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Background and Purpose: The κ opioid receptor antagonists demonstrate potential for maintaining abstinence from psychostimulant abuse, but existing non-peptide κ-receptor selective antagonists show exceptionally long activity. We hypothesized that the L- and D-Trp isomers of CJ-15,208, a natural cyclic tetrapeptide reported to be a κ-receptor antagonist in vitro, would demonstrate short-acting, dose-dependent antagonism in vivo, preventing reinstatement of cocaine-seeking behaviour.Experimental Approach: Affinity, selectivity and efficacy of the L-Trp and D-Trp isomers for opioid receptors were assessed in vitro in radioligand and GTPγS binding assays. Opioid receptor agonist and antagonist activities were characterized in vivo following i.c.v. administration with the 55°C warm water tail-withdrawal assay. The D-Trp isomer, which demonstrated primarily κ-receptor selective antagonist activity, was further evaluated for its prevention of stress- and drug-induced reinstatement of extinguished cocaine conditioned place preference (CPP).Key Results: The two isomers showed similar affinity and selectivity for κ receptors (K(i)  30-35 nM) as well as κ receptor antagonism in vitro. As expected, the D-Trp cyclic tetrapeptide exhibited minimal agonist activity and induced dose-dependent κ-receptor selective antagonism lasting less than 18 h in vivo. Pretreatment with this peptide prevented stress-, but not cocaine-induced, reinstatement of extinguished cocaine CPP. In contrast, the L-Trp cyclic tetrapeptide unexpectedly demonstrated mixed opioid agonist/antagonist activity.Conclusions and Implications: The L-Trp and the D-Trp isomers of CJ-15,208 demonstrate stereospecific opioid activity in vivo. The relatively brief κ opioid receptor antagonism, coupled with the prevention of stress-induced reinstatement of extinguished cocaine-seeking behaviour, suggests the D-Trp isomer could be used therapeutically to maintain abstinence from psychostimulant abuse. [ABSTRACT FROM AUTHOR]

Kappa opioid receptor (KOR) antagonists have potential therapeutic applications in the treatment of stress-induced relapse to substance abuse and mood disorders. The dynorphin A analog arodyn (Ac[Phe 1,2,3 ,Arg 4 ,D-Ala 8 ]dynorphin A-(1-11)-NH 2 ) exhibits potent and selective kappa opioid receptor antagonism. Multiple cyclizations in longer peptides, such as dynorphin and its analogs, can extend the conformational constraint to additional regions of the peptide beyond what is typically constrained by a single cyclization. Here, we report the design, synthesis, and pharmacological evaluation of a bicyclic arodyn analog with two constraints in the opioid peptide sequence. The peptide, designed based on structure-activity relationships of monocyclic arodyn analogs, was synthesized by solid-phase peptide synthesis and cyclized by sequential ring-closing metathesis (RCM) in the C- and N-terminal sequences. Molecular modeling studies suggest similar interactions of key aromatic and basic residues in the bicyclic peptide with KOR as found in the cryoEM structure of KOR-bound dynorphin, despite substantial differences in the backbone conformations of the two peptides. The bicyclic peptide's affinities at KOR and mu opioid receptors (MOR) were determined in radioligand binding assays, and its KOR antagonism was determined in the [ 35 S]GTPγS assay in KOR-expressing cells. The bicyclic analog retains KOR affinity and selectivity (K i = 26 nM, 97-fold selectivity over MOR) similar to arodyn and exhibits potent KOR antagonism in the dynorphin-stimulated [ 35 S]GTPγS assay. This bicyclic peptide represents a promising advance in preparing cyclic opioid peptide ligands and opens avenues for the rational design of additional bicyclic opioid peptide analogs.

The macrocyclic tetrapeptide CJ-15,208 ( cyclo [Phe-D-Pro-Phe-Trp]) and its D-Trp isomer exhibit kappa opioid receptor (KOR) antagonism which prevents stress-induced reinstatement of extinguished cocaine-conditioned place preference. Here, we evaluated the effects of substitution of Trp and D-Trp on the peptides' opioid activity, antinociceptive tolerance, and the ability to prevent relapse to extinguished drug-CPP. Six analogs were synthesized using a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, efficacy in the [ 35 S]GTPγS assay, metabolic stability in mouse liver microsomes, and for opioid activity and selectivity in vivo in the mouse 55 °C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance, and conditioned place preference (CPP) were also assessed in vivo, and the ameliorating effect of analogs on the reinstatement of extinguished cocaine-place preference was assessed. Substitutions of other D-amino acids for D-Trp did not affect (or in one case increased) KOR affinity, while two of the three substitutions of an L-amino acid for Trp decreased KOR affinity. In contrast, all but one substitution increased mu opioid receptor (MOR) affinity in vitro. The metabolic stabilities of the analogs were similar to those of their respective parent peptides, with analogs containing a D-amino acid being much more rapidly metabolized than those containing an L-amino acid in this position. In vivo, CJ-15,208 analogs demonstrated antinociception, although potencies varied over an 80-fold range and the mediating opioid receptors differed by substitution. KOR antagonism was lost for all but the D-benzothienylalanine analog, and the 2'-naphthylalanine analog instead demonstrated significant delta opioid receptor (DOR) antagonism. Introduction of DOR antagonism coincided with reduced acute opioid antinociceptive tolerance and prevented stress-induced reinstatement of extinguished cocaine-CPP.

Opioid use disorder (OUD) relapse rates are discouragingly high, underscoring the need for new treatment options. The macrocyclic tetrapeptide natural product CJ-15,208 and its stereoisomer [d-Trp]CJ-15,208 demonstrate kappa opioid receptor (KOR) antagonist activity upon oral administration which prevents stress-induced reinstatement of cocaine-seeking behavior. In order to further explore the structure-activity relationships and expand the potential therapeutic applications of KOR antagonism for the treatment of OUD, we screened a series of 24 analogs of [d-Trp]CJ-15,208 with the goal of enhancing KOR antagonist activity. From this screening, analog 22 arose as a compound of interest, demonstrating dose-dependent KOR antagonism after central and oral administration lasting at least 2.5 h. In further oral testing, analog 22 lacked respiratory, locomotor, or reinforcing effects, consistent with the absence of opioid agonism. Pretreatment with analog 22 (30 mg/kg, p.o.) prevented stress-induced reinstatement of extinguished morphine conditioned place preference and reduced some signs of naloxone-precipitated withdrawal in mice physically dependent on morphine. Collectively, these data support the therapeutic potential of KOR antagonists to support abstinence in OUD and ameliorate opioid withdrawal., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Ligands for kappa opioid receptors (KOR) have potential uses as non-addictive analgesics and for the treatment of pruritus, mood disorders, and substance abuse. These areas continue to have major unmet medical needs. Significant advances have been made in recent years in the preclinical development of novel opioid peptides, notably ones with structural features that inherently impart stability to proteases. Following a brief discussion of the potential therapeutic applications of KOR agonists and antagonists, this review focuses on two series of novel opioid peptides, all-D-amino acid tetrapeptides as peripherally selective KOR agonists for the treatment of pain and pruritus without centrally mediated side effects, and macrocyclic tetrapeptides based on CJ-15,208 that can exhibit different opioid profiles with potential applications such as analgesics and treatments for substance abuse., (© 2021. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

We explored the utility of the real-time FLIPR Membrane Potential (FMP) assay as a method to assess kappa opioid receptor (KOR)-induced hyperpolarization. The FMP Blue dye was used to measure fluorescent signals reflecting changes in membrane potential in KOR expressing CHO (CHO-KOR) cells. Treatment of CHO-KOR cells with kappa agonists U50,488 or dynorphin [Dyn (1-13)NH 2 ] produced rapid and concentration-dependent decreases in FMP Blue fluorescence reflecting membrane hyperpolarization. Both the nonselective opioid antagonist naloxone and the κ-selective antagonists nor-binaltorphimine (nor-BNI) and zyklophin produced rightward shifts in the U50,488 concentration-response curves, consistent with competitive antagonism of the KOR mediated response. The decrease in fluorescent emission produced by U50,488 was blocked by overnight pertussis toxin pretreatment, indicating the requirement for PTX-sensitive G proteins in the KOR mediated response. We directly compared the potency of U50,488 and Dyn (1-13)NH 2 in the FMP and [ 35 S]GTPγS binding assays, and found that both were approximately 10 times more potent in the cellular fluorescence assay. The maximum responses of both U50,488 and Dyn (1-13)NH 2 declined following repeated additions, reflecting receptor desensitization. We assessed the efficacy and potency of structurally distinct KOR small molecule and peptide ligands. The FMP assay reliably detected both partial agonists and stereoselectivity. Using KOR-selective peptides with varying efficacies, we found that the FMP assay allowed high throughput quantification of peptide efficacy. These data demonstrate that the FMP assay is a sensitive method for assessing κ-opioid receptor induced hyperpolarization, and represents a useful approach for quantification of potency, efficacy and desensitization of KOR ligands., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Kappa opioid receptor (KOR) antagonists have recently shown potential for treating drug addiction and mood disorders. The linear acetylated dynorphin A analog arodyn (Ac[Phe 1,2,3 ,Arg 4 ,d-Ala 8 ]dynorphin A-(1-11)NH 2 ), synthesized in our laboratory, demonstrated potent and selective KOR antagonism. Cyclization of arodyn could potentially stabilize the bioactive conformation and enhance its metabolic stability. The cyclization strategy employed involved ring closing metathesis between adjacent meta - or para -substituted Tyr(allyl) residues in the "message" sequence that were predicted in a docking study to yield analogs that would bind to the KOR with binding poses similar to arodyn. Consistent with the modeling, the resulting analogs retained KOR affinity similar to arodyn; the peptides involving cyclization between para O -allyl groups also retained high KOR selectivity, with one analog exhibiting KOR antagonist potency ( K B = 15 nM) similar to arodyn. These promising cyclized analogs with constrained aromatic residues represent novel leads for further exploration of KOR pharmacology.

The macrocyclic tetrapeptide cyclo [Phe-d-Pro-Phe-Trp] (CJ-15,208) and its stereoisomer cyclo [Phe-d-Pro-Phe-d-Trp] exhibit different opioid activity profiles in vivo. The present study evaluated the influence of the Phe residues' stereochemistry on the peptides' opioid activity. Five stereoisomers were synthesized by a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, and for opioid activity and selectivity in vivo in the mouse 55 °C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance development, and place conditioning were also assessed in vivo. All of the stereoisomers exhibited antinociception following either intracerebroventricular or oral administration differentially mediated by multiple opioid receptors, with kappa opioid receptor (KOR) activity contributing for all of the peptides. However, unlike the parent peptides, KOR antagonism was exhibited by only one stereoisomer, while another isomer produced DOR antagonism. The stereoisomers of CJ-15,208 lacked significant respiratory effects, while the [d-Trp]CJ-15,208 stereoisomers did not elicit antinociceptive tolerance. Two isomers, cyclo [d-Phe-d-Pro-d-Phe-Trp] ( 3 ) and cyclo [Phe-d-Pro-d-Phe-d-Trp] ( 5 ), did not elicit either preference or aversion in a conditioned place preference assay. Collectively, these stereoisomers represent new lead compounds for further investigation in the development of safer opioid analgesics.

Background and Purpose: The macrocyclic tetrapeptide natural product CJ-15,208 (cyclo[Phe-d-Pro-Phe-Trp]) is a multifunctional μ-opioid receptor and κ-opioid receptor agonist and κ-opioid receptor antagonist that produces antinociception and prevents stress-induced reinstatement of extinguished cocaine-conditioned place preference (CPP). We hypothesized that an analogue of CJ-15,208, cyclo[Pro-Sar-Phe-d-Phe], would demonstrate multifunctional μ-opioid receptor and κ-opioid receptor ligand activity, producing potent antinociception with fewer liabilities than selective μ-opioid receptor agonists, while preventing both drug- and stress-induced reinstatement of morphine-induced CPP., Experimental Approach: The opioid receptor agonist and antagonist activity of cyclo[Pro-Sar-Phe-d-Phe] was characterized after i.c.v. and i.p. administration to C57BL/6J or transgenic opioid receptor "knockout" mice using the 55°C warm-water tail-withdrawal assay. Liabilities of locomotor coordination, respiration and spontaneous ambulation, and direct rewarding or aversive properties were assessed. Finally, the ability of cyclo[Pro-Sar-Phe-d-Phe] to block morphine- and stress-induced reinstatement of extinguished CPP was determined., Key Results: cyclo[Pro-Sar-Phe-d-Phe] demonstrated dose-dependent, short-lasting antinociception, with an ED 50 (and 95% confidence interval) of 0.15 (0.05-0.21) nmol i.c.v. and 1.91 (0.40-3.54) mg·kg -1 i.p., mediated by μ- and κ-opioid receptors. The macrocyclic tetrapeptide also demonstrated potent dose-dependent κ-opioid receptor antagonist-like activity at 2.5, but not at 4.5, h after administration. cyclo[Pro-Sar-Phe-d-Phe] displayed reduced liabiities compared with morphine, attributed to its additional activity at κ-receptors. Pretreatment with cyclo[Pro-Sar-Phe-d-Phe] prevented stress- and drug-induced reinstatement of extinguished morphine-place preference responses in a time-dependent manner., Conclusions and Implications: These data suggest that cyclo[Pro-Sar-Phe-d-Phe] is a promising lead compound for both the treatment of pain with reduced sideeffects and preventing both drug- and stress-induced relapse in morphine-abstinent subjects., (© 2020 The British Pharmacological Society.)

Substance abuse remains a serious public health crisis, affecting millions of people worldwide. Macrocyclic tetrapeptides like CJ-15,208 and [d-Trp]CJ-15,208 demonstrate opioid activity shown to attenuate the rewarding effects of cocaine in conditioned place preference assays in mice, making them promising lead compounds for treating substance abuse. In the present study, we report the rational design, synthesis, conformational analysis, and continued pharmacological evaluation of the novel macrocyclic tetrapeptide cyclo [Pro-Sar-Phe-d-Phe] to further explore this unique molecular scaffold. This peptide was rationally designed based on X-ray and NMR structures of related macrocyclic tetrapeptides. Following synthesis, its solution-phase conformations were determined by NMR and molecular modeling. The peptide adopted multiple conformations in polar solvents, but a single conformation in chloroform that is stabilized by intramolecular hydrogen bonding. The peptide is orally bioavailable, producing antinociception and antagonism of kappa opioid receptor (KOR) stimulation following oral administration in a mouse 55 °C warm-water tail-withdrawal assay. Notably, cyclo [Pro-Sar-Phe-d-Phe] blocked both stress- and drug-induced reinstatement of cocaine and morphine conditioned place preference in mice following oral administration, and displayed a decreased side-effect profile compared to morphine. Thus, cyclo [Pro-Sar-Phe-d-Phe] is a promising lead compound for the treatment of substance abuse.

We are exploring constraining aromatic residues in the kappa opioid receptor selective antagonist arodyn (Ac[Phe 1,2,3 ,Arg 4 ,d-Ala 8 ]dynorphin A(1-11)-NH 2 ) by ring closing metathesis (RCM) involving tyrosine( O -allyl) (Tyr(All)), but desallyl products limited the yields of the desired cyclic peptide. The model dipeptide Fmoc-Tyr(All)-Tyr(All) was used to explore different reaction conditions, including the use of isomerization suppressants, to minimize formation of the desallyl products and enhance formation of the desired RCM product. Reaction conditions were identified that enhanced the RCM product yield while suppressing desallyl products using both second-generation Grubbs and second-generation Hoveyda-Grubbs catalysts. These optimized reaction conditions were then applied to the cyclization of a tripeptide and an arodyn analog resulting in ≥70% conversion to the desired cyclic peptides. These strategies should be applicable to RCM involving Tyr(All) and similar residues in peptide and peptidomimetic cyclizations performed on solid phase.

Kappa (κ) opioid receptor selective antagonists are useful pharmacological tools in studying κ opioid receptors and have potential to be used as therapeutic agents for the treatment of a variety of diseases including mood disorders and drug addiction. Arodyn (Ac[Phe 1-3 ,Arg 4 ,d-Ala 8 ]Dyn A-(1-11)NH 2 ) is a linear acetylated dynorphin A (Dyn A) analog that is a potent and selective κ opioid receptor antagonist (Bennett et al. J Med Chem 2002;45:5617-5619) and prevents stress-induced reinstatement of cocaine-seeking behavior following central administration (Carey et al. Eur J Pharmacol 2007;569:84-89). To restrict its conformational mobility, explore possible bioactive conformations and potentially increase its metabolic stability we synthesized cyclic arodyn analogs on solid phase utilizing a novel ring-closing metathesis (RCM) reaction involving allyl-protected Tyr (Tyr(All)) residues. This approach preserves the aromatic functionality and directly constrains the side chains of one or more of the Phe residues. The novel cyclic arodyn analog 4 cyclized between Tyr(All) residues incorporated in positions 2 and 3 exhibited potent κ opioid receptor antagonism in the [ 35 S]GTPγS assay (K B  = 3.2 nM) similar to arodyn. Analog 3 cyclized between Tyr(All) residues in positions 1 and 2 also exhibited nanomolar κ opioid receptor antagonist potency (K B  = 27.5 nM) in this assay. These are the first opioid peptides cyclized via RCM involving aromatic residues, and given their promising pharmacological activity represent novel lead peptides for further exploration., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

To date structure-activity relationship (SAR) studies of the dynorphins (Dyn), endogenous peptides for kappa opioid receptors (KOR), have focused almost exclusively on Dyn A with minimal studies on Dyn B. While both Dyn A and Dyn B have identical N-terminal sequences, their C-terminal sequences differ, which could result in differences in pharmacological activity. We performed an alanine scan of the non-glycine residues up through residue 11 of Dyn B amide to explore the roles of these side chains in the activity of Dyn B. The analogs were synthesized by fluorenylmethyloxycarbonyl (Fmoc)-based solid phase peptide synthesis and evaluated for their opioid receptor affinities and opioid potency and efficacy at KOR. Similar to Dyn A the N-terminal Tyr 1 and Phe 4 residues of Dyn B amide are critical for opioid receptor affinity and KOR agonist potency. The basic residues Arg 6 and Arg 7 contribute to the KOR affinity and agonist potency of Dyn B amide, while Lys 10 contributes to the opioid receptor affinity, but not KOR agonist potency, of this peptide. Comparison to the Ala analogs of Dyn A (1-13) suggests that the basic residues in the C-terminus of both peptides contribute to KOR binding, but differences in their relative positions may contribute to the different pharmacological profiles of Dyn A and Dyn B. The other unique C-terminal residues in Dyn B amide also appear to influence the relative affinity of this peptide for KOR versus mu and delta opioid receptors. This SAR information may be applied in the design of new Dyn B analogs that could be useful pharmacological tools., (© 2017 Wiley Periodicals, Inc.)

The oncoprotein c-Myc is often overexpressed in cancer cells, and the stability of this protein has major significance in deciding the fate of a cell. Thus, targeting c-Myc levels is an attractive approach for developing therapeutic agents for cancer treatment. In this study, we report the anti-cancer activity of the macrocyclic peptides [D-Trp]CJ-15,208 (cyclo[Phe-D-Pro-Phe-D-Trp]) and the natural product CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]). [D-Trp]CJ-15,208 reduced c-Myc protein levels in prostate cancer cells and decreased cell proliferation with IC 50 values ranging from 2.0 to 16 µM in multiple PC cell lines. [D-Trp]CJ-15,208 induced early and late apoptosis in PC-3 cells following 48 hours treatment, and growth arrest in the G2 cell cycle phase following both 24 and 48 hours treatment. Down regulation of c-Myc in PC-3 cells resulted in loss of sensitivity to [D-Trp]CJ-15,208 treatment, while overexpression of c-Myc in HEK-293 cells imparted sensitivity of these cells to [D-Trp]CJ-15,208 treatment. This macrocyclic tetrapeptide also regulated PP2A by reducing the levels of its phosphorylated form which regulates the stability of cellular c-Myc protein. Thus [D-Trp]CJ-15,208 represents a new lead compound for the potential development of an effective treatment of prostate cancer.

Selective kappa opioid receptor (KOR) antagonists may have therapeutic potential as treatments for substance abuse and mood disorders. Since [D-Trp]CJ-15,208 (cyclo[Phe-d-Pro-Phe-d-Trp]) is a novel potent KOR antagonist in vivo, it is imperative to evaluate its pharmacokinetic properties to assist the development of analogs as potential therapeutic agents, necessitating the development and validation of a quantitative method for determining its plasma levels. A method for quantifying [D-Trp]CJ-15,208 was developed employing high performance liquid chromatography-tandem mass spectrometry in mouse plasma. Sample preparation was accomplished through a simple one-step protein precipitation method with acetonitrile, and [D-Trp]CJ-15,208 analyzed following HPLC separation on a Hypersil BDS C8 column. Multiple reaction monitoring (MRM), based on the transitions m/z 578.1→217.1 and 245.0, was specific for [D-Trp]CJ-15,208, and MRM based on the transition m/z 566.2→232.9 was specific for the internal standard without interference from endogenous substances in blank mouse plasma. The assay was linear over the concentration range 0.5-500ng/mL with a mean r(2)=0.9987. The mean inter-day accuracy and precision for all calibration standards were 93-118% and 8.9%, respectively. The absolute recoveries were 85±6% and 81±9% for [D-Trp]CJ-15,208 and the internal standard, respectively. The analytical method had excellent sensitivity with a lower limit of quantification of 0.5ng/mL using a sample volume of 20μL. The method was successfully applied to an initial pharmacokinetic study of [D-Trp]CJ-15,208 following intravenous administration to mice., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We have shown that dysregulation of the dynorphin/kappa-opioid receptor (DYN/KOR) system contributes to escalated alcohol self-administration in alcohol dependence and that KOR antagonists with extended durations of action selectively reduce escalated alcohol consumption in alcohol-dependent animals. As KOR antagonism has gained widespread attention as a potential therapeutic target to treat alcoholism and multiple neuropsychiatric disorders, we tested the effect of zyklophin (a short-acting KOR antagonist) on escalated alcohol self-administration in rats made alcohol-dependent using intermittent alcohol vapor exposure. Following dependence induction, zyklophin was infused centrally prior to alcohol self-administration sessions and locomotor activity tests during acute withdrawal. Zyklophin did not impact alcohol self-administration or locomotor activity in either exposure condition. To investigate the neurobiological basis of this atypical effect for a KOR antagonist, we utilized a κ-, μ-, and δ-opioid receptor agonist-stimulated GTPyS coupling assay to examine the opioid receptor specificity of zyklophin in the rat brain and mouse brain. In rats, zyklophin did not affect U50488-, DAMGO-, or DADLE-stimulated GTPyS coupling, whereas the prototypical KOR antagonist nor-binaltorphimine (norBNI) attenuated U50488-induced stimulation in the rat brain tissue at concentrations that did not impact μ- and δ-receptor function. To reconcile the discrepancy between the present rat data and published mouse data, comparable GTPyS assays were conducted using mouse brain tissue; zyklophin effects were consistent with KOR antagonism in mice. Moreover, at higher concentrations, zyklophin exhibited agonist properties in rat and mouse brains. These results identify species differences in zyklophin efficacy that, given the rising interest in the development of short-duration KOR antagonists, should provide valuable information for therapeutic development efforts., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Prototypical long-acting kappa opioid receptor (KOPR) antagonists [e.g., norbinaltorphimine (norBNI)] have been reported to exert anxiolytic-like effects in several commonly used anxiety tests in rodents including the novelty-induced hypophagia (NIH) and elevated plus maze (EPM) tests. It remains unknown if the short-acting KOPR antagonists (e.g., zyklophin and LY2444296) have similar effects. In this study effects of zyklophin and LY2444296 (s.c.) were investigated in the NIH and EPM tests in mice 1h post-injection and compared with norBNI (i.p.) 48h post-administration. In the NIH test, zyklophin at 3 and 1mg/kg, but not 0.3mg/kg, or LY2444296 at 30mg/kg decreased the latency of palatable food consumption in novel cages, but had no effect in training cages, similar to norBNI (10mg/kg). Zyklophin at 3 or 1mg/kg increased or had a trend of increasing the amount of palatable food consumption in novel cages, with no effects in training cages, further indicating its anxiolytic-like effect, but norBNI (10mg/kg) and LY2444296 (30mg/kg) did not. In the EPM test, norBNI (10mg/kg) increased open arm time and % open arm entries or time, but zyklophin at all three doses and LY2444296 (30mg/kg) had no effects. In addition, zyklophin at 3mg/kg increased numbers of close and total arm entries on EPM, suggesting increased activity; however, norBNI and LY2444296 had no effects on close and total arm entries. Thus, all three KOPR antagonists had anxiolytic-like effects in the NIH test. However, only the long-acting one (norBNI), but not the short-acting ones (zyklophin and LY2444296), demonstrated anti-anxiety like effects in the EPM test. It remains to be investigated if the differences are due to the differences in their durations of action and/or pharmacodynamic properties., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The dynorphin (Dyn) A analogue zyklophin ([N-benzyl-Tyr(1)-cyclo(d-Asp(5),Dap(8))]dynorphin A(1-11)NH2) is a kappa opioid receptor (KOR)-selective antagonist in vitro, is active in vivo, and antagonizes KOR in the CNS after systemic administration. Hence, we synthesized zyklophin analogues to explore the structure-activity relationships of this peptide. The synthesis of selected analogues required modification to introduce the N-terminal amino acid due to poor solubility and/or to avoid epimerization of this residue. Among the N-terminal modifications, the N-phenethyl and N-cyclopropylmethyl substitutions resulted in analogues with the highest KOR affinities. Pharmacological results for the alanine-substituted analogues indicated that Phe(4) and Arg(6), but interestingly not the Tyr(1) phenol, are important for zyklophin's KOR affinity and that Arg(7) was important for KOR antagonist activity. In the GTPγS assay, while all of the cyclic analogues exhibited negligible KOR efficacy, the N-cyclopropylmethyl-Tyr(1) and N-benzyl-Phe(1) analogues were 28- and 11-fold more potent KOR antagonists, respectively, than zyklophin.

Background and Purpose: The novel macrocyclic peptide cyclo[Phe-D-Pro-Phe-D-Trp] ([D-Trp]CJ-15,208) exhibits κ opioid (KOP) receptor antagonist activity in both in vitro and in vivo assays. The four alanine analogues of this peptide were synthesized and characterized both in vitro and in vivo to assess the contribution of different amino acid residues to the activity of [D-Trp]CJ-15,208., Experimental Approach: The peptides were synthesized by a combination of solid phase peptide synthesis and cyclization in solution. The analogues were evaluated in vitro in receptor binding and functional assays, and in vivo with mice using a tail-withdrawal assay for antinociceptive and opioid antagonist activity. Mice demonstrating extinction of cocaine conditioned-place preference (CPP) were pretreated with selected analogues to evaluate prevention of stress or cocaine-induced reinstatement of CPP., Key Results: The alanine analogues displayed pharmacological profiles in vivo distinctly different from [D-Trp]CJ-15,208. While the analogues exhibited varying opioid receptor affinities and κ and μ opioid receptor antagonist activity in vitro, they produced potent opioid receptor-mediated antinociception (ED50 = 0.28-4.19 nmol, i.c.v.) in vivo. Three of the analogues also displayed KOP receptor antagonist activity in vivo. Pretreatment with an analogue exhibiting both KOP receptor agonist and antagonist activity in vivo prevented both cocaine- and stress-induced reinstatement of cocaine-seeking behaviour in the CPP assay in a time-dependent manner., Conclusions and Implications: These unusual macrocyclic peptides exhibit in vivo opioid activity profiles different from the parent compound and represent novel compounds for potential development as therapeutics for drug abuse and possibly as analgesics., (© 2014 The British Pharmacological Society.)

It has been shown previously that norbinaltorphimine (norBNI) and 5'-guanidinonaltrindole (5'-GNTI), long-acting kappa opioid receptor (KOPR) antagonists, cause frenzied scratching in mice [1,2]. In the current study, we examined if zyklophin, a short-acting cyclic peptide KOPR antagonist, also elicited scratching behavior. When injected s.c. in the nape of the neck of male Swiss-Webster mice, zyklophin at doses of 0.1, 0.3 and 1mg/kg induced dose-related hindleg scratching of the neck between 3 and 15 min after injection. Pretreating mice with norBNI (20mg/kg, i.p.) at 18-20 h before challenge with zyklophin (0.3mg/kg) did not markedly affect scratching. Additionally, KOPR-/- mice given 0.3mg/kg of zyklophin displayed similar levels of scratching as wild-type animals. The absence of KOPR in KOPR-/- mice was confirmed with ex vivo radioligand binding using [(3)H]U69,593. Taken together, our data suggest that the presence of kappa receptors is not required for the excessive scratching caused by zyklophin. Thus, zyklophin, similar to the structurally different KOPR antagonist 5'-GNTI, appears to act at other targets to elicit scratching and potentially the sensation of itch., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Inhibitor 2 of protein phosphatase 2A (I2PP2A), a biological inhibitor of the cellular serine/threonine protein phosphatase PP2A, is associated with numerous cellular processes that often lead to the formation and progression of cancer. In this study we hypothesized that targeting the inhibition of I2PP2A's multiple functions in prostate cancer cells might prevent cancer progression. We have investigated the effect of the small chain C6-ceramide, known to be a bioactive tumor suppressor lipid, on I2PP2A function, thereby affecting c-Myc signaling and histone acetylation in cells. Our data indicated that C6-ceramide treatment of prostate cancer cells induces cell death in PC-3, DU145, and LNCaP cells, but not normal prostate epithelial cells. C6-ceramide was able to disrupt the association between PP2A and I2PP2A. C6-ceramide inhibits I2PP2A's upregulation of c-Myc and downregulation of histone acetylation in prostate cancer cells. Our data indicated that targeting cancer related signaling pathways through I2PP2A using ceramide as an anti-I2PP2A agent could have beneficial effects as a therapeutic approach to prevent prostate cancer.

Background and Purpose: Cyclic peptides are resistant to proteolytic cleavage, therefore potentially exhibiting activity after systemic administration. We hypothesized that the macrocyclic κ opioid receptor (KOR)-selective antagonist [D-Trp]CJ-15,208 would demonstrate antagonist activity after systemic, that is, s.c. and oral (per os, p. o.), administration., Experimental Approach: C57BL/6J mice were pretreated with [D-Trp]CJ-15,208 s.c. or p.o. before administration of the KOR-selective agonist U50,488 and the determination of antinociception in the warm-water tail-withdrawal assay. The locomotor activity of mice treated with [D-Trp]CJ-15,208 was determined by rotorod testing. Additional mice demonstrating cocaine conditioned place preference and subsequent extinction were pretreated daily with vehicle or [D-Trp]CJ-15,208 and then exposed to repeated forced swim stress or a single additional session of cocaine place conditioning before redetermining place preference., Key Results: Pretreatment with [D-Trp]CJ-15,208 administered s.c. or p.o. dose-dependently antagonized the antinociception induced by i.p. administration of U50,488 in mice tested in the warm-water tail-withdrawal assay for less than 12 and 6 h respectively. [D-Trp]CJ-15,208 also produced limited (<25%), short-duration antinociception mediated through KOR agonism. Orally administered [D-Trp]CJ-15,208 dose-dependently antagonized centrally administered U50,488-induced antinociception, and prevented stress-, but not cocaine-induced, reinstatement of extinguished cocaine-seeking behaviour, consistent with its KOR antagonist activity, without affecting locomotor activity., Conclusions and Implications: The macrocyclic tetrapeptide [D-Trp]CJ-15,208 is a short-duration KOR antagonist with weak KOR agonist activity that is active after oral administration and demonstrates blood-brain barrier permeability. These data validate the use of systemically active peptides such as [D-Trp]CJ-15,208 as potentially useful therapeutics., (© 2013 The Authors. British Journal of Pharmacology © 2013 The British Pharmacological Society.)

The macrocyclic tetrapeptide natural product CJ-15,208 (cyclo[Phe-d-Pro-Phe-Trp]) exhibited both dose-dependent antinociception and kappa opioid receptor (KOR) antagonist activity after oral administration. CJ-15,208 antagonized a centrally administered KOR selective agonist, providing strong evidence it crosses the blood-brain barrier to reach KOR in the CNS. Orally administered CJ-15,208 also prevented both cocaine- and stress-induced reinstatement of extinguished cocaine-seeking behavior in the conditioned place preference assay in a time- and dose-dependent manner. Thus, CJ-15,208 is a promising lead compound with a unique activity profile for potential development, particularly as a therapeutic to prevent relapse to drug-seeking behavior in abstinent subjects.

Dynorphin A 1-17 (Dyn A 1-17) is an endogenous neuropeptide known to act at the kappa opioid receptor; it has been implicated in a number of neurological disorders, including neuropathic pain, stress, depression, and Alzheimer's and Parkinson's diseases. The investigation of Dyn A 1-17 metabolism at the blood-brain barrier (BBB) is important since the metabolites exhibit unique biological functions compared to the parent compound. In this work, Dyn A 1-6 is identified as a metabolite of Dyn A 1-17 in the presence of bovine brain microvessel endhothelial cells (BBMECs), using LC-MS/MS. The transport of Dyn A 1-6 at the BBB was examined using this in vitro cell culture model of the BBB. Furthermore, the permeation of the BBB by the low molecular weight permeability marker fluorescein was characterized in the presence and absences of Dyn A 1-6., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Opioid receptors are important targets for the treatment of pain and potentially for other disease states (e.g. mood disorders and drug abuse) as well. Significant recent advances have been made in identifying opioid peptide analogs that exhibit promising in vivo activity for treatment of these maladies. This review focuses on the development and evaluation of opioid peptide analogs demonstrating activity after systemic administration, and recent clinical evaluations of opioid peptides for possible therapeutic use.

The blood-brain barrier (BBB) is an important interface between the peripheral and central nervous systems. It protects the brain against the infiltration of harmful substances and regulates the permeation of beneficial endogenous substances from the blood into the extracellular fluid of the brain. It can also present a major obstacle in the development of drugs that are targeted for the central nervous system. Several methods have been developed to investigate the transport and metabolism of drugs, peptides, and endogenous compounds at the BBB. In vivo methods include intravenous injection, brain perfusion, positron emission tomography, and microdialysis sampling. Researchers have also developed in vitro cell-culture models that can be employed to investigate transport and metabolism at the BBB without the complication of systemic involvement. All these methods require sensitive and selective analytical methods to monitor the transport and metabolism of the compounds of interest at the BBB.

An alanine scan was performed on the novel κ opioid receptor (KOR) peptide ligand CJ-15,208 to determine which residues contribute to the potent in vivo agonist activity observed for the parent peptide. These cyclic tetrapeptides were synthesized by a combination of solid-phase peptide synthesis of the linear precursors, followed by cyclization in solution. Like the parent peptide, each of the analogues exhibited agonist activity and KOR antagonist activity in an antinociceptive assay in vivo. Unlike the parent peptide, the agonist activity of the potent analogues was mediated predominantly, if not exclusively, by μ opioid receptors (MOR). Thus analogues 2 and 4, in which one of the phenylalanine residues was replaced by alanine, exhibited both potent MOR agonist activity and KOR antagonist activity in vivo. These peptides represent novel lead compounds for the development of peptide-based opioid analgesics., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mouse embryonic spinal cord neurons in culture exhibit spontaneous calcium oscillations from day in vitro (DIV) 6 through DIV 10. Such spontaneous activity in developing spinal cord contributes to maturation of synapses and development of pattern-generating circuits. Here we demonstrate that these calcium oscillations are regulated by κ opioid receptors (KORs). The κ opioid agonist dynorphin (Dyn)-A (1-13) suppressed calcium oscillations in a concentration-dependent manner, and both the nonselective opioid antagonist naloxone and the κ-selective blocker norbinaltorphimine eliminated this effect. The KOR-selective agonist (+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U69593) mimicked the effect of Dyn-A (1-13) on calcium oscillations. A κ-specific peptide antagonist, zyklophin, was also able to prevent the suppression of calcium oscillations caused by Dyn-A (1-13). These spontaneous calcium oscillations were blocked by 1 μM tetrodotoxin, indicating that they are action potential-dependent. Although the L-type voltage-gated calcium channel blocker nifedipine did not suppress calcium oscillations, the N-type calcium channel blocker ω-conotoxin inhibited this spontaneous response. Blockers of ionotropic glutamate receptors, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline and dizocilpine maleate (MK-801), also suppressed calcium oscillations, revealing a dependence on glutamate-mediated signaling. Finally, we have demonstrated expression of KORs in glutamatergic spinal neurons and localization in a presynaptic compartment, consistent with previous reports of KOR-mediated inhibition of glutamate release. The KOR-mediated inhibition of spontaneous calcium oscillations may therefore be a consequence of presynaptic inhibition of glutamate release.

Arodyn (Ac[Phe¹,²,³,Arg⁴,D-Ala⁸]Dyn A(1-11)NH₂) is an acetylated dynorphin A (Dyn A) analog that is a potent and selective κ opioid receptor antagonist (Bennett et al., J Med Chem 2002, 45, 5617), and its analog [NMePhe¹]arodyn shows even higher affinity and selectivity for κ opioid receptors (Bennett et al., J Pept Res 2005, 65, 322). However, the latter compound is prone to deletion of the Ac-NMePhe moiety from the N-terminus of the peptide during acidic cleavage as described in the accompanying paper. Several stable analogs of [NMePhe¹]arodyn and [NMePhe¹,Trp³]arodyn where the acetyl group was substituted with a heteroatom-containing group were evaluated for their opioid receptor affinity, selectivity, and efficacy. Methoxycarbonyl derivatives exhibited the highest κ opioid receptor affinity among the analogs. Additional [CH₃OCO-NMePhe¹]arodyn analogs where position 3 was substituted with other aromatic or nonaromatic residues were also evaluated for κ receptor affinity, selectivity, and efficacy. [CH₃OCO-NMePhe¹]arodyn has similar κ opioid receptor affinity as [NMePhe¹]arodyn, retains high κ opioid receptor selectivity, and is a potent κ opioid receptor antagonist.

Peptide C-terminal N-alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C-terminal N-alkyl amides using the well-known Fukuyama N-alkylation reaction on a standard resin commonly used for the synthesis of peptide C-terminal primary amides, the peptide amide linker-polyethylene glycol-polystyrene (PAL-PEG-PS) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N-alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N-alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N-alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N-alkyl amides., (Copyright © 2011 Wiley Periodicals, Inc.)

Peptides containing N-methylamino acids can exhibit improved pharmacodynamic and pharmacokinetic profiles compared to nonmethylated peptides, and therefore interest in these N-methylated peptides has been increasing in recent years. Arodyn (Ac[Phe¹,²,³,Arg⁴,D-Ala⁸]Dyn A(1-11)NH₂) is an acetylated dynorphin A(Dyn A) analog that is a potent and selective κ opioid receptor antagonist (Bennett et al., J Med Chem 2002, 45, 5617), and its analog [NMePhe¹]arodyn shows even higher affinity and selectivity for κ opioid receptors (Bennett et al., J Pept Res 2005, 65, 322). During the synthesis of [NMePhe¹]arodyn analogs, the arodyn-(2-11) derivatives were obtained as major products. Analysis indicated that Ac-NMePhe was lost from the completed peptide sequence during acidic cleavage of the peptides from the resin and that the acetyl group played an important role in this side reaction. Different cleavage conditions were evaluated to minimize this side reaction and maximize the yield of pure [NMePhe¹]arodyn analogs. Modifications to the N-terminus of the peptides to prevent the side reaction were also explored. The incorporation of a heteroatom-containing group such as methoxycarbonyl as the N-terminal functionality prevented this side reaction, while the incorporation of a bulky acyl group could not. Substituting NMePhe with the conformationally constrained analog Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) also prevented the side reaction.

The tryptophan isomers of the cyclic tetrapeptide CJ-15,208, reported to be a kappa opioid receptor (KOR) antagonist [Saito, T.; Hirai, H.; Kim, Y. J.; Kojima, Y.; Matsunaga, Y.; Nishida, H.; Sakakibara, T.; Suga, O.; Sujaku, T.; Kojima, N. J. Antibiot. (Tokyo)2002, 55, 847-854.], were synthesized to determine the tryptophan stereochemistry in the natural product. A strategy was developed to select linear precursor peptides that favor cyclization using molecular modeling, and optimized cyclization conditions are reported. The optical rotation of the l-Trp isomer is consistent with that of the natural product. Unexpectedly both isomers exhibit similar nanomolar affinity for KOR.

A series of potent electrophilic affinity labels (IC(50) = 0.1-5 nM) containing either a bromoacetamide or isothiocyanate based on the mu opioid receptor (MOR) selective peptide dermorphin were prepared. All four analogues exhibited wash resistant inhibition of [(3)H]DAMGO binding at subnanomolar to nanomolar concentrations, suggesting that these analogues bind covalently to MOR. To our knowledge, these peptides are the highest affinity peptide-based affinity labels for MOR reported to date.

Structural modifications affecting the efficacy of analogues of the endogenous opioid peptide dynorphin (Dyn) A have focused on the N-terminal "message" sequence based on the "message-address" concept. To test the hypothesis that changes in the C-terminal "address" domain could affect efficacy, modified amino acids and cyclic constraints were incorporated into this region of the partial agonist [N-benzylTyr(1)]Dyn A-(1-11). Modifications in the C-terminal domain of [N-benzylTyr(1)]Dyn A-(1-11)NH(2) resulted in increased kappa opioid receptor (KOR) affinity for all of the linear analogues but did not affect the efficacy of these peptides at KOR. Cyclization between positions 5 and 8 yielded [N-benzylTyr(1),cyclo(d-Asp(5),Dap(8))]Dyn A-(1-11)NH(2) (zyklophin, 13) ( J. Med. Chem. 2005 , 48 , 4500 - 4503 ) with high selectivity for KOR. In contrast to the linear peptides, this peptide exhibits negligible efficacy in the adenylyl cyclase (AC) assay and is a KOR antagonist. These data are consistent with our hypothesis that appropriate modifications in the "address" domain of Dyn A analogues may affect efficacy.

The cyclic peptide zyklophin {[N-benzylTyr(1),cyclo(D-Asp(5),Dap(8))-dynorphin A-(1-11)NH(2), Patkar KA, et al. (2005) J Med Chem 48: 4500-4503} is a selective peptide kappa opioid receptor (KOR) antagonist that shows activity following systemic administration. Systemic (1-3 mg/kg s.c.) as well as central (0.3-3 nmol intracerebroventricular, i.c.v.) administration of this peptide dose-dependently antagonizes the antinociception induced by the selective KOR agonist U50,488 in C57BL/6J mice tested in the 55 degrees C warm water tail withdrawal assay. Zyklophin administration had no effect on morphine- or SNC-80-mediated antinociception, suggesting that zyklophin selectively antagonizes KOR in vivo. Additionally, the antagonism of antinociception induced by centrally (i.c.v.) administered U50,488 following peripheral administration of zyklophin strongly suggests that the peptide crosses the blood-brain barrier to antagonize KOR in the CNS. Most importantly, the antagonist activity of zyklophin (3 mg/kg s.c.) lasts less than 12 h, which contrasts sharply with the exceptionally long duration of antagonism reported for the established small-molecule selective KOR antagonists such as nor-binaltorphimine (nor-BNI) that last weeks after a single administration. Systemically administered zyklophin (3 mg/kg s.c.) also prevented stress-induced reinstatement of cocaine-seeking behavior in a conditioned place preference assay. In conclusion, the peptide zyklophin is a KOR-selective antagonist that exhibits the desired shorter duration of action, and represents a significant advance in the development of KOR-selective antagonists.

Dynorphin A (Dyn A) is an endogenous ligand for kappa opioid receptors. To restrict the conformational mobility, we synthesized several cyclic Dyn A-(1-11)NH(2) analogues on solid phase utilizing ring-closing metathesis (RCM) between the side chains of allylglycine (AllGly) residues incorporated in positions 2, 5, and/or 8. Cyclizations between the side chains of AllGly gave reasonable yields (56-74%) of all of the desired cyclic peptides. Both the cis and trans isomers were obtained for all of the cyclic peptides, with the ratio of cis to trans isomers depending on the position and stereochemistry of the AllGly. Most of the cyclic Dyn A-(1-11)NH(2) analogues examined exhibit low nanomolar binding affinity for kappa opioid receptors (K(i) = 0.84-11 nM). In two of the three cases, the configuration of the double bond has a significant influence on the opioid receptor affinities and agonist potency. All of the peptides inhibited adenylyl cyclase activity in a concentration-dependent manner with full or close to full agonist activity. These potent Dyn A analogues are the first ones cyclized by RCM.

While narcotic analgesics such as morphine, which act preferentially through mu opioid receptors, remain the gold standard in the treatment of severe pain, their use is limited by detrimental liabilities such as respiratory depression and drug dependence. Thus, there has been considerable interest in developing ligands for kappa opioid receptors (KOR) as potential analgesics and for the treatment of a variety of other disorders. These include effects mediated both by central receptors, such as antidepressant activity and a reduction in cocaine-seeking behavior, and activity resulting from the activation of peripheral receptors, such as analgesic and anti-inflammatory effects. While the vast majority of opioid receptor ligands that have progressed in preclinical development have been small molecules, significant advances have been made in recent years in identifying opioid peptide analogs that exhibit promising in vivo activity. This review will focus on possible therapeutic applications of ligands for KOR and specifically on the potential development of peptide ligands for these receptors.

Herein, we describe a general strategy for the facile synthesis of a multifunctional amino acid derivative bearing both fluorescent and photolabile groups such as the lysine derivative NvocLys(CO(CH2)5NH-NBD)OCH2CN (1) that can be used as a biophysical tool for studying protein structure. The synthetic strategy involves functionalization of the amine groups while the amino acid is attached to a solid support, followed by esterification of the carboxylic acid in solution. The solid support protects the caboxylic acid, preventing a side reaction associated with the synthesis in solution and obviating the need for chromatographic purification of several intermediates. This synthetic strategy can be used for the preparation of a variety of amino acid derivatives with unusual alpha-amine and side chain functionalities.

A general strategy for the design of dual labeled peptides was developed, and derivatives of the delta opioid receptor (DOR) selective antagonist TIPP (Tyr-Tic-Phe-PheOH) containing both an affinity label and biotin were prepared by solid-phase synthesis. Tyr-Tic-Phe-Phe(p-X)-Asp-NH(CH2CH2O)2-CH2CH2NH-biotin (where X = N=C=S or NHCOCH2Br) exhibit nanomolar DOR affinity. The ability to detect receptors labeled with these peptides following solubilization and SDS-PAGE demonstrate the applicability of this design approach for dual labeled peptide derivatives.

Solid phase synthetic methodology has been developed in our laboratory to incorporate an affinity label (a reactive functionality such as isothiocyanate or bromoacetamide) into peptides (Leelasvatanakij, L. and Aldrich, J. V. (2000) J. Peptide Res. 56, 80), and we have used this synthetic strategy to prepare affinity label derivatives of a variety of opioid peptides. To date side reactions have been detected only in two cases, both involving intramolecular cyclization. We have identified several peptide-based affinity labels for delta opioid receptors that exhibit wash-resistant inhibition of binding to these receptors and are valuable pharmacological tools to study opioid receptors. Even in cases where the peptide derivatives do not bind covalently to their target receptor, studying their binding has revealed subtle differences in receptor interactions with particular opioid peptide residues, especially Phe residues in the N-terminal "message" sequences. Solid phase synthetic methodology for the incorporation of other labels (e.g. biotin) into the C-terminus of peptides has also been developed in our laboratory (Kumar, V. and Aldrich, J. V. (2003) Org. Lett. 5, 613). These two synthetic approaches have been combined to prepare peptides containing multiple labels that can be used as tools to study peptide ligand-receptor interactions. These solid phase synthetic methodologies are versatile strategies that are applicable to the preparation of labeled peptides for a variety of targets in addition to opioid receptors.

A series of cyclic analogues with a lactam linkage were prepared by solid phase peptide synthesis to explore possible biologically active conformation(s) of nociceptin/orphanin FQ (N/OFQ). cyclo[D-Asp(7),Lys(10)]- and cyclo[Asp (6),Lys(10)]N/OFQ(1-13)NH2 exhibit high affinity (Ki = 0.27 and 0.34 nM, respectively) and high potency in the GTPgammaS assay (EC 50 = 1.6 and 4.1 nM, respectively) at human nociceptin/orphanin FQ peptide (NOP) receptors. These analogues exhibit 2- to 3-fold higher affinity and 2- to 5-fold higher potency than the parent peptide.

Stress contributes to the reinstatement of cocaine-seeking behavior in abstinent subjects. Kappa-opioid receptor antagonists attenuate the behavioral effects of stress, potentially providing therapeutic value in treating cocaine abuse. Presently, the peptide arodyn produced long-lasting kappa-opioid receptor antagonism, suppressing kappa-opioid receptor agonist-induced antinociception at least 3 days after intracerebroventricular administration of 0.3 nmol. C57Bl/6J mice demonstrated cocaine-conditioned place preference, extinction over 3 weeks, and a subsequent reinstatement of place preference. Arodyn pretreatment suppressed stress-induced, but not cocaine-exposed, reinstatement of cocaine place preference. These results verify that arodyn and other kappa-opioid receptor antagonists may be useful therapeutics for cocaine abuse.

Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.