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1.  Synthesis and evaluation of 4-substituted piperidines and piperazines as balanced affinity µ opioid receptor (MOR) agonist/δ opioid receptor (DOR) antagonist ligands 
In this report, we describe a series of 4-substituted piperidine and piperazine compounds based on tetrahydroquinoline 1, a compound that shows balanced, low nanomolar binding affinity for the mu opioid receptor (MOR) and the delta opioid receptor (DOR). We have shown that by changing the length and flexibility profile of the side chain in this position, binding affinity is improved at both receptors by a significant degree. Furthermore, several of the compounds described herein display good efficacy at MOR, while simultaneously displaying DOR antagonism. The MOR agonist/DOR antagonist has shown promise in the reduction of negative side effects displayed by selective MOR agonists, namely the development of dependence and tolerance.
doi:10.1016/j.bmcl.2013.12.021
PMCID: PMC3919453  PMID: 24365161
2.  Translation of Structure-Activity Relationships from Cyclic Mixed Efficacy Opioid Peptides to Linear Analogues 
Biopolymers  2014;102(1):107-114.
Most opioid analgesics used in the treatment of pain are mu opioid receptor (MOR) agonists. While effective, there are significant drawbacks to opioid use, including the development of tolerance and dependence. However, the co-administration of a MOR agonist with a delta opioid receptor (DOR) antagonist slows the development of MOR-related side effects, while maintaining analgesia. We have previously reported a series of cyclic mixed efficacy MOR agonist/DOR antagonist ligands. Here we describe the transfer of key features from these cyclic analogs to linear sequences. Using the linear MOR/DOR agonist, Tyr-DThr-Gly-Phe-Leu-Ser-NH2 (DTLES), as a lead scaffold, we replaced Phe4 with bulkier and/or constrained aromatic residues shown to confer DOR antagonism in our cyclic ligands. These replacements failed to confer DOR antagonism in the DTLES analogs, presumably because the more flexible linear ligands can adopt binding poses that will fit in the narrow binding pocket of the active conformations of both MOR and DOR. Nonetheless, the pharmacological profile observed in this series, high affinity and efficacy for MOR and DOR with selectivity relative to KOR, has also been shown to reduce the development of unwanted side effects. We further modified our lead MOR/DOR agonist with a C-terminal glucoserine to improve bioavailability. The resulting ligand displayed high efficacy and potency at both MOR and DOR and no efficacy at KOR.
doi:10.1002/bip.22437
PMCID: PMC4132888  PMID: 24436042
3.  Exploring pharmacological activities and signaling of morphinans substituted in position 6 as potent agonists interacting with the μ opioid receptor 
Molecular Pain  2014;10:48.
Background
Opioid analgesics are the most effective drugs for the treatment of moderate to severe pain. However, they also produce several adverse effects that can complicate pain management. The μ opioid (MOP) receptor, a G protein-coupled receptor, is recognized as the opioid receptor type which primarily mediates the pharmacological actions of clinically used opioid agonists. The morphinan class of analgesics including morphine and oxycodone are of main importance as therapeutically valuable drugs. Though the natural alkaloid morphine contains a C-6-hydroxyl group and the semisynthetic derivative oxycodone has a 6-carbonyl function, chemical approaches have uncovered that functionalizing position 6 gives rise to a range of diverse activities. Hence, position 6 of N-methylmorphinans is one of the most manipulated sites, and is established to play a key role in ligand binding at the MOP receptor, efficacy, signaling, and analgesic potency. We have earlier reported on a chemically innovative modification in oxycodone resulting in novel morphinans with 6-acrylonitrile incorporated substructures.
Results
This study describes in vitro and in vivo pharmacological activities and signaling of new morphinans substituted in position 6 with acrylonitrile and amido functions as potent agonists and antinociceptive agents interacting with MOP receptors. We show that the presence of a 6-cyano group in N-methylmorphinans has a strong influence on the binding to the opioid receptors and post-receptor signaling. One 6-cyano-N-methylmorphinan of the series was identified as the highest affinity and most selective MOP agonist, and very potent in stimulating G protein coupling and intracellular calcium release through the MOP receptor. In vivo, this MOP agonist showed to be greatly effective against thermal and chemical nociception in mice with marked increased antinociceptive potency than the lead molecule oxycodone.
Conclusion
Development of such novel chemotypes by targeting position 6 provides valuable insights on ligand-receptor interaction and molecular mode of action, and may aid in identification of opioid therapeutics with enhanced analgesic properties and fewer undesirable effects.
doi:10.1186/1744-8069-10-48
PMCID: PMC4121618  PMID: 25059282
Opioid receptors; Agonist; Morphine; Oxycodone; Pain; Analgesia; Signaling; G protein; Calcium mobilization
4.  Differential Control of Opioid Antinociception to Thermal Stimuli in a Knock-In Mouse Expressing Regulator of G-Protein Signaling-Insensitive Gαo Protein 
Regulator of G-protein signaling (RGS) proteins classically function as negative modulators of G-protein-coupled receptor signaling. In vitro, RGS proteins have been shown to inhibit signaling by agonists at the μ-opioid receptor, including morphine. The goal of the present study was to evaluate the contribution of endogenous RGS proteins to the antinociceptive effects of morphine and other opioid agonists. To do this, a knock-in mouse that expresses an RGS-insensitive (RGSi) mutant Gαo protein, GαoG184S (Gαo RGSi), was evaluated for morphine or methadone antinociception in response to noxious thermal stimuli. Mice expressing Gαo RGSi subunits exhibited a naltrexone-sensitive enhancement of baseline latency in both the hot-plate and warm-water tail-withdrawal tests. In the hot-plate test, a measure of supraspinal nociception, morphine antinociception was increased, and this was associated with an increased ability of opioids to inhibit presynaptic GABA neurotransmission in the periaqueductal gray. In contrast, antinociception produced by either morphine or methadone was reduced in the tail-withdrawal test, a measure of spinal nociception. In whole-brain and spinal cord homogenates from mice expressing Gαo RGSi subunits, there was a small loss of Gαo expression and an accompanying decrease in basal G-protein activity. Our results strongly support a role for RGS proteins as negative regulators of opioid supraspinal antinociception and also reveal a potential novel function of RGS proteins as positive regulators of opioid spinal antinociceptive pathways.
doi:10.1523/JNEUROSCI.5470-12.2013
PMCID: PMC3740968  PMID: 23467353
5.  Opioid Peptidomimetics: Leads for the Design of Bioavailable Mixed Efficacy Mu Opioid Receptor (MOR) Agonist/Delta Opioid Receptor (DOR) Antagonist Ligands 
Journal of medicinal chemistry  2013;56(5):2139-2149.
We have previously described opioid peptidomimetic, 1, employing a tetrahydroquinoline scaffold and modeled on a series of cyclic tetrapeptide opioid agonists. We have recently described modifications to these peptides that confer a mu opioid receptor (MOR) agonist, delta opioid receptor (DOR) antagonist profile, which has been shown to reduce the development of tolerance to the analgesic actions of MOR agonists. Several such bifunctional ligands have been reported, but none has been demonstrated to cross the blood brain barrier. Here we describe the transfer of structural features that evoked MOR agonist/DOR antagonist behavior in the cyclic peptides to the tetrahydroquinoline scaffold and show that the resulting peptidomimetics maintain the desired pharmacological profile. Further, the 4R diastereomer of 1 was fully efficacious and approximately equipotent to morphine in the mouse warm water tail withdrawal assay following intraperitoneal administration and thus a promising lead for the development of opioid analgesics with reduced tolerance.
doi:10.1021/jm400050y
PMCID: PMC3618660  PMID: 23419026
6.  Modulation of Opioid Receptor Ligand Affinity and Efficacy Using Active and Inactive State Receptor Models 
Chemical biology & drug design  2012;80(5):763-770.
Mu opioid receptor (MOR) agonists are widely used for the treatment of pain; however chronic use results in the development of tolerance and dependence. It has been demonstrated that co-administration of a MOR agonist with a delta opioid receptor (DOR) antagonist maintains the analgesia associated with MOR agonists, but with reduced negative side effects. Using our newly refined opioid receptor models for structure-based ligand design, we have synthesized several pentapeptides with tailored affinity and efficacy profiles. In particular, we have obtained pentapeptides 8, Tyr-c(S-S)[DCys-1Nal-Nle-Cys]NH2, and 12, Tyr-c(S-S)[DCys-1Nal-Nle-Cys]OH, which demonstrates high affinity and full agonist behavior at MOR, high affinity but very low efficacy for DOR, and minimal affinity for the kappa opioid receptor (KOR). Functional properties of these peptides as MOR agonists/DOR antagonists lacking undesired KOR activity make them promising candidates for future in vivo studies of MOR/DOR interactions. Subtle structural variation of 12, by substituting D-Cys5 for L-Cys5, generated analog 13 which maintains low nanomolar MOR and DOR affinity, but which displays no efficacy at either receptor. These results demonstrate the power and utility of accurate receptor models for structure-based ligand design, as well as the profound sensitivity of ligand function on its structure.
doi:10.1111/cbdd.12014
PMCID: PMC3465523  PMID: 22882801
Delta opioid receptor; G protein-coupled receptors; mixed efficacy ligand; mu opioid receptor; opioid; peptide; structure-based design
7.  Increased agonist affinity at the mu-opioid receptor induced by prolonged agonist exposure 
Prolonged exposure to high-efficacy agonists results in desensitization of the mu opioid receptor (MOR). Desensitized receptors are thought to be unable to couple to G-proteins, preventing downstream signaling, however the changes to the receptor itself are not well characterized. In the current study, confocal imaging was used to determine whether desensitizing conditions cause a change in agonist-receptor interactions. Using rapid solution exchange, the binding kinetics of fluorescently labeled opioid agonist, dermorphin Alexa594 (derm A594), to MORs was measured in live cells. The affinity of derm A594 binding increased following prolonged treatment of cells with multiple agonists that are known to cause receptor desensitization. In contrast, binding of a fluorescent antagonist, naltrexamine Alexa 594, was unaffected by similar agonist pre-treatment. The increased affinity of derm A594 for the receptor was long-lived and partially reversed after a 45 min wash. Treatment of the cells with pertussis toxin did not alter the increase in affinity of the derm A594 for MOR. Likewise the affinity of derm A594 for MORs expressed in mouse embryonic fibroblasts derived from arrestin 1 and 2 knockout animals increased following treatment of the cells with the desensitization protocol. Thus, opioid receptors were “imprinted” with a memory of prior agonist exposure that was independent of G-protein activation or arrestin binding that altered subsequent agonist-receptor interactions. The increased affinity suggests that acute desensitization results in a long lasting but reversible conformational change in the receptor.
doi:10.1523/JNEUROSCI.4187-12.2013
PMCID: PMC3711766  PMID: 23447620
8.  Orvinols with Mixed Kappa/Mu Opioid Receptor Agonist Activity 
Journal of Medicinal Chemistry  2013;56(8):3207-3216.
Dual-acting kappa opioid receptor (KOR) agonist and mu opioid receptor (MOR) partial agonist ligands have been put forward as potential treatment agents for cocaine and other psychostimulant abuse. Members of the orvinol series of ligands are known for their high binding affinity to both KOR and MOR, but efficacy at the individual receptors has not been thoroughly evaluated. In this study, it is shown that a predictive model for efficacy at KOR can be derived, with efficacy being controlled by the length of the group attached to C20 and by the introduction of branching into the side chain. In vivo evaluation of two ligands with the desired in vitro profile confirms both display KOR, and to a lesser extent MOR, activity in an analgesic assay suggesting that, in this series, in vitro measures of efficacy using the [35S]GTPγS assay are predictive of the in vivo profile.
doi:10.1021/jm301543e
PMCID: PMC3646402  PMID: 23438330
9.  Differential modulation of mu-opioid receptor signaling to adenylyl cyclase by RGS proteins 4 or 8 and 7 in permeabilised C6 cells is Gα subtype dependent 
Journal of neurochemistry  2009;112(4):1026-1034.
Regulators of G protein signaling (RGS) proteins act as GTPase accelerating proteins (GAPs) to negatively modulate G protein signaling and are defined by a conserved RGS domain with considerable amino acid diversity. To determine the effects of specific, purified RGS proteins on mu-opioid signaling, C6 cells stably expressing a mu-opioid receptor were rendered permeable to proteins by treatment with digitonin. Mu-opioid inhibition of forskolin-stimulated adenylyl cyclase (AC) by DAMGO, a mu-specific opioid peptide, remained fully intact in permeabilized cells. Purified RGS domain of RGS4 added to permeabilized cells resulted in a two-fold loss in DAMGO potency but had no effect in cells expressing RGS-insensitive G proteins. The inhibitory effect of DAMGO was reduced to the same extent by purified RGS4 and RGS8. In contrast, the RGS domain of RGS7 had no effect and inhibited the action of RGS8 due to weak physical association with Gαi2 and minimal GAP activity in C6 cell membranes. These data suggest that differences in conserved RGS domains of specific RGS proteins contribute to differential regulation of opioid signaling to AC and that a permeabilized cell model is useful for studying the effects of specific RGS proteins on aspects of G protein-coupled receptor signaling.
doi:10.1111/j.1471-4159.2009.06519.x
PMCID: PMC2947325  PMID: 20002516
RGS proteins; mu-opioid; adenylyl cyclase; permeabilization; Gα proteins
10.  Pentapeptides displaying mu opioid receptor agonist and delta opioid receptor partial agonist/antagonist properties 
Journal of medicinal chemistry  2009;52(23):7724-7731.
Chronic use of mu-opioid agonists has been shown to cause neurochemical adaptations resulting in tolerance and dependence. While the analgesic effects of these drugs are mediated by mu-opioid receptors (MOR), several studies have shown that antagonism or knockdown of delta-opioid receptors (DOR) can lessen or prevent development of tolerance and dependence. Based on computational modeling of putative active and inactive conformations of MOR and DOR, we have synthesized a series of pentapeptides with the goal of developing a MOR agonist/DOR antagonist peptide with similar affinity at both receptors as a tool to probe functional opioid receptor interaction(s). The eight resulting naphthylalanine-substituted cyclic pentapeptides displayed variable mixed-efficacy profiles. The most promising peptide (9; Tyr-c(S-CH2-S)[D-Cys-Phe-2-Nal-Cys]NH2) displayed a MOR agonist and DOR partial agonist/antagonist profile and bound with equipotent affinity (Ki ~ 0.5 nM) to both receptors, but also showed kappa opioid receptor (KOR) agonist activity.
doi:10.1021/jm9007483
PMCID: PMC2788680  PMID: 19788201
11.  Regulators of G protein signaling (RGS) Proteins as Drug Targets: Modulating GPCR Signal Transduction 
Journal of medicinal chemistry  2011;54(21):7433-7440.
doi:10.1021/jm101572n
PMCID: PMC3208131  PMID: 21916427
RGS; G protein; serotonin; dopamine; academic drug discovery; novel target; flow cytometry
12.  Mu-Opioid Receptor Coupling to Gαo Plays an Important Role in Opioid Antinociception 
Neuropsychopharmacology  2011;36(10):2041-2053.
Opioid analgesics elicit their effects via activation of the mu-opioid receptor (MOR), a G protein-coupled receptor known to interact with Gαi/o-type G proteins. Work in vitro has suggested that MOR couples preferentially to the abundant brain Gαi/o isoform, Gαo. However, studies in vivo evaluating morphine-mediated antinociception have not supported these findings. The aim of the present work was to evaluate the contribution of Gαo to MOR-dependent signaling by measuring both antinociceptive and biochemical endpoints in a Gαo null transgenic mouse strain. Male wild-type and Gαo heterozygous null (Gαo +/−) mice were tested for opioid antinociception in the hot plate test or the warm-water tail withdrawal test as measures of supraspinal or spinal antinociception, respectively. Reduction in Gαo levels attenuated the supraspinal antinociception produced by morphine, methadone, and nalbuphine, with the magnitude of suppression dependent on agonist efficacy. This was explained by a reduction in both high-affinity MOR expression and MOR agonist-stimulated G protein activation in whole brain homogenates from Gαo +/− and Gαo homozygous null (Gαo −/−) mice, compared with wild-type littermates. On the other hand, morphine spinal antinociception was not different between Gαo +/− and wild-type mice and high-affinity MOR expression was unchanged in spinal cord tissue. However, the action of the partial agonist nalbuphine was compromised, showing that reduction in Gαo protein does decrease spinal antinociception, but suggesting a higher Gαo protein reserve. These results provide the first in vivo evidence that Gαo contributes to maximally efficient MOR signaling and antinociception.
doi:10.1038/npp.2011.91
PMCID: PMC3158321  PMID: 21654736
morphine; antinociception; mu-opioid receptor; G protein; signaling; transgenic mice; neuropharmacology, opioids, pain/analgesics, animal models; morphine; antinociception; mu-opioid receptor; g protein; signaling
13.  Opioids and Efflux Transporters. Part 2: P-Glycoprotein Substrate Activity of 3- and 6-Substituted Morphine Analogs 
Journal of Medicinal Chemistry  2008;51(7):2316-2320.
Continuing our studies investigating opioids with reduced P-glycoprotein (P-gp) substrate activity, a series of known 3- and 6-hydroxy, -methoxy, and -desoxymorphine analogs was synthesized and analyzed for P-gp substrate activity and opioid binding affinity. 6-Desoxymorphine (7) showed high affinity for opioid receptors and did not induce P-gp-mediated ATP hydrolysis. Additionally, 7 demonstrated morphine-like antinociceptive potency in mice, indicating this compound as an ideal lead to further evaluate the role of P-gp in opioid analgesic tolerance development.
doi:10.1021/jm701457j
PMCID: PMC3401598  PMID: 18311899
14.  The selective delta opioid agonist SNC80 enhances amphetamine-mediated efflux of dopamine from rat striatum 
Neuropharmacology  2008;55(5):755-762.
The highly selective delta opioid agonist, SNC80, elicits dopamine-related behaviors including locomotor stimulation and conditioned place-preference. In contrast, it has been reported that SNC80 fails to promote dopamine efflux from the striatum of freely moving rats. However, SNC80 does enhance behavioral responses to the stimulants, amphetamine and cocaine, suggesting an interaction between delta opioids and psychostimulants. Since the increase in locomotor activity elicited by amphetamine and related stimulants acting at the dopamine transporter is associated with increases in extracellular concentrations of dopamine within the striatum, we hypothesized that SNC80 enhances this activity by potentiating the overflow of dopamine through the transporter. To test this hypothesis, striatal preparations from Sprague-Dawley rats were assayed for dopamine efflux in response to amphetamine challenge. SNC80 was given either in vivo or in vitro directly to rat striatal tissue, prior to in vitro amphetamine challenge. Both in vivo and in vitro administration of SNC80 enhanced amphetamine-mediated dopamine efflux in a concentration- and time-dependent manner. However, SNC80 in either treatment paradigm produced no stimulation of dopamine efflux in the absence of amphetamine. The effect of SNC80 on amphetamine-mediated dopamine overflow, but not the effect of amphetamine alone, was blocked by the delta selective antagonist, naltrindole and was also observed with other delta agonists. The results of this study demonstrate that even though SNC80 does not stimulate dopamine efflux alone, it is able to augment amphetamine-mediated dopamine efflux through a delta opioid receptor mediated action locally in the striatum.
doi:10.1016/j.neuropharm.2008.06.017
PMCID: PMC2659683  PMID: 18602932
SNC80; amphetamine; delta opioid; dopamine; striatum; naltrindole
15.  RGS9-2: probing an intracellular modulator of behavior as a drug target 
Regulators of G-protein signaling (RGS proteins) comprise a large family of signal transduction molecules that modulate G-protein-coupled-receptor (GPCR) function. Among the RGS proteins expressed in the brain, RGS9-2 is very abundant in the striatum, a brain region involved in movement, motivation, mood and addiction. This protein negatively modulates signal transduction thus playing a key part in striatal function and resultant behavioral responses. In particular, there is evidence of important interactions with μ-opioid- and dopamine D2-receptor signaling pathways. Several studies indicate that manipulations of RGS9-2 levels in the striatum might greatly affect pharmacological responses. These findings indicate that treatment strategies targeting RGS9-2 levels or activity might be used to enhance responses to drugs acting at GPCRs and/or prevent undesired drug actions.
doi:10.1016/j.tips.2008.11.006
PMCID: PMC3394094  PMID: 19211160
16.  Comparison of Peptidic and Nonpeptidic δ-Opioid Agonists on Guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) Binding in Brain Slices from Sprague-Dawley Rats 
Previous studies have demonstrated that peptidic and nonpeptidic δ-opioid receptor agonists have different effects depending on the measure. For example, nonpeptidic δ-opioid agonists, but not peptidic agonists, produce convulsions in rats, and in vitro studies suggested that peptidic and nonpeptidic δ-opioid agonists might have differential mechanisms of receptor down-regulation. The present study evaluated potential differences between peptidic and nonpeptidic δ-opioid agonists in their ability to activate G proteins using guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) autoradiography experiments in rat brain slices. The peptidic agonist [D-Pen2,D-Pen5]-enkephalin and the nonpeptidic agonist (+)BW373U86 [(+)-4-[α(R)-α-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-hydroxyphenyl)methyl]-N,N-diethylbenzamide] demonstrated concentration-dependent increases in [35S]GTPγS binding that were attenuated by the δ-opioid antagonist naltrindole. (+)BW373U86 was more potent and efficacious than the peptidic agonist, and this difference remained consistent across brain regions where significant stimulation was observed. In addition, multiple δ-opioid compounds were evaluated for their agonist activity in this assay. These data suggested that differences between peptidic and nonpeptidic δ-opioid agonists in behavioral studies were most likely caused by differences in agonist efficacy. Finally, these data also revealed that [35S]GTPγS autoradiography could be used to compare efficacy differences among agonists across various brain regions in rat brain slices.
doi:10.1124/jpet.104.078741
PMCID: PMC1780166  PMID: 15574687
[35S]GTPγS, 5′-O-(3-[35S]thio)triphosphate; DPDPE, [D-Pen2,D-Pen5]-enkephalin; SNC80, (+)-4-[α(R)-α-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-methoxyphenyl)methyl]-N,N-diethylbenzamide; OMI, oxymorphindole; SIOM, spiroindanyloxymorphone; (+)BW373U86, (+)-4-[α(R)-α-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-hydroxyphenyl)methyl]-N,N-diethylbenzamide; NSB, nonspecific binding; NTI, naltrindole; TAN67, 2-methyl-4aa-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aα-octahydroquinolino[2,3,3-g]isoquinoline dihydrobro-mide (SB205607); FrCtx, frontal cortex; PCtx, prelimbic cortex; CCtx, cingulate cortex; NAcc nucleus accumbens; CP, caudate putamen; HPC, hippocampus; Amyg, amygdala; PirCtx, piriform cortex; Thal, thalamus; Hypothal, hypothalamus; SN, substantia nigra; PAG, periaqueductal gray; PN, pontine nuclei
17.  DEVELOPMENT AND IN VITRO CHARACTERIZATION OF A NOVEL BIFUNCTIONAL MU-AGONIST/DELTA-ANTAGONIST OPIOID TETRAPEPTIDE 
ACS chemical biology  2011;6(12):1375-1381.
The development of tolerance to and dependence on opioid analgesics greatly reduces their long-term usefulness. Previous studies have demonstrated that co-administration of a mu opioid receptor (MOR) agonist and delta opioid receptor (DOR) antagonist can decrease MOR agonist induced tolerance and dependence development after chronic exposure. Clinically, a single ligand displaying multiple efficacies (e.g. MOR agonism concurrently with DOR antagonism) would be of increased value over two drugs administered simultaneously. Guided by modeling of receptor-ligand complexes we have developed a series of potent non-selective opioid tetrapeptides that have differing efficacy at MOR and DOR. In particular, our lead peptide (KSK-103) binds with equal affinity to MOR and DOR but acts as a MOR agonist with similar efficacy but greater potency than morphine and a DOR antagonist in cellular assays measuring both G protein stimulation and adenylyl cyclase inhibition.
doi:10.1021/cb200263q
PMCID: PMC3241856  PMID: 21958158
18.  Synthesis and In Vivo Brain Distribution of Carbon-11 Labeled δ-Opioid Receptor Agonists1 
Nuclear medicine and biology  2010;37(8):989-996.
Three new radiolabeled compounds, [11C]SNC80 ((+)-4-[(αR)- α-{(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl}-3-[11C]methoxybenzyl-N,N-diethylbenzamide), N,N-diethyl-4-[3-methoxyphenyl-1-[11C]methylpiperidin-4-ylidenemethyl)benzamide, and N,N-diethyl-4-[(1-[11C]methylpiperidin-4-ylidene)phenylmethyl]benzamide, were prepared as potential in vivo radiotracers for the δ opioid receptor. Each compound was synthesized by alkylation of the appropriate desmethyl compounds using [11C]methyl triflate. In vivo biodistribution studies in mice showed very low initial brain uptake of all three compounds, and no regional specific binding for [11C]SNC80. A monkey PET study of [11C]SNC80 confirmed low brain permeability and uniform regional distribution of this class of opioid agonists in a higher species. Opioid receptor ligands of this structural class are thus unlikely to succeed as in vivo radiotracers, likely due to efficient exclusion from the brain by the P-glycoprotein efflux transporter.
doi:10.1016/j.nucmedbio.2010.06.002
PMCID: PMC3018154  PMID: 21055630
Opioid; Tomography, emission computed; SNC80 carbon radioisotopes
19.  Mixed Kappa/Mu Opioid Receptor Agonists: The 6β-Naltrexamines 
Journal of medicinal chemistry  2009;52(6):1546-1552.
Ligands from the naltrexamine series have consistently demonstrated agonist activity at kappa opioid receptors (KOR), with varying activity at the mu opioid receptor (MOR). Various 6β-cinnamoylamino derivatives were made with the aim of generating ligands with a KOR agonist/MOR partial agonist profile, as ligands with this activity may be of interest as treatment agents for cocaine abuse. The ligands all displayed the desired high affinity, non-selective binding in vitro and in the functional assays were high efficacy KOR agonists with some partial agonist activity at MOR. Two of the new ligands (12a, 12b) have been evaluated in vivo, with 12a acting as a KOR agonist, and therefore somewhat similar to the previously evaluated analogues 3–6, while 12b displayed predominant MOR agonist activity.
doi:10.1021/jm8015552
PMCID: PMC3070388  PMID: 19253970
20.  Comparison of the opioid receptor antagonist properties of naltrexone and 6β-naltrexol in morphine-naïve and morphine-dependent mice 
European journal of pharmacology  2008;583(1):48-55.
It has been proposed that on chronic morphine treatment the μ-opioid receptor becomes constitutively active, and as a consequence, the opioid withdrawal response arises from a reduction in the level of this constitutively active receptor. In support of this, the putative μ-opioid receptor inverse agonist naltrexone has been shown to precipitate more severe withdrawal behavior in mice than the putative neutral receptor antagonist 6β-naltrexol. In the present study naltrexone and 6β-naltrexol were compared in NIH Swiss mice to test the hypothesis that their differential ability to precipitate withdrawal is due to differences in their in vivo opioid receptor antagonist potencies caused by differential access to μ-opioid receptors in the central nervous system and not necessarily by intrinsic differences in their opioid receptor activity. In naïve mice both compounds had similar potencies to antagonize morphine-induced antinociception in the hot plate and warm-water tail-withdrawal assays when measured under equilibrium conditions and afforded similar calculated apparent in vivo μ-opioid receptor affinities. In morphine-dependent mice both compounds precipitated withdrawal jumping but naltrexone was between 10- and 100-fold more potent than 6β-naltrexol. A similar potency difference was seen for other withdrawal behaviors. Both naltrexone and 6β-naltrexol at 1 mg/kg reversed antinociception induced by the long-lasting μ-opioid receptor agonist BU72 in the warm-water tail-withdrawal assay, but antagonism by naltrexone was 6-fold more rapid in onset at equal doses. Since the compounds have similar affinity for the μ-opioid receptor in vivo, the results suggest that the differences observed between the ability of naltrexone and 6β-naltrexol to precipitate withdrawal in the mouse may be explained by differential onset of receptor antagonist action.
doi:10.1016/j.ejphar.2008.01.004
PMCID: PMC3010382  PMID: 18275956
Morphine; withdrawal; dependence; mice; naltrexone; 6β-naltrexol
21.  14β-Arylpropiolylamino-17-cyclopropylmethyl-7,8-dihydronormorphinones and related opioids. Further examples of pseudo-irreversible mu opioid receptor antagonists 
Journal of medicinal chemistry  2009;52(21):6926-6930.
14β-4’-Chlorocinnamoylaminodihydronormorphinone (2a), and analogues, are selective pseudoirreversible antagonists of the mu opioid receptor (MOR). The preparation of analogues with ethynic bonds, replacing the ethenic bond of 2a, is described. The new ligands, in mouse antinociceptive assays, had pseudoirreversible MOR antagonist activity which, in the case of 8b was of longer duration than that of 2a. The related codeinone (9b) had only antagonist activity in vivo, in contrast to 2a’s codeinone equivalent 3a, which had potent antinociceptive activity.
doi:10.1021/jm901074a
PMCID: PMC2788771  PMID: 19842669
22.  Collision coupling, crosstalk, and compartmentalization in G-protein coupled receptor systems: Can a single model explain disparate results? 
Journal of theoretical biology  2008;255(3):278-286.
The collision coupling model describes interactions between receptors and G-proteins as first requiring the molecules to find each other by diffusion. A variety of experimental data on G-protein activation have been interpreted as suggesting (or not) the compartmentalization of receptors and/or G-proteins in addition to a collision coupling mechanism. In this work, we use a mathematical model of G-protein activation via collision coupling but without compartmentalization to demonstrate that these disparate observations do not imply the existence of such compartments. In experiments with GTP analogs (commonly GTPγS), the extent of G-protein activation is predicted to be a function of both receptor number and the rate of GTP analog hydrolysis. The sensitivity of G-protein activation to receptor number is shown to be dependent upon the assay used, with the sensitivity of phosphate production assays (GTPase) > GTPγS binding assays > cAMP inhibition assays. Finally, the amount of competition or crosstalk between receptor species activating the same type of G-proteins is predicted to depend on receptor and G-protein number, but in some (common) experimental regimes this dependence is expected to be minimal. Taken together, these observations suggest that the collision coupling model, without compartments of receptors and/or G-proteins, is sufficient to explain a variety of observations in literature data.
doi:10.1016/j.jtbi.2008.08.003
PMCID: PMC2917770  PMID: 18761019
mathematical model; adrenergic; opioid; cannabinoid; muscarinic
23.  Differentiation of Kappa Opioid Agonist-Induced Antinociception by Naltrexone Apparent pA2 Analysis in Rhesus Monkeys1,2 
Naltrexone (NTX) exhibited approximately 3-fold higher affinity for sites labeled by [3H]U69,593 (putative κ1-selective ligand) than [3H]bremazocine (non-selective ligand) in the presence of mu and delta receptor blockade in monkey brain membranes. This led us to test an hypothesis that NTX could display in vivo antagonist selectivity for κ1- versus non-k1-mediated effects. Six opioid agonists were characterized by NTX apparent pA2 analysis in a 50°C water tail-withdrawal assay in rhesus monkeys. Constrained NTX pA2 values (95% confidence limits) were: alfentanil, 8.66 (8.47– 8.85); ethylketocyclazocine, 7.97 (7.93– 8.01); U69,593, 7.64 (7.49 –7.79); U50,488, 7.55 (7.42–7.67); bremazocine, 6.92 (6.73–7.12); enadoline, 6.87 (6.69– 7.05). Pretreatment with clocinnamox, an irreversible mu antagonist, confirmed that mu receptors were not involved in the antinociception produced by the kappa agonists, U69,593, U50,488, bremazocine and enadoline; however, both mu and kappa receptors mediated the antinociceptive effects of ethyl-ketocyclazocine. The apparent NTX pA2 profile of opioid agonists correlated highly with the radioligand binding studies, which indicates that U69,593 and U50,488 produced antinociception by acting on kappa-1 receptors, whereas bremazocine and enadoline probably acted via non-kappa-1 receptors. This study provides further functional evidence of kappa opioid receptor multiplicity in primates and suggests that NTX may be a useful tool to study this phenomenon in vivo.
PMCID: PMC2882849  PMID: 9580592
24.  The effects of the phyllolitorin analogue [desTrp3,Leu8]phyllolitorin on scratching induced by bombesin and related peptides in rats 
Brain research  1999;839(1):194-198.
Bombesin along with several closely related neuropeptides elicit scratching behavior when administered centrally. The first part of the study was designed to determine the antagonistic effects of a novel phyllolitorin analogue wdesTrp3,Leu8]phyllolitorin (DTP) on scratching induced by three peptides (bombesin, neuromedin-C, and [Leu8]phyllolitorin). In addition, the binding affinity of each peptide for the bombesin receptor site was determined. DTP (30 μg) inhibited scratching induced by these peptides, but unlike the peptides, DTP had no affinity for the bombesin site, thereby suggesting that DTP is displaying physiological antagonism through an unknown mechanism.
PMCID: PMC2863106  PMID: 10482814
Bombesin; Neuromedin-C; GRP(18-27); Phyllolitorin; Scratching behavior; Bombesin antagonist
25.  Nitrile analogs of meperidine as high affinity and selective sigma-1 receptor ligands 
A series of N-substituted-4-cyano-4-phenylpiperidine analogs were synthesized and evaluated for binding affinity at opioid receptors and showed no affinity. The series similarity to previously reported σ ligands prompted analysis at σ receptors to determine the SAR for affinity at σ receptors. Within the N-substituent series the saturated analogs showed increased affinity at both σ receptors. Optimal chain length in the N-arylalkyl series for σ1 and σ2 receptors proved to be N-propylphenyl; extension to a four carbon chain dramatically decreased affinity at both receptors. Substituents in the 4-position affects only σ1 affinity; no change in affinity at σ2 was shown. The N-isobutyl, N-phenylpropyl, and N-benzyl analogs are worth pursuing due to their good affinity and selectivity at the σ1 receptor, whereas the N-benzyl analog exhibits the greatest selectivity for σ1.
doi:10.1016/j.ejmech.2007.09.026
PMCID: PMC2486414  PMID: 17988766
σ receptor; AC927; UMB24; N-substituted piperidines

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