon the opiate type and concentration. Thus, an opiate selective for kappa receptors, U-50,488H, significantly augmented IM. The kappa-selective agonists dynorphin A and dynorphin B, which exist in mossy fiber afferents to CA3 pyramidal neurons, also markedly augmented IM at low concentrations (20-100 nM). By contrast, dynorphin A at higher concentrations (1-1.5 microM) often reduced IM. Similarly, several opiates [e.g., D-Ala2,D-Leu5-enkephalin: (DADL), [D-Pen2,5]-enkephalin (DPDPE)] known to act on the delta receptor subtypes reduced the M-current, with partial reversal of this effect by naloxone. Neither the selective mu-receptor agonist [D-Ala2, NMe-Phe4, Gly-ol]-enkephalin (DAMGO) nor the nonopioid fragment of dynorphin, des-Tyr-dynorphin, consistently altered IM. These opiate effects on IM were accompanied by changes in conductance and holding current consistent with their respective effects on IM. Dynorphin A did not measurably affect the Q-current, a conductance known to contribute to inward rectification in hippocampal pyramidal neurons. The opiate effects on IM were not altered by pretreatment with Cs+ (which blocks IQ) or Ca2+ channel blockers. The opposing effects of the dynorphins (both A and B) and DADL on IM were antagonized by naloxone (1-3 microM), and the dynorphin-induced augmentations of IM were usually reversed by the kappa receptor antagonist norbinaltorphimine. These results suggest that the opiates can have opposing effects on the same voltage-dependent K+ channel type (the M channel) in the rat CA3 pyramidal neuron, with the direction of the response depending on which receptor subtype is activated. These data not only help explain the mixed effects of opiates seen in other studies, but also suggest a potential postsynaptic function for the endogenous opiates contained in the CA3 mossy fibers.

Combinatorial chemistry--applications of light-directed chemical synthesis.

Combinatorial methods in biology and chemistry are proving to be powerful methods for generating molecular diversity. One approach, light-directed chemical synthesis, combines semiconductor-based photolithography technologies with solid-phase organic chemistry to synthesize large arrays of molecules with potential biological activity. This novel technology has the potential to provide libraries of both natural and synthetic molecules that might be screened rapidly for biological activity.

Evidence for the expression of dynorphin gene in the prolactin-immunoreactive neurons of the rat lateral hypothalamus.

Dynorphin B (DYN B) immunoreactivity was recently reported in a population of prolactin (PRL)-immunoreactive neurons of the rat lateral hypothalamus (LH). By coupling immunocytochemistry and in situ hybridization using two synthetic oligonucleotide probes complementary to DYN mRNA, a hybridization signal was observed over the neurons of the LH exhibiting both DYN- and PRL-like immunoreactivities. Our results clearly demonstrate that these neurons contain the mRNA encoding preproDYN and are able to synthesize authentic DYN B in colocalization with a peptide related to PRL.

Comparative effects of intrahippocampal injection of dynorphin A(1-8), dynorphin A(1-13), dynorphin A(1-17), U-50,488H, and dynorphin B on blood pressure and heart rate in spontaneously hypertensive and normotensive Wistar-Kyoto rats.

We previously demonstrated centrally mediated hypotensive and bradycardic effects of dynorphin A(1-8) (DA1-8) on microinjection into various areas of the hippocampal formation (HF) of both anesthetized and conscious male normotensive and spontaneously hypertensive rats (SHR). The purpose of the present study was to determine whether other dynorphin fragments also had this activity. We microinjected DA1-8, dynorphin A(1-13), dynorphin A(1-17), dynorphin B (DB), and the nonpeptide kappa-opioid agonist U-50,488H into HF areas previously found to react to DA1-8, at doses ranging from 0.05 to 50 nmol. The subjects were male SHR and normotensive Wistar-Kyoto (WKY) rats in which arterial pressure and heart rate were monitored. Dose-related centrally mediated hypotension and bradycardia were found in both strains with all agents used, except for DB, which had no effects. Similarly injected drug vehicle was also without effect. In general, the responses were greater in SHR than in WKY rats. Preinjection of the active HF areas with 2 nmol of nor-binaltorphimine (nor-BNI), a selective kappa-opioid receptor antagonist, which itself had no blood pressure or heart rate effects, abolished both the decrease in blood pressure and heart rate of all dynorphins and U-50,488H. The results demonstrated the equivalent abilities of all the dynorphin fragments studied, except DB, to cause HF-mediated hypotension and bradycardia. The results with U-50,488H and nor-BNI strongly implicate kappa-opiate receptor activation of the HF in these effects.

Blockade of morphine reward through the activation of kappa-opioid receptors in mice.

The effects of systemic (s.c.) treatment with the kappa-agonists U-50,488H and E-2078 (a stable dynorphin analog) on the morphine-induced place preference were examined in mice. Morphine (s.c.) caused a dose-related preference for the drug-associated place; the effects at doses of 3 and 5 mg/kg were significant. On the other hand, U-50,488H or E-2078 produced a dose-related conditioned place aversion. Both U-50,488H (1 mg/kg, s.c.) and E-2078 (0.1 mg/kg, s.c.) induced a slight, nonsignificant place aversion. Pretreatment with U-50,488H (1 mg/kg) abolished the morphine (3 mg/kg)-induced place preference. The morphine-induced place preference was also significantly decreased by pretreatment with E-2078 (0.1 mg/kg). The inhibitory effects of the kappa-agonists were antagonized by the kappa-antagonist nor-binaltorphimine (nor-BNI; 3 mg/kg, s.c.). In contrast, pretreatment with U-50,488H did not affect the place preference induced by the dopamine (DA) receptor agonist apomorphine (1 mg/kg, s.c.). In addition, morphine (3 mg/kg, s.c.) significantly increased the levels of the DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the limbic forebrain (nucleus accumbens and olfactory tubercle) but not in the striatum, implying that activation of the mesolimbic DA system may play an important role in the morphine-induced place preference in mice. Pretreatment with U-50,488H significantly reduced the morphine-induced elevation of DA metabolites in the limbic forebrain.(ABSTRACT TRUNCATED AT 250 WORDS)

Syntheses, opioid binding affinities, and potencies of dynorphin A analogues substituted in positions, 1, 6, 7, 8 and 10.

Structural, stereochemical, stereoelectronic and conformational requirements for biological activity of dynorphin A1-11-NH2 analogues at opioid receptors were explored by substitution of Tyr1, Arg6, Arg7, Ile8 and Pro10 with other amino acid residues. Interestingly, substitution of Tyr1 with N alpha-Ac-Tyr1, D-Tyr1, Phe1 or p-BrPhe1 led to analogues that were quite potent at kappa opioid receptors, and additional substitution of Ile8 with D-Ala8 and/or Pro10 with D-Pro10 retained high potency in brain binding assay: [N alpha-Ac-Tyr1]- (1), [D-Tyr1]-(2) [Phe1]- (3), [Phe1,D-Ala8]- (5), [-BrPhe1, D-Ala8]- (6), [Phe1, D-Pro10]- (7) and [Phe1,D-Ala8, D-Pro10]- Dyn A1-11-NH2 (8) had IC50 (nM) binding affinities of 13.2, 18.6, 1.64, 1.26, 1.84, 2.44 and 1.62 nM, respectively. The D-Phe1 analogue 4, however, was only weakly active (610 nM). All of the analogues except 4 were modestly selective for kappa vs. mu guinea pig brain opioid receptor (11- to 88-fold) and quite selective for kappa vs. delta receptors (65-576). However, all of the analogues appeared to have very low or essentially no activity in the guinea pig ileum and mouse vas deference functional bioassays, and one analogue, 5, appeared to have weak antagonist activities. On the other hand, if constrained amino acids such as beta-methylphenylalanine or 1,2,3,4-tetrahydroisoquinoline carboxylic acid, and hydroxyproline were placed in the 1 position, inactive analogues or analogues with greatly reduced potency and biological activity were obtained (compounds 12-14). It had previously been suggested that the Arg6 and Arg7 residues were critical for biological activity.(ABSTRACT TRUNCATED AT 250 WORDS)

[The serotonin-dependent suppression of the immune response by kappa-opiate receptors]. / Serotoninzavisimaia supressiia immunnogo otveta kappa-opiatnymi retseptorami.

The modulating effect of the kappa-opiate agonist, rimorphin, on the immune response was studied. This opioid in all the doses tested (1, 10 and 100 mkg/kg) was found to cause inhibition of the immune reaction as compared to the control CBA mice which received saline only. The dissection of the pituitary stalk prevented the inhibitory effect of rimorphin on the immunity. The opioid-induced immunosuppression was shown to be due to interaction with the serotoninergic system since it had no effect after preliminary blockade of S-2 serotonin postsynaptic receptors with cyproheptadine.

Expression of prodynorphin-derived peptides and mRNA in guinea-pig cortex.

The distributions and extent of processing of four prodynorphin-derived peptides (dynorphin A (1-17), dynorphin A (1-8), dynorphin B, and alpha-neoendorphin) were determined in ten regions of the cortex as well as in the striatum of the guinea-pig. There were significant differences between concentrations of these peptides in most cortical regions, with alpha-neoendorphin being several times more abundant than the other peptides, and dynorphin A (1-17) being present in the least amount. There were significant between-region differences in concentration for each peptide, although most regions had concentrations similar to those seen in the striatum. Concentrations of each peptide tended to be higher in piriform, entorhinal, motor, and auditory cortex than in other cortical regions. The extent of processing of prodynorphin varied across cortical regions as well, primarily due to the extent of processing to alpha-neoendorphin. Prodynorphin mRNA levels were not significantly different between cortical regions or from the amount observed in the striatum. Although specific regional variation exists, it appears that in general prodynorphin is expressed and processed in a similar manner in the cortex as in the striatum.

Suppression by dynorphin A and [des-Tyr1]dynorphin A peptides of the expression of opiate withdrawal and tolerance in morphine-dependent mice.

Previously, we demonstrated that the expression of opiate withdrawal and antinociceptive tolerance can be suppressed by dynorphin (dyn) A-(1-13) in morphine-dependent mice. In this study, it was shown that the normal, endogenous dyn, dyn A-(1-17) also possessed this suppressive property. While using the nonopioid dyn analog, [des-Tyr1]dyn A [dyn A-(2-17)] as a negative control, we discovered unexpectedly that this peptide fragment also suppressed naloxone-induced withdrawal and the expression of morphine tolerance in morphine-dependent mice. Thus, an extensive structure activity relationship was studied using 11 peptide fragments. It was determined that the amino acid sequence of dyn A was required for the suppressive activity because dyn B and alpha-neoendorphin both failed to suppress naloxone-precipitated withdrawal jumping. Of the [des-Tyr1]dyn fragments, the minimal amino acid sequence required to suppress naloxone-induced withdrawal was determined to be dyn A-(2-8), containing the sequence G-G-F-L-R-R-I.

Design and synthesis of highly potent and specific dynorphin derivatives for affinity labeling of kappa opioid receptors.

Dynorphin A derivatives containing S-(3-nitro-2-pyridinesulfenyl)-cysteine(Cys(Npys)) were designed and synthesized for affinity labeling of kappa opioid receptors. Derivatives with Cys(Npys) residue at the 8 position were highly potent and specific to kappa receptors and bound to receptors through disulfide bond formation.

Apomorphine-susceptible and apomorphine-unsusceptible Wistar rats differ in novelty-induced changes in hippocampal dynorphin B expression and two-way active avoidance: a new key in the search for the role of the hippocampal-accumbens axis.

The present study examines two characteristic traits of the hippocampus in apomorphine-susceptible (APO-SUS) and apomorphine-unsusceptible (APO-UNSUS) Wistar rat lines. Since hippocampal mossy fibers contain among others dynorphin B as transmitter, a radioimmunoassay was used to analyze the hippocampal dynorphin B expression in response to novelty in these lines. Dynorphin B expression at the end of the baseline condition was greater in APO-SUS rats than in APO-UNSUS rats, while exposure to novelty decreased and increased the dynorphin B expression in APO-SUS and APO-UNSUS rats, respectively. These interline differences in dynorphin B expression could be due to (a) an interline difference in the size of the mossy fiber terminal fields, (b) an interline difference in the regulation of the firing rate of mossy fibers by corticosteroids, and/or (c) an interline difference in the release of corticosteroids in response to novelty. Since the size of the mossy fiber infra/intrapyramidal terminal field is inversely related to two-way active avoidance performance, APO-SUS and APO-UNSUS rats (n = 9 per line) were given this task: APO-UNSUS rats performed much better than APO-SUS rats. It is concluded that the neurochemical and behavioural function of the hippocampus significantly differs between lines. Given the already known interline differences in the function of the nucleus accumbens, the present results provide a new avenue in search for the functional relationship between the hippocampus and the nucleus accumbens.

Transduction of a protein kinase C-generated signal into the long-lasting facilitation of glutamate release.

The present study investigated the delayed and persistent effects of 4 beta-phorbol 12,13-dibutyrate (PDBu) on the K(+)-evoked release of endogenous glutamate and dynorphin B-like immunoreactivity from a subcellular fraction (P3) that is enriched in hippocampal mossy fiber synaptosomes. It is demonstrated that the alpha, beta, gamma, epsilon, and zeta isoforms of protein kinase C (PKC) are present in the P3 fraction obtained using the guinea pig hippocampus as starting tissue. The K(+)-evoked release of glutamate was found to be selectively enhanced when mossy fiber-enriched synaptosomes were preincubated with PDBu for 15 minutes and extensively washed with a PDBu-free medium. The persistent enhancement of glutamate release observed under this condition was not reversed by the protein kinase inhibitor staurosporine and was desensitized to the potentiating effects of an acute reexposure to PDBu. The overall content and activity of PKC was not substantially altered during the initial 15 minutes of treatment with PDBu (10 microM). More prolonged pretreatments with PDBu altered the substrate specificity of PKC and decreased the content of all PKC isoforms, but did not reverse the facilitation of glutamate release that followed preincubation in the presence of PDBu. It is concluded that the persistent activation of PKC enhances K(+)-evoked glutamate release from hippocampal mossy fiber-enriched synaptosomes and that, once established, this presynaptic facilitation is sustained by a process that is no longer directly dependent on continued PKC phosphotransferase activity.

Design of potent and selective dynorphin A related peptides devoid of supraspinal motor effects in mice.

Dynorphin A-(1-13)-Tyr-Leu-Phe-Asn-Gly-Pro (Dyn Ia) was previously shown to be a highly potent and selective kappa opioid peptide. Four analogs of Dyn Ia are synthesized by the solid-phase procedure, introducing pseudo CH2NH linkage between positions 6 and 7 as follows: analog 1, [6 psi 7 (CH2NH)]Dyn Ia; analog 2, [6 psi 7 (CH2NH), D-Leu8]Dyn Ia; analog 3, [N(Me)-Tyr1, 6 psi 7 (CH2NH)]Dyn Ia; and analog 4, [N(Me)-Tyr1, 6 psi 7 (CH2NH), D-Leu8]Dyn Ia. The purified peptides are compared in vitro with Dyn Ia for their ability to compete with the binding of selective kappa, mu, and delta opioid ligands using membrane preparations of guinea pig cerebellum (kappa) and rat brain (mu and delta). The synthetic compounds are also compared in vivo in mice (intracerebroventricularly administered) for their analgesic activity against acetic acid induced writhing and their ability to produce motor dysfunction. All compounds display a high affinity (Ki = 0.5-1.8 nM) and a good selectivity for the kappa opioid receptor, and their rank order of potency on the kappa site (analog 2 > analog 1 > analog 3 > analog 4) closely parallels their potency (AD50 = 1.57-5 nmol/mouse) in inhibiting acetic acid induced writhing in mice (analog 2 > analog 1 > analog 4 > analog 3). On the other hand, all the synthetic analogs are less potent than Dyn Ia in producing motor effects, analog 2 being the least potent (CD50 = 15.4 nM as compared with 2.9 nM for Dyn Ia).(ABSTRACT TRUNCATED AT 250 WORDS)

Design and synthesis of highly potent and selective cyclic dynorphin A analogs. 2. New analogs.

We have designed and synthesized several cyclic disulfide-containing peptide analogs of dynorphin A (Dyn A) which are conformationally constrained in the putative "address" segment of the opioid ligand. Several of these Dyn A analogs exhibit unexpected apparent selectivities for the kappa and mu opioid receptors(s) of the central vs peripheral nervous systems. Thus, incorporation of conformational constraint in the putative "address" segment of Dyn A analogs has resulted in the kappa/mu opioid receptor ligands [L-Pen5,Cys11]Dyn A1-11-NH2 (4), [Cys5,Cys10]Dyn A1-11-NH2 (5), [Cys5,Cys9]DynA1-11-NH2 (6), and [Cys4,Cys9,Arg10]DynA1-11-NH2(7). All of these analogs possess high kappa and mu opioid receptor affinities for the central receptor (guinea pig brain), but effect only weak potency at peripheral kappa and mu opioid receptors (GPI). In fact cyclic dynorphin A analog 4 shows > 19,000-fold differences between central kappa opioid affinity and potency in the guinea pig ileum (GPI). Additionally analog 4 is not an antagonist in the GPI, suggesting possible receptor differences between these sites. Substitution of Tyr1 by Phe1 in the cyclic 1-11 series gave the analog [Phe1,Cys5,Cys11]Dyn A1-11-NH2 (1) that was surprisingly potent in the guinea pig brain binding assay (IC50 = 15.1 nM) at the kappa receptor, but was inactive in the GPI and mouse vas deferens bioassays. D-Ala2 and Tic4 analogs of 1 had lower affinity at brain kappa receptors and had very weak potencies in the GPI and MVD bioassays. On the other hand, [Cys6,Cys10]DynA1-11-NH2 (8), [Cys8,D-Cys13]DynA1-13-NH2 (9), [D-Cys8,D-Cys12]DynA1-13-NH2 (10), and [D-Pro10,Cys5,Cys13]-Dyn A1-13-NH2 (11) were surprisingly potent in the GPI bioassay, though considerable apparent selectivity for central receptors is still retained. The apparent lack of correlation between the pharmacological profiles observed in smooth muscle and in the brain binding assays, particularly with 1 and 4, may suggest the existence of different subtypes of the kappa and mu opioid receptors in the brain and peripheral systems.