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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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Probe Reports from the NIH Molecular Libraries Program [Internet].

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Optimization and characterization of an opioid kappa receptor (OPRK1) antagonist

, , , , , , , , and .

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Received: ; Last Update: September 18, 2014.

The opioid receptors are a subfamily of the family A G protein-coupled opioid receptor superfamily and consist of mu (OPRM1), delta (OPRD1), and kappa (OPRK1), all of which activate inhibitory G proteins. The dynorphins act as endogenous agonists of OPRK to activate a variety of signaling transduction pathways including those involving mitogen activated protein kinases (MAPK). Activation of OPRK leads to a number of physiological effects implicating a role for these receptors in addiction, dysphoria and reward. Hence, OPRK antagonists are being explored for their effects in the treatment of cocaine addiction, depression, and feeding behavior and have been proposed as a treatment for psychosis and schizophrenia. While a number of OPRK1 antagonists have been identified, all of the prototypic antagonists are very long-acting, exhibit unusual pharmacology, exhibit delayed onset of action, and are associated with serious safety concerns. Very few drug-like OPRK antagonists have been developed. New OPRK antagonists possessing novel scaffolds and improved selectivity are needed as pharmacological tools to better understand the OPRK- dynorphin system and as potential pharmacotherapies. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), reports ML350 as a highly potent OPRK1 antagonist with an IC50 of 9-16 nM, with high selectivity (selectivities vs. OPRD1 and OPRM1 of 219-382–fold and 20-35–fold, respectively). ML350 was identified by high-throughput screening using a cell-based Tango™-format assay. A set of pharmacokinetic analyses show that ML350 has high passive membrane permeability, good brain penetration, no significant activity at three of four human cytochrome P450 subtypes, high binding for rodent plasma protein and modest binding for human plasma protein, and an encouraging in vivo pharmacokinetic profile in rats. ML350 was submitted to CEREP for broad panel screening against a panel of receptors, transporters, and ion channels; the data suggest that ML350 is generally inactive against a broad array of off targets and does not likely exert unwanted effects. Importantly, ML350 was shown to have a reversible analgesic effect when challenged with an OPRK agonist in a tail flick assay in mice. ML350 serves as a novel OPRK antagonist that can be developed as a therapeutic for the treatment of a variety of disorders involving the OPRK1-dynorphin system.

Assigned Assay Grant #: R03 NS053751

Screening Center Name & PI: The Scripps Research Institute Molecular Screening Center (SRIMSC), Hugh Rosen

Chemistry Center Name & PI: SRIMSC, Hugh Rosen

Assay Submitter & Institution: Lakshmi Devi, Mount Sinai School of Medicine

PubChem Summary Bioassay Identifier (AID): 652045

Probe Structure & Characteristics

Image ml350f1
CID/ML#Target NameIC50 (nM) [SID, AID]Anti-target Name(s)IC50 (μM) [SID, AID]Fold SelectiveSecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID 60156214/ML350OPRK19.16 [SID 144087319, AID 652084]
16.4 nM [SID 144087319, AID 652032]
OPRD1; OPRM1OPRD1: 3.5 uM [SID 144087319, AID 652033]
OPRM1: 323 nM [SID 144087319, AID 652034]
OPRD1: 219-382 fold
OPRM1: 20-35 fold
Cytotoxicity: [SID 144087319, AID 652086]
Plasma protein binding: [SID 144087319, AID 652078]
Hepatic microsome stability: [SID 144087319, AID 652086]
Cytochrome P450 inhibition: [SID 144087319, AID 652076]
CEREP panel counterscreen: [SID 144087319, AID 652083]
CEREP hERG counterscreen: [SID 144087319, AID 662075]
Plasma and brain levels: [SID 144087319, AID 662085]
Tail Flick assay: [SID 144087319, AID 652108]
PAMPA permeability: [SID 144087319, AID 652113]

Recommendations for scientific use of the probe

Prototypic OPRK antagonists are very long-acting, exhibit delayed onset of action, and are associated with serious safety concerns. Very few drug-like OPRK antagonists have been developed. The phase I studies with PF-04455242 were terminated due to toxicity issues. Eli Lilly advanced LY2456302 to a phase I clinical trial study (oral treatment of alcohol dependence) to measure the occupancy of brain OPRK after single oral doses. The last update in ClinicalTrials.gov reported for this study is May 5, 2011; no information on any progress in further Phase I studies or progression to Phase II is available. Such a delay is unusual due to the costs involved. Hence, there is a need for additional drug-like highly potent and selective OPRK antagonists with a good safety profile and CNS exposure.

Selective OPRK antagonists are being explored for their effects in the treatment of a wide variety of areas including cocaine addiction [1], depression [2], and feeding behavior [3] and have been proposed as a treatment for psychosis and schizophrenia [4]. ML350 will be used in a stress-induced migraine model assay that measures cutaneous (tactile) allodynia in rats as a measurable translational endpoint for headache-related pain [5], an alcohol withdrawal assay that measures working memory performance and anxiety-like behavior in rats after withdrawal from alcohol [6], and a cocaine reward assay that measures the effect on drug-seeking behavior in a cocaine addiction model.

The probe will be of use to researchers interested in the role of the OPRK in neuropathic pain, drug addiction, or affective psychiatric disorders.

New OPRK antagonists possessing novel scaffolds and improved selectivity are needed as pharmacological tools to better understand the OPRK-dynorphin system. Signaling through OPRK results in the activation of multiple signal transduction pathways including activation of PI3-kinase, PKCζ, and EERK-1 and ERK-2 [7]. In addition, signaling through OPRK results in activation of the p38 MAPK [8-15] and the JNK1 [16-17] signaling pathways, and the JAK2/STAT3 and IRF2 signaling cascade [18]. Different OPRK1 ligands are reported to activate distinct signal transduction pathways; this activation of multiple signal transduction pathways are thought to be due to ‘ligand-directed signal trafficking’ by OPRK1 [7-17, 19-21]. It is thought that ligands that are selective for one pathway over the other will help elucidate the role of each pathway in specific responses. This will help in the development of reagents and/or therapeutics that can target pathway responsible for the wanted effects versus unwanted side effects.

1. Introduction

Opioids are the most widely used class of analgesics [22]. The opioid receptors are a subfamily of the G protein-coupled opioid receptor (GPCR) superfamily and the three major types, mu (OPRM1), delta (OPRD1), and kappa (OPRK1) opioid receptors have been pharmacologically characterized and cloned [23-25]. All activate inhibitory G proteins. Signaling through OPRK results in the activation of a number of signal transduction pathways [7-21] and this, in turn, leads to a number of physiological effects implicating a role for these receptors in addiction, dysphoria and reward [1-2, 4]. Hence, kappa opioid antagonists are being explored for their effects in the treatment of cocaine addiction, depression, and feeding behavior and have been proposed as a treatment for psychosis and schizophrenia. While a number of OPRK1 antagonists have been identified, all of the prototypic antagonists very long-acting, exhibit unusual pharmacology, exhibit delayed onset of action, and are associated with serious safety concerns. Very few drug-like OPRK antagonists have been developed. New OPRK antagonists possessing novel scaffolds and improved selectivity are needed as pharmacological tools to better understand the OPRK system and as potential pharmacotherapies.

Neuropathic pain: Studies have identified a role for dynorphin and OPRK in neuropathic pain [26]. The dynorphins act as endogenous agonists of OPRK1 [27]. The OPRK1-dynorphin system mediates astrocyte proliferation through the activation of p38 MAPK that is required for the effects of neuropathic pain on analgesic responses [15, 28]. Increased dynorphin expression in neuropathic pain leads to a sustained activation of OPRK [26, 29-30].

There is evidence that the endogenous dynorphin-derived opioids may produce either a sustained reduction or an increase in sensitivity to painful stimuli [15, 31]. Thus, dynorphin can elicit multiple effects to modulate analgesic responses. Antinociceptive effects of intrathecal and systemic administration of selective OPRK1 agonists have been documented [31-32]. It has also been reported that the OPRK1 antagonist norbinaltorphimine (nor-BNI) significantly lowers pain thresholds and increases pain sensation after sciatic nerve ligation [33]. Thus, the endogenous dynorphin-derived opioids may have both antinociceptive and pronociceptive actions. How sustained activation of opioid receptors by endogenous dynorphins contributes to the neuropathic pain state is not clear. One of the side effects of OPRK1-mediated analgesia is depression [30, 34]; the development of novel analgesics that bypass this side effect would be therapeutically beneficial.

Drug addiction: About 90 million people worldwide suffer from drug addiction [35]. A role for the OPRK1-dynorphin system in modulating drug addiction has been proposed; however its function appears to be diverse, and may modulate drug-seeking behavior depending on factors such as drug history, pattern of intake, and stress (for review see [36-37]). Multiple exposures to cocaine result in complex molecular changes in the brain, and, ultimately, in addiction [38]. Although a single exposure to cocaine in rats does not affect brain dynorphin levels, repeated exposures increase dynorphin concentrations in the striatum and substantia nigra [39].

The role of OPRK1 in cocaine addiction has been actively studied [40-50]. Results clearly demonstrate an activation of the OPRK1 system following chronic cocaine exposure. As OPRK1 stimulation in the brain produces aversive effects in animals and humans [51-53], it is likely that cocaine-induced up-regulation of OPRK1 in regions associated with reward might be part of a protective compensatory neuroadaptive mechanism to counteract the rewarding effect of cocaine and might contribute to the emergence of persistent dysphoria, an emotional state marked by anxiety, depression, and restlessness that is often reported in humans after the withdrawal of the drug [54]. Studies with OPRK1 knockout mice [55-62] suggest that the receptor antagonists might be useful in preventing stress-induced relapse in cocaine-dependent individuals and therefore help to prevent drug use relapse.

It has been reported that chronic nicotine exposure affects OPRKs modulation of neurotransmission, leading to enhanced negative affect and increased anxiogenic effects [63]. Furthermore, spontaneous nicotine withdrawal-induced anxiety-like behavior and somatic signs of withdrawal are blocked by pretreatment with the OPRK antagonists nor-BNI or JDTic [63].

Several studies have implicated the role of OPRK1 signaling in stress-induced reoccurrence of ethanol self-administration [64-65]. In addition, OPRK1-dynorphin systems have been shown to be altered by opiate treatment in reward-related neural circuits in animal models [64, 66-67]. Taken together, these studies suggest that antagonism of OPRK1 may mitigate negative reinforcing behavioral states associated with drug withdrawal.

Affective psychiatric disorders: Affective psychiatric disorders comprise a worldwide health challenge; about 120 and 25 million people suffer from depression and schizophrenia, respectively [35]. These afflictions are all characterized by changes in emotion, motivation, cognition, and stress reactivity. Imaging studies have consistently shown altered activity in the amygdala, hippocampus, basal ganglia, and prefrontal cortex of psychiatric patients [68]; areas involved in stress responsiveness, emotional reactivity, goal-directed behavior, motivation, and executive function. OPRK1 and the dynorphin peptides are enriched in these brain regions, where they play a role in modulating neurotransmission. This has been an increasingly active area of research in recent years, and resulting data suggest that dysregulation of this system may contribute to the development and maintenance of various affective psychiatric disorders [69]; for review, see [36, 70]. However, solid evidence from clinical studies is lacking.

There is increasing evidence for a potential involvement of OPRK1-dynorphin in schizophrenia; OPRK1 agonists appear to induce symptoms in humans and animals that are present in schizophrenia [70-72]. The potent OPRK1 agonist Salvinorin A produces hallucinations in humans, supporting the idea of a OPRK1-dynorphin involvement in disorders characterized by disturbed perception [72]. Potential evidence for a role of dynorphin in psychotic disorders also comes from animal experiments. In rats, the selective OPRK1 agonist U- 50488H induced a dose-dependent reduction of pre-pulse inhibition [73], which is seen as a readout of sensorimotor gating and is impaired in schizophrenics. Pre-pulse inhibition was restored by the selective OPRK1 antagonist nor-BNI [73].

The role of OPRK1 in stress has been an active area of study, linking OPRK signaling and behavior [74-75]. Stress-induced opioid peptide release resulting in stress-induced analgesia through action at opioid receptors has been reported for all of the major opioid systems. It has been reported that OPRK1 activation after stress can also modulate numerous behaviors, including reward and depression [20, 76-77]. OPRK1 antagonist administration produces anxiolytic effects in several rat models of stress (elevated plus-maze, open-field, and fear potentiated startle paradigms) [78], suggesting a role for endogenous dynorphin release in the expression of anxiety-like behavior in these stress models. OPRK1 antagonists produce effects similar to that of traditional anti-depressants [2, 51, 79]. In an animal model widely used to model social defeat stress, wild-type mice treated with the selective OPRK1 antagonist Nor-BNI exhibit decreased social defeat postures [80].

In the forced swim test, a rodent model of depression and a procedure that identifies in rats treatments with antidepressant efficacy in humans [81], effects produced by OPRK1 agonists and antagonists have been interpreted as “prodepressive” and “anti-depressant”, respectively [2, 82]. The antidepressant-like effects of OPRK1 antagonists have also been observed in other studies [51, 83-84]. Interestingly, standard antidepressant drugs often cause anxiety [85-87]. The antidepressant and anxiety-relieving effects of OPRK1 antagonists is notable, and suggests that this class drug might be particularly efficacious for the treatment of concomitant depressive and anxiety disorders [88].

The physiological and pathophysiological mechanisms of OPRK1-dynorphin systems and their roles in neuropathic pain, drug addiction, and affective psychiatric disease in humans are active areas of study. The availability of new research tools such as potent and selective OPRK1 antagonists will facilitate understanding of the mechanisms involved in these processes and potentially have therapeutic value as novel therapies with an improved side effect profile to currently available drugs.

Several OPRK1 antagonists have been described in the literature. Norbinaltorphimine (nor-BNI) [89-90], 5′-guanidinonaltrindole (GNTI) [89, 91], and JDTic [89, 92] all exhibit a delay in the onset of action of approximately 24 hours [89, 93-95], and have very long lasting in vivo effects of up to 56 days [89, 95-96]. Due to their pharmacodynamics/pharmacokinetics and poor unbound brain/plasma ratio, morphine-like derivatives, nor-BNI and GNTI, are only used as pharmacological tools. The non-opioid compound JDTic exhibited a poor brain exposure but had an extraordinary persistence in brain (mean brain concentration declined by only 56% over 24 hours, and the drug was still detectable at 1 week in mice) despite its P-glycoprotein-mediated efflux, and its moderate lipophilicity and low affinity for cell homogenates. The presence of two basic nitrogens and entrapment in cellular compartments such as lysosomes have been proposed as a possible mechanism of this persistence [89, 97]. A Phase 1 clinical trial of JDTic for cocaine dependence was terminated in 2012 due to adverse events. Several short-acting compounds from distinct chemotypes have been developed with greater CNS exposure compared to the prototypic ligands (nor-BNI, GNTI, JDTic). AstraZeneca disclosed a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides. AZ-MTAB was efficacious in animal models of mood disorders, but was associated with a significant hERG liability (IC50 of 260 nM) [97-98]. Another OPRK antagonist PF-04455242 [99] was developed by Pfizer. However, on January 6, 2010 the phase 1 study for bipolar disorder and depression was terminated due to toxicology findings in animals exposed to PF-04455242 for three months. Eli Lilly identified a subnanomolar OPRK antagonist with selectivity of 21 and 135 over OPRM and OPRD respectively [100]. In 2010 LY2456302 was advanced to Phase I clinical trial for the oral treatment of alcohol dependence. The clinical study was to assess the brain OPRK1 occupancy after single oral doses of LY2456302 as measured by positron emission tomography with radioligand LY2879788 (11C PKAB) in healthy subjects. The last update of the study was May 5, 2011, and no new studies for the compound are listed in ClinicalTrials.gov.

Even though a few drug-like OPRK antagonists with more favorable pharmacokinetics than nor-BNI, GNTI, JDTic have been developed, new OPRK antagonists possessing novel scaffolds and improved selectivity are needed both as pharmacological tools to better understand the OPRK1-dynorphin system and as potential pharmacotherapies.

2. Materials and Methods

2.1. Assays

Probe Characterization Assays

Solubility

The solubility of compounds was tested in phosphate buffered saline, pH 7.4. Test tubes containing 1-2 mg compound in 1 mL PBS were inverted for 24. The samples were centrifuged and analyzed by HPLC (Agilent 1100 with diode-array detector). Peak area was compared to a standard of known concentration.

Stability

Demonstration of stability in PBS was conducted under conditions likely to be experienced in a laboratory setting. The compound was dissolved in 1 mL of PBS at a concentration of 10 μM, unless its maximum solubility was insufficient to achieve this concentration. Low solubility compounds were tested between ten and fifty percent of their solubility limit. The solution was immediately aliquoted into seven standard polypropylene microcentrifuge tubes which were stored at ambient temperature in a block microcentrifuge tube holder. Individual tubes were frozen at -80 °C at 0, 1, 2, 4, 8, 24, and 48 hours. The frozen samples were thawed at room temperature and an equal volume of acetonitrile was added prior to determination of concentration by LC-MS/MS.

LC-MS/MS for stability assay

All analytical methods are in MRM mode where the parent ion is selected in Q1 of the mass spectrometer. The parent ion is fragmented and a characteristic fragment ion is monitored in Q3. MRM mass spectroscopy methods are particularly sensitive because additional time is spent monitoring the desired ions and not sweeping a large mass range. Methods are rapidly set up using Automaton® (Applied Biosystems), where the compounds are listed with their name and mass in an Excel datasheet. Compounds are submitted in a 96-well plate to the HPLC autosampler and are slowly injected without a column present. A narrow range centered on the indicated mass is scanned to detect the parent ion. The software then evaluates a few pre-selected parameters to determine conditions that maximize the signal for the parent ion. The molecule is then fragmented in the collision cell of the mass spectrometer and fragments with m/z larger than 70 but smaller than the parent mass are determined. Three separate collision energies are evaluated to fragment the parent ion and the largest three ions are selected. Each of these three fragment ions is further optimized and the best fragment is chosen. The software then inserts the optimized masses and parameters into a template method and saves it with a unique name that indicates the individual compound being optimized. Spectra for the parent ion and the fragmentation pattern are saved and can be reviewed later.

Determination of glutathione reactivity

One μL of a 10 mM compound stock solution was added to 1 mL of a freshly prepared solution of 50 μM reduced glutathione. Final compound concentration is 10 μM unless limited by solubility. The solution was allowed to incubate at 37°C for 6 hours prior to being directly analyzed for glutathione adduct formation. LC-MS/MS analysis of GSH adducts was performed on an API 4000 Q-TrapTM mass spectrometer equipped with a Turboionspray source (Applied Biosystems, Foster City, CA). Two methodologies were utilized: a negative precursor ion (PI) scan of m/z 272, corresponding to GSH fragmenting at the thioether bond, and a neutral loss scan of -129 AMU to detect GSH adducts. This triggered positive ion enhanced resolution and enhanced product ion scans [101]).

Primary Assays

Primary HTS OPRK1 antagonists assay of Maybridge Collection (AID 652031, AID 652082, AID 65077)

Assay Overview: The purpose of this assay is to identify compounds from the Maybridge Library that act as antagonists of OPRK1. This assay uses Tango OPRK1-BLA U2OS cells which contain OPRK1 linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the OPRK1 receptor by agonist U-50488 causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as OPRK1 antagonists will inhibit OPRK1 activation and migration of the fusion protein, thus preventing proteolysis of GAL4-VP16 and BLA transcription, leading to no increase in well FRET. Compounds were tested in singlicate (AID 652031) or triplicate (AID 652082) at a final nominal concentration of 9 μM, or in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar (AID 652077).

Protocol Summary: U2OS cells were cultured in T-175 sq cm flasks at 37°C and 95% relative humidity (RH). The growth media consisted of McCoy's 5A Medium supplemented with 10% v/v dialyzed fetal bovine serum, 0.1 mM NEAA, 25 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 100 U/mL penicillin-streptomycin, 200 μg/mL Zeocin, 50 μg/mL Hygromycin, and 100 μg/mL Geneticin. Prior to the start of the assay, cells were suspended at a concentration of 250,000/mL in Assay Medium (McCoy's 5A Medium supplemented with 10% v/v charcoal dextran stripped fetal bovine serum, 0.1 mM NEAA, 25 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 100 U/mL penicillin-streptomyci). The assay was started by dispensing 10 μL of cell suspension to each well, followed by overnight incubation at 37°C in 5% CO2 and 95% RH. The next day, 50 nL of test compound (9 μM final nominal concentration) in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to control wells. Next, U-50488 in Assay Medium (8 nM final nominal EC80 concentration) was added to the appropriate wells. Plates were then incubated at 37°C in 5% CO2 for 4 hours. After the incubation, 2.2 μL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer's protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm. Assay Cutoff: Compounds that inhibited OPRK >50% (AIDs 652031 and 652082), or with an IC50 of ≤10 μM (AID 652077) were considered active.

S1P1 Counterscreen HTS assay of Maybridge Collection (AID 652087, AID 652079)

Assay Overview: The purpose of this assay is to determine whether compounds from the Maybridge Library that act as antagonists of OPRK1 are nonselective due to inhibition of S1P1. The Tango EDG-1-bla U2OS cells express S1P1 (EDG1) linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the S1P1 receptor by agonist S1P causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as S1P1 antagonists will inhibit S1P1 activation and migration of the fusion protein, thus preventing proteolysis of GAL4-VP16 and BLA transcription, leading to no increase in well FRET. Compounds were tested in triplicate at a final nominal concentration of 9 μM (AID 652087) or in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 μM (AID 652079).

Protocol Summary: U2OS cells were cultured in T-175 sq cm flasks at 37°C and 95% RH. The growth media consisted of McCoy's 5A Medium supplemented with 10% v/v dialyzed fetal bovine serum, 0.1 mM NEAA, 25 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 100 U/mL penicillin-streptomycin-neomycin, 200 μg/mL Zeocin, 50 μg/mL Hygromycin, and 100 μg/mL Geneticin. Prior to the start of the assay, cells were suspended at a concentration of 1,000,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 10 μL of cell suspension to each well in 384-well plates, followed by overnight incubation at 37°C in 5% CO2 and 95% RH. The next day, 50 nL of test compound in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to control wells. Next, S1P prepared in 2% BSA (0.22 μM final nominal EC80 concentration) was added to the appropriate wells. Plates were then incubated at 37°C in 5% CO2 for 4 hours. After the incubation, 2.2 μL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer's protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm. Assay Cutoff: Compounds that inhibited S1P1 >30% (AID652087) or with an IC50 of ≤10 μM (AID 652079) were considered active.

OPRD1 Counterscreen HTS assay of Maybridge Collection (AID 652080)

Assay Overview: The purpose of this counterscreen assay is to test the selectivity of OPRK1 antagonist compounds against the OPRD1 receptor. This assay uses Tango OPRD1-bla U2OS cells which express OPRD1 linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a Beta-arrestin/TEV protease fusion protein and a Beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the OPRD1 receptor by agonist SNC80 causes migration of the Beta-arrestin fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as OPRD1 antagonists will inhibit agonist activation and migration of the fusion protein, thus preventing proteolysis of GAL4-VP16 and BLA transcription, leading to no increase in well FRET. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 μM.

Protocol Summary: The Tango OPRD1-U20S dividing cell line was routinely cultured in 150 mm dishes at 37°C, 5% CO2 and 95% RH. The growth medium consisted of McCoys 5A Media supplemented with 10% v/v dialyzed fetal bovine serum, 25 mM HEPES, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, and 1X antibiotic mix (penicillin streptomycin). On day 1 of the assay, 16,000 cells in 10 μL of assay media (DMEM-Glutamax with sodium pyruvate, 10% fetal bovine serum stripped with charcoal-dextran, 25 mM HEPES, 0.1 mM non-essential amino acids, and antibiotic mix (penicillin streptomycin) were seeded into each well of a 384-well plate. 50 nl of test compound in DMSO were added to the appropriate wells and plates were incubated for 30 minutes at 37°C, 5% CO2 and 95% RH. Next, 1.1 μL of 3.7 uM SNC80 (OPRD1 agonist EC80 Challenge; final concentration 370 nM) or DMSO in assay medium was added to appropriate wells and incubated 16-24 hours at 37°C, 5% CO2 and 95% RH. On day 2, 2.5 μL of LiveBLazer trade mark FRET B/G (CCF4-AM) loading mix (prepared according to manufacturer's instructions; 6 μL solution A, 60 μL Solution B, 904 μL Solution C, and 30 μL Solution D) were added to each well, and plates incubated at room temperature in the dark for 2 hours. Well fluorescence was measured on Perkin Elmer's Envision using an Excitation filter 405 nm, Emission filters at 460 nm and 590 nm, bottom read. Assay Cutoff: Compounds that inhibited OPRD1 >30% were considered active.

OPRM1 Counterscreen HTS assay of Maybridge Collection (AID 652081)

Assay Overview: The purpose of this counterscreen assay is to test the selectivity of OPRK1 antagonist compounds against the OPRM1 receptor. The assay monitors GPCR-Beta-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). The reconstituted holoenzyme catalyzes the hydrolysis of a substrate which yields a chemiluminescent signal. This assay employs U2OS cells which express OPRM1 fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compounds and an agonist DAMGO (EC80 challenge), followed by measurement of well luminescence. As designed, compounds that inhibit OPRM1 will decrease the level of beta-arrestin recruitment elicited by DAMGO, resulting in a decrease in the level of reconstitution of the beta-gal holoenzyme, and decreased well luminescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 uM.

Protocol Summary: The PathHunter® DiscoverX OPRM1-U20S cell line was routinely cultured in 150 mm dishes at 37°C, 5% CO2 and 95% RH. The growth medium consisted of DMEM/F12 1:1 Media supplemented with 10% v/v heat inactivated fetal bovine serum, 25 mM HEPES, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 1x antibiotic mix (penicillin streptomycin). On Day 1 of the assay, 5000 cells in 20 μL of assay buffer (Discover X's Cell Plating Reagent 5) were seeded into each well of a 384-well plate, and incubated 16-24 hours at 37°C, 5% CO2 and 95% RH. On Day 2, 100 nL of test compound in DMSO were added to the appropriate wells and plates were incubated for 30 minutes at 37°C, 5% CO2 and 95% RH. Next, 2.2 μL of DAMGO OPRM1 agonist (EC80 Challenge; 1.8 μL of 3.7 μM DAMGO and 0.4 μL assay buffer; final assay concentration 303 nM) or DMSO in assay media were added. After incubation for 3 hours at 37°C, 5% CO2 and 95% RH, 10 μL of Path Hunter Detection Mix prepared according to manufacturer's protocol; 1 part Galacton Star:5 parts Emerald II:19 parts PH Cell Assay Buffer) was added to each well, and plates were incubated at room temperature in the dark for 1 hour. Well luminescence was measured on Perkin Elmer's Envision. Assay Cutoff: Compounds that inhibited OPRM1 >30% were considered active.

Secondary Assays

OPRK1 antagonist assay of SAR compounds (AID 652032, AID 652084)

Assay Overview: The purpose of this assay is to confirm the potency of test synthesized compounds. The assay is as described above (AID 652031, AID 652082, AID 65077). Compounds were tested triplicate (except for SID 144087324, which was tested in quadruplicate) using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 μM (AID 652032) or in either quadulplicate or octuplet using a 12-point, 1:3 dilution series starting at a nominal concentration of 10 μM (AID 652084).

Protocol Summary: The Tango OPRK1-U20S dividing cell line was cultured as described above (AIDs 652031, 652082, 65077). On day 1 of the assay, 16,000 cells in 10 μL of assay media (DMEM-Glutamax with sodium pyruvate, 10% fetal bovine serum stripped with charcoal-dextran, 25 mM HEPES, 0.1 mM non-essential amino acids, and antibiotic mix (penicillin streptomycin) were seeded into each well of a 384-well plate. On Day 2, 50 nL of test compound in DMSO were added to the appropriate wells and plates were incubated for 30 minutes at 37°C, 5% CO2 and 95% RH. Next, 0.6 uL of 111 nM U50488 (OPRK1 agonist EC80 Challenge; final concentration 6 nM) or DMSO in assay medium was added to appropriate wells and incubated 4 hours at 37°C, 5% CO2 and 95% RH. 2.5 μL of LiveBLazer (trade mark) FRET B/G (CCF4-AM) loading mix (prepared according to manufacturer's instructions; 6 μL solution A, 60 μL Solution B, 904 μL Solution C, and 30 μL Solution D) were added to each well, and plates incubated at room temperature in the dark for 2 hours. Well fluorescence was measured on Perkin Elmer's Envision using an Excitation filter 409 nm, Emission filters at 460 nm and 590 nm, bottom read. Assay Cutoff: Compounds with an IC50 of ≤10 μM were considered active.

OPRD1 Counterscreen assay of SAR compounds (AID 652033)

Assay Overview: The assay is as described above (AID 652080). Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 μM.

Protocol Summary: The Tango OPRD1-U20S dividing cell line was cultured and the assay was performed as described above (AID 652080). Assay Cutoff: Compounds with an IC50 of ≤10 μM were considered active.

OPRM1 Counterscreen assay of SAR compounds (AID 652034)

Assay Overview: The assay is as described above (AID 652081). Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 μM.

Protocol Summary: The PathHunter® DiscoverX OPRM1-U20S cell line was cultured and the assay was performed as described above (AID 652034). Assay Cutoff: Compounds with an IC50 of ≤10 μM were considered active.

Cytotoxicity assay (AID 652086)

Assay Overview: The purpose of this assay is to determine cytotoxicity of a powder compound that inhibits OPRK1. In this assay, U2OS cells are incubated with test compound, followed by determination of cell viability. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Compounds were tested in quadruplicate in a 12-point 1:3 dilution series starting at a nominal test concentration of 10 μM.

Protocol Summary: This assay was started by dispensing OPRK1-bla U20S cells in McCoy's 5A medium plus 10% FBS, penicillin 100 U/mL and streptomycin 100 μg/mL (20 μL, 4000 cells/well) into the wells of a 384-well plate. Twelve 1:3 serial dilutions of compound (100 μM in growth media) were made. 5 μL of diluted compound or media were added to wells. The plate was incubated at 37°C in a humidified incubator for 24 hours, then equilibrated to room temperature for 30 minutes. 25 μL CellTitre-Glo reagent was added to each well, followed by incubation of the plate in the dark for 10 minutes. Well luminescence was measured on the Envision plate reader. Assay Cutoff: Compounds with a CC50 value ≤10 μM were considered active (cytotoxic).

Pharmacokinetic assay: plasma protein binding (AID 652078)

Assay Overview: The purpose of this assay is to assess the percentages of a lead OPRK1 antagonist test compound that bind to human, mouse, and rat plasma proteins.

Protocol Summary: Plasma protein binding to human, mouse, and rat plasma was evaluated using equilibrium dialysis (Pierce RED system). Compound (1.0 μM) was added to the plasma compartment and after eight hours the concentration of drug was measured in the plasma and buffer compartments by LC-MS/MS. The concentration in the buffer compartment is considered to be the free fraction of compound. Assay Cutoff: For each plasma species, compounds that exhibited > 50% binding to plasma protein were considered active.

Pharmacokinetic assay: hepatic microsome stability (AID 652088)

Assay Overview: The purpose of this assay is to assess the stability of a lead OPRK1 antagonist test compound in the presence of pooled human, rat, mouse, monkey, and dog microsomes.

Protocol Summary: Test compound was incubated (separately) with 0.2 mg/mL pooled human, mouse, rat, monkey, and dog hepatic microsomes and cofactors to determine the rate of metabolism. Samples were collected at multiple time points and the concentration of test compound was determined using HPLC coupled to a triple quadrupole mass spectrometer (LC/MS-MS), allowing the calculation of half-life for each compound. Assay Cutoff: For each microsome species, compounds with a half-life of > 60 minutes were considered active.

Pharmacokinetic assay: cytochrome P450 inhibition (AID 652076)

Assay Overview: The purpose of this assay is to obtain information regarding potential drug-drug interactions for a lead OPRK1 antagonist test compound. Inhibition of the four major human isoforms of cytochrome P450 are evaluated by following the metabolism of specific marker substrates CYP1A2, CYP2C9, CYP2D6, and CYP3A4 in the presence test compound.

Protocol Summary: The metabolism of CYP1A2 (phenaceten demethylated to acetaminophen), CYP2C9 (tolbutamide hydroxylated to hydroxytolbutamide), CYP2D6 (bufuralol hydroxylated to 4′-hydroxybufuralol), and CYP3A4 (midazolam hydroxylated to 1′-hydroxymidazolam) in the presence or absence of 10 μM test compound were evaluated. The concentration of each marker substrate is approximately its Km. Specific inhibitors for each isoform were used to validate the system. Compound concentrations were determined by LC-MS/MS. Assay Cutoff: For each P450 isoform, compounds that inhibited ≤50% were considered active.

CEREP broad panel counterscreen of receptors, transporters and ion channels (AID 652083)

Assay Overview: The purpose of this panel of binding assays performed by CEREP is to identify a subset of potential receptors, transporters, ion channels, etc. for which a lead OPRK1 antagonist compound displays affinity. Assays were run in duplicate.

Protocol Summary: The panel assays were radioligand binding assays. Specific ligand binding to the targets was defined as the difference between the total binding in the presence of 10 μM test compound and the nonspecific binding determined in the presence of 10 μM compound and an excess of labeled ligand. The results are expressed as a percent inhibition of control specific binding. Assay Cutoff: For each assay target, inhibition of ≥50% was considered active.

CEREP hERG counterscreen assay (AID 652075)

Assay Overview: The purpose of this binding assay performed by CEREP is to determine whether a lead OPRK1 antagonist compound has activity against the potassium voltage-gated channel, hERG. The assay was run in duplicate.

Protocol Summary: The assay was a radioligand binding assay. Specific ligand binding to the target was defined as the difference between the total binding in the presence of 10 μM test compound and the nonspecific binding determined in the presence of 10 μM compound and an excess of unlabeled ligand. The results are expressed as a percent inhibition of control specific binding. Assay Cutoff: Inhibition of ≥50% was considered active.

In vivo pharmacokinetic assay: plasma and brain levels (AID 652085)

Assay Overview: The purpose of this assay is to assess the level of a lead OPRK1 antagonist compound in mouse plasma and brain at 30 minutes and 120 minutes after dosing.

Protocol Summary: Compounds were dosed IP at 10 mg/kg in a 1 mg/ml solution containing 1 part DMSO, 1 part Tween 80, and 8 parts water into C57Bl6 mice (n = 6). Blood and brain were taken at 30 minutes and 120 minutes. Blood was collected into EDTA-containing tubes and plasma was generated using standard centrifugation techniques. Brain was homogenized and proteins were precipitated with acetonitrile and compound concentrations were determined by LC-MS/MS. Data were fit by WinNonLin using a noncompartmental model and compound concentration in plasma and brain is calculated. Assay Cutoff: Compounds that exhibited a brain to plasma ratio of > 1 were considered active.

In vivo pharmacokinetic assay: parallel artificial membrane permeability assay (PAMPA) (AID 652113)

Assay Overview: The purpose of this assay is to assess the permeability of a lead OPRK1 antagonist test compound using a commercial Parallel Artificial Membrane Permeability Assay (PAMPA) kit.

Protocol Summary: An assessment of permeability was done using a commercial PAMPA kit. Compound was evaluated over a range of concentrations in 300 μL of PBS containing the compound in a well of the receiver plate, which is coupled to the bottom donor plate. The plates were allowed to incubate at room temperature. After 5 hours, aliquots were taken from the donor and receiver plates and the concentration of drug was determined. Propanolol and antiprine were used as positive controls; BHF177 and BLK998 were used as negative controls. Compound permeability was calculated.

In vivo tail flick assay (AID 652108)

Assay Overview: The purpose of this assay is to assess the effect of a lead OPRK antagonist test compound in the Tail Flick assay in mice. The Tail Flick assay is a pain receptive assay in which a mouse is placed within a restraining tube with its tail protruding. The tail is placed on a level surface, radiant heat is applied to the tail and the latency of the mouse to remove its tail from the heat is recorded. This latency is used as a measure to indicate neurological pathology. In this assay, the mice are administered an OPRK1 agonist (U-69593) and test compound, and the ability of test compound to block the analgesic effect of the agonist compound is measured.

Protocol Summary: This assay was performed by the Mouse Behavioral Assessment Core of The Scripps Research Institute. Ten mice each were pre-treated with test compound (administered i.p. at 10 mg/kg), OPRK1 antagonist NOR-BNI (administered s.c. 10 mg/kg), or vehicle. Mice were subsequently challenged with OPRK1 agonist U-69593 (administered i.p. at 2 mg/kg) at one hour, 24 hours, and 1 week post pre-treatment. After each agonist challenge, each moue was tested by application of a heat source three times and the latency time of the mouse to remove its tail from the heat was measured and reported in seconds.

2.2. Probe Chemical Characterization

Image ml350f2

The probe structure was verified by 1H and 13C NMR (see Section 2.3) and high resolution LC-MS (Figure 1). Purity was assessed to be greater than 95% by LC-MS.

Figure 1. LC/MS analysis of ML350.

Figure 1

LC/MS analysis of ML350.

Solubility (at room temperature) for ML350 in PBS was determined to be 557 μM. Solubility in water and saline was determined to be 1.1 mM. ML335 has a half-life of >48 hours in PBS at room temperature (79% compound remaining at 48 hours) (Figure 2).

Figure 2. Stability of ML350 (CYM50202) in PBS.

Figure 2

Stability of ML350 (CYM50202) in PBS.

No Michael acceptor adducts were observed when a sample of the probe was incubated with 50 μM glutathione and analyzed by LC-MS.

The following compounds have been submitted to the SMR collection (Table 1).

Table 1. Compounds submitted to the SMR collection (2-21-2013).

Table 1

Compounds submitted to the SMR collection (2-21-2013).

2.3. Probe Preparation

Figure 3. Synthetic scheme for ML350 (CYM50202).

Figure 3Synthetic scheme for ML350 (CYM50202)

To a stirred solution of trans-rac-N-Boc-4amino-3-hydroxy piperidine I and cyclohexanone in DCE were added NaBH(OAc)3 and AcOH. The reaction mixture was stirred overnight at room temperature. The mixture was diluted with ethyl acetate and washed with brine (2X). The organic phase was concentrated, and the product II purified by column chromatography using CH2Cl2/MeOH (9:1).

To a solution of II in CH2Cl2 was added TFA and the reaction mixture was stirred for 30 minutes at room temperature. The mixture was concentrated under reduced pressure. The residue was dissolved in ethanol followed by the addition of DIPEA and pyridine III. The reaction mixture was heated at 145°C for 35 minutes under microwave irradiation. The crude was concentrated under reduced pressure and the product purified by HPLC furnishing the pure compound ML350 (CYM50202; CID 60156214).

1H NMR and 13C NMR of methyl 5-bromo-2-(4-(cyclohexylamino)-3-hydroxypiperidin-1-yl)nicotinate are as follows: 1H NMR (600 MHz, CDCl3): δ 8.20 (s, 1H), 8.07 (dd, J = 9.5, 2.4 Hz, 1H), 3.95-3.90 (m, 2H), 3.83 (s, 3H), 3.79 (d, J = 13.3 Hz, 1H), 3.15 (bs, 2H), 2.89 (t, J = 12.4 Hz, 1H), 2.81 (t, J = 12.1 Hz, 1H), 2.07-2.02 (m, 3H), 1.90-1.82 (m, 3H), 1.67 (d, J = 12.4 Hz, 1H), 1.51 (q, J = 11.5 Hz, 1H), 11.4 (q, J = 11.4 Hz, 1H), 1.29-1.16 (m, 3H); 13C NMR (125 MHz, CDCl3): δ 166.63, 157.96, 152.16, 143.80, 115.87, 110.44, 68.27, 59.70, 56.08, 55.39, 53.34, 48.62, 30.61, 29.16, 27.24, 25.67, 25.47, 25.31. MS (EI) m/z: 412, 414 (M+H). The purity was assessed to be greater than 95% by LC-MS.

3. Results

3.1. Summary of Screening Results

Prior to implementing the OPRM1-OPRD1 agonist HTS screen of the MLPCN library, a series of pilot screens of the Maybridge Library was done at the SRIMSC Screening Center (Figure 4). Screens for each opioid receptor were run in agonist and antagonist mode. An OPRK1 antagonist screen with an activity cutoff of >50% inhibition identified 72 hits out of 16,000 compounds screened (1X%INH; AID 652031). A confirmation OPRK1 antagonist screen (3X%INH; AID 652082) and an S1P1 counterscreen (3X%INH; AID 652087) identified 11 compounds that confimed OPRK1 antagonism and were inactive at S1P1; these were repurchased as powders. These 11 compounds were screened in four dose response assays: OPRK1 antagonist (AID 652077), OPRM1 antagonist counterscreen (AID 652081), OPRD1 antagonist counterscreen (AID 652080), and S1P1 antagonist counterscreen (AID 652079). From these screens a hit compound that appeared to exhibit potency and selectivity as an OPRK antagonist was identified (CID 2796048) (Figure 5). Structural integrity and potency of CID 2796048 were verified by independent synthesis. Chemical archeology was performed on and around the structure. Medicinal chemistry optimization by SAR by purchase and synthesis was begun.

Figure 4. Flow chart describing Maybridge screening results.

Figure 4

Flow chart describing Maybridge screening results.

Figure 5. Compound identified from Maybridge library; CID 2796048.

Figure 5

Compound identified from Maybridge library; CID 2796048.

3.2. Dose Response Curve for Probe

Figure 6. Dose response curve for ML350.

Figure 6Dose response curve for ML350

3.3. Scaffold/Moiety Chemical Liabilities

A resolution of the trans-diastereomeric mixture will need to be performed in order to establish the individual antagonist activity and PK profile. The methyl ester metabolic “soft spot” may underlie rapid metabolism. Addressing these issues is fundamental in further development of this chemotype.

3.4. SAR Table

The original screening hit CID 2796048 can be conceptualized as consisting of three regions A, B, and C. Table 2 shows structures of compounds used for probe optimization.

Table 2. SAR Table for optimization of antagonist probe ML350 for OPRK.

Table 2

SAR Table for optimization of antagonist probe ML350 for OPRK.

The HTS hit compound (entry 1) was purchased (SID 26534319) with confirmed IC50s of 410 nM at OPRK1, 4.59 μM at OPRM1 and no demonstrated OPRD1 activity at concentrations up to 50 μM. Compound 1 does not contain reactive functional groups, is chemically amiable and is moderately and highly selective against the OPRM1 and OPRD1, thus making this compound suitable for medicinal chemistry optimization.

Pyridine region (A): We started our SAR studies by modifying the two metabolic soft spots (ester groups) in region A using the piperidine-4-yl and cyclohexylamine as regions B and C. 4-cyclohexylamine piperidine was the constant moiety to simplify the synthetic work and allow rapid screening of region A. First, we sought to minimize the structure of the hit (Andrews' analysis) by reducing the number of substituents on the pyridine ring. Interestingly, the 5-acetamide (entry 2) was found less that 2-fold less potent than compound 1. Changing the acetamide group for halogens led to an interesting series of compounds. The increase in potency was inversely related to the electronegativity of the halogen atoms: the fluoride (entry 6) was ∼16-fold less potent than compound 1, while the iodine (entry 3), bromide (entry 4) and chloride (entry 5) were ∼5-, ∼3-, ∼2.5-fold more potent, respectively. Conversely, the selectivity against the OPRM1 increased directly proportionally to their electronegativity: iodide, bromide and chloride were ∼6-, 7.5- and 12.5-fold selective against the OPRM. Interestingly the selectivity against the OPRD1 was higher for the bromide (143-fold) followed by the iodide (32-fold) and chloride (25-fold). Installing a phenyl ring (entry 7) on the 5-position led to complete loss of potency. Replacing the methyl ester of compound 4 for an isopropyl ester furnished compound 8 with ∼3-fold loss in potency at the OPRK1. A decrease in selectivity against the OPRD (7.5-fold) and an increase in selectivity against the OPRM1 (50-fold) were observed, indicating that steric factors at the 3-position of ring A are important for modulating potency and selectivity. A 39-fold loss in potency was observed for the carboxylic acid (entry 9) compared to compound 4.

Cyclohexylamine region (C): The cyclohexylamine analog of compound 1 (entry 10) was ∼6-fold less potent at OPRK1. To continue our SAR studies we selected the methyl 5-bromopyridine-3-carboxylate moiety from compound 4 due to its potency (19.5-fold more potent than compound 10) and selectivity profile. The 1,2-trans-aminoalcohol (entry 11), 1,2-cis-aminoalcohol (entry 12) and diastereomeric 1,3-aminoalcohol (entry 13) were ∼2-, ∼4-, and ∼5-fold less potent than the un-substituted compound 4, respectively. The selectivity against the OPRM1 of trans- and cis-aminoalchol were 12- and 33-fold, respectively, while the selectivity of the 1,3-aminoalcohol was 25-fold. Drastic loss of potency resulted from the noncyclic 1,2-aminoalcohol (entry 14). The diasteromeric 2-methoxycycloxylamine (entry 15) was nearly equipotent to the trans-1,2-aminoalcohol (entry 11), but the selectivity against the OPRM1 was 3.7-fold bigger. Introducing a methyl group at the 4-position of the cyclohexyl group (entry 16) led to 4-fold loss of potency at the OPRK, 2-fold increase in selectivity against OPRM1 and ∼4-fold decrease in selectivity against the OPRD. Introducing an oxygen atom into the cyclohexyl ring (entry 17) led to 4.5-fold loss in potency at the OPRK1, but the selectivity against OPRM1 was ∼11-fold higher than for compound 4. Increasing (cyloheptyl, entry 18) or decreasing (cyclopentyl, entry 19) the ring size did not affect the potency at the OPRK1, whereas it increased the selectivity against the OPRM (4-5-fold) and decreased the selectivity against the OPRD1 (1.2-2.4-fold). Moving the cyclohexyl ring (entry 20) or the cyclohexylamine (entry 21) one methylene further from ring B led to 51-and 26-fold loss in potency, respectively. Methylation of the amino group (entry 22) led to 20-fold loss in potency. When the cyclohexyl group was removed (entry 23), the basicity of the amine reduced (entry 24) or the nitrogen replaced by oxygen (entry 25), the potency was completely lost. Taken together, it can be concluded that the basicity of the amine and its position in relation to the pyridine ring are fundamental for the activity at the OPRK1, while the installation of polar groups decreases the potency for the OPRK1, has small impact on the selectivity against OPRD1 but a greater impact against the OPRM1. A small lipophilic portion is important for the activity at the OPRK, however long lipophilic moieties lead to a decrease in potency on both OPRK1 and OPRM1.

Piperidine region (B): When the cyclohexylamine was moved from 4- to 3-position (entry 26) within the piperidine ring a complete loss of potency was observed. Interestingly, when a pyrrolidine system (entry 27) was introduced the potency slightly decreased. Adding a methyl group on position 3 (entry 28) led to small loss in potency. Interestingly, installing a hydroxyl group oriented in trans to the amino group (entry 29) led to a substantial increase of potency at OPRK (8-14.5-fold) and the selectivity against the OPRM1 and OPRD1 were 20-35- and 219-382-fold, respectively.

Complementary SAR on compound 29: Removing the cyclohexyl group (entry 30) led to a 368-644–fold loss in potency, as similarly observed for compound 23. Removing the bromine from position 5 of the pyridine ring led to 137-240-fold loss in potency.

3.5. Cellular Activity

ML350 has been evaluated in a series of cell-based assays (the OPRK1 primary and OPRD1, OPRM1, and S1P1 counterscreen assays, and a cytotoxicity assay) and shown to have activity in a cell-based system.

3.6. Profiling Assays

ML350 was submitted to CEREP for broad panel screening of 52 protein targets (AID 652083), including hERG (AID 662075). The purpose of this panel of binding assays was to identify potential receptors, transporters, or ion channels for which ML350 displays affinity. ML350 exhibits 67% and 71% inhibition of the Na+ channel (site 2) and the NOP receptor, respectively (Table 3). The IC50s were determined to be 1.8 μM and 3 μM for these two targets, respectively. Thus, ML350 is >100-fold selective for OPRK1 over the Na+ channel (site 2) and approximately 200-fold selective over the NOP receptor. These data suggest that ML350 is generally inactive against a broad array of off targets and does not likely exert unwanted effects.

Table 3. Targets that exhibit ≥ 50% inhibition by ML350 in CEREP screen.

Table 3

Targets that exhibit ≥ 50% inhibition by ML350 in CEREP screen.

3.7. Pharmacokinetic Assays

Permeability: ML350 has high passive membrane permeability as measured in a PAMPA assay (AID 652113) as compared to standard compounds (Figure 7).

Figure 7. ML350 has high passive membrane permeability, similar to positive controls propanolol and antipirine.

Figure 7

ML350 has high passive membrane permeability, similar to positive controls propanolol and antipirine.

Brain penetration: ML350 exhibited good brain penetration when administered by intraperitoneal (IP) injection in mice (AID 662085). Thus, dosed at 10 mg/kg in mice, ML350 (1 mg/mL in 10/10/80 DMSO/Tween/Water), reached brain at levels 3 times higher than plasma (Table 4).

Table 4. Levels of ML350 in plasma and brain of mice 30 and 120 minutes after IP administration.

Table 4

Levels of ML350 in plasma and brain of mice 30 and 120 minutes after IP administration.

Cytochrome P450 inhibition: ML350 had no significant activity at 3 of 4 human cytochrome P450 subtypes (Table 5, AID 652076). ML350 inhibited CYP2D6 by 89% at 10 µM.

Table 5. Inhibition of human cytochrome P450 subtypes 1A2, 2C9, 2D6, and 3A4 by ML350.

Table 5

Inhibition of human cytochrome P450 subtypes 1A2, 2C9, 2D6, and 3A4 by ML350. Furafylline, sulfaphenazole, quinidine, and ketoconazone were the positive controls used for CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively.

Plasma protein binding: ML350 (1uM) had modest plasma protein binding in human (73%), but was higher in rodent, 96% in mouse and 99% in rat (AID 652078, Table 6).

Table 6. Binding of ML350 to human, mouse, and rat plasma proteins.

Table 6

Binding of ML350 to human, mouse, and rat plasma proteins.

Hepatic microsome stability: In vitro hepatic microsomal stability of ML350 was measured (AID 652086, Table 7). The half life in 0.2 mg/mL hepatic microsomes was >120, 20, 5, 13 and >120 minutes in human, rat, mouse, monkey and dog hepatic microsomes, respectively.

Table 7. Hepatic microsome stability of ML350.

Table 7

Hepatic microsome stability of ML350.

Rat pharmacokinetics: ML350 has an encouraging in vivo pharmacokinetic profile in rats (Table 8). The oral results are particularly exciting with, concentrations in plasma 10 times the cell-based IC50 after the relatively modest dose of 2 mg/kg.

Table 8. In vivo pharmacokinetic profile of ML350 in rats.

Table 8

In vivo pharmacokinetic profile of ML350 in rats. ML350 was administered by IV at 1 mg/kg, or by mouth at 2 mg/kg

3.8. Reversibility of Analgesic Effects in Mice

In order to assess the reversibility of analgesic effects of ML350 in mice, a tail flick assay was used (AID 652108, Figure 8). This is a pain receptive assay in which a mouse is placed within a restraining tube with its tail protruding. The tail is placed on a level surface, radiant heat is applied to the tail and the latency of the mouse to remove its tail from the heat is recorded. This latency is used as a measure to indicate neurological pathology. In this assay, the mice are administered ML350, vehicle, or the OPRK1 antagonist Nor-BNI, and subsequently challenged with OPRK agonist U-69,593 at one hour, 24 hours, and 1 week post pre-treatment. The ability of ML350 to block the analgesic effect of the agonist compound is measured. After each agonist challenge, each moue was tested by application of a heat source and the latency time of the mouse to remove its tail from the heat is measured and reported in seconds. The ability of ML350 to block the analgesic effect of the agonist is gone after 24 hours, whereas Nor-BNI is still efficacious after 1 week.

Figure 8. Results of Tail Flick assay to determine the reversibility of the analgesic effect of ML350.

Figure 8

Results of Tail Flick assay to determine the reversibility of the analgesic effect of ML350. Mice received ML350 (5 mg/kg) administered IP, Nor-BNI (10 mg/kg) administered SC, or vehicle/. Mice were then challenged with agonist U-69,593 (2mg/kg) administered (more...)

4. Discussion

ML350 was identified by high-throughput screening using a cell-based Tango™-format assay. A set of pharmacokinetic analyses show that ML350 has high passive membrane permeability, good brain penetration, no significant activity at three of four human cytochrome P450 subtypes, high binding for rodent plasma protein and modest binding for human plasma protein, and an encouraging in vivo pharmacokinetic profile in rats. ML350 was submitted to CEREP for broad panel screening against a panel of receptors, transporters, and ion channels; the data suggest that ML350 is generally inactive against a broad array of off targets and does not likely exert unwanted effects. Importantly, ML350 was shown to have a reversible analgesic effect when challenged with an OPRK1 agonist in a tail flick assay in mice.

4.1. Comparison to existing art and how the new probe is an improvement

Several OPRK1 antagonists have been described in the literature. Norbinaltorphimine (nor-BNI) [89-90], 5′-guanidinonaltrindole (GNTI) [89, 91], and JDTic [89, 92] all exhibit a delay in the onset of action of approximately 24 hours [89, 93-95], and have very long lasting in vivo effects of up to 56 days [89, 95-96]. Due to their pharmacodynamics/pharmacokinetics and poor unbound brain/plasma ratio, morphine-like derivatives, nor-BNI and GNTI, are only used as pharmacological tools. The non-opioid compound JDTic exhibited a poor brain exposure but had an extraordinary persistence in brain (mean brain concentration declined by only 56% over 24 hours, and the drug was still detectable at 1 week in mice) despite its P-glycoprotein-mediated efflux, and its moderate lipophilicity and low affinity for cell homogenates. The presence of two basic nitrogens and entrapment in cellular compartments such as lysosomes have been proposed as a possible mechanism of this persistence [89, 97]. A Phase 1 clinical trial of JDTic for cocaine dependence was terminated in 2012 due to adverse events. Several short-acting compounds from distinct chemotypes have been developed with greater CNS exposure compared to the prototypic ligands (nor-BNI, GNTI, JDTic). AstraZeneca disclosed a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides. AZ-MTAB was efficacious in animal models of mood disorders, but was associated with a significant hERG liability (IC50 of 260 nM) [97-98]. Another OPRK1 antagonist PF-04455242 [99] was developed by Pfizer. However, on January 6, 2010 the phase 1 study for dipolar disorder and depression was terminated due to toxicology findings in animals exposed to PF-04455242 for three months. Eli Lilly identified a subnanomolar OPRK1 antagonist with selectivity of 21 and 135 over OPRM1 and OPRD1 respectively [100]. In 2010 LY2456302 was advanced to phase I clinical trial for the oral treatment of alcohol dependence. The clinical study was to assess the brain OPRK1 occupancy after single oral doses of LY2456302 as measured by positron emission tomography with radioligand LY2879788 (11C PKAB) in healthy subjects. The last update of the study was May 5, 2011, and no new studies for the compound are listed in ClinicalTrials.gov.

Even though a few drug-like OPRK1 antagonists with more favorable pharmacokinetics than nor-BNI, GNTI, JDTic have been developed, new OPRK1 antagonists possessing novel scaffolds and improved selectivity are still needed both as pharmacological tools to better understand the OPRK1-dynorphin system and as potential pharmacotherapies.

Table 9Prior Art OPRK Antagonist Compounds

Compound NameStructureOPRK
IC50 (nM)
Selectivity (nM)B/P*Ref(s)
Norbinaltorphimine (nor-BNI)
Image ml350fu95.jpg
0.07 ± 0.03OPRM: IC50 15.8 ± 5.7; OPRD: IC50 12.1 ± 3.1< 0.05[92, 97]
5′-guanidinonaltrindole (GNTI)
Image ml350fu96.jpg
0.04OPRM: IC50 3.2; OPRD: IC50 15.5< 0.0007[97, 102]
JDTic
Image ml350fu97.jpg
0.006 ± 0.001OPRM: IC50 3.42 ± 0.83; OPRD: IC50 > 100< 0.05[92, 97]
AZ-MTAB
Image ml350fu98.jpg
20 ± 3OPRM: IC50 722 ± 47; OPRD: IC50 8306 ± 1635; (hERG: IC50 260 nM)∼ 1[97-98]
PF-04455242
Image ml350fu99.jpg
1.23OPRM: IC50 10 nM; OPRD: Ki > 4000∼ 1[99, 103]
LY2456302
Image ml350fu100.jpg
0.813 ± 0.285OPRM: IC50 17.4 ± 6.33; OPRD: IC50 110 ± 33.6∼ 1[97, 100]
*

Unbound brain/plasma ratio

4.2. Mechanism of Action Studies

Planned future studies (see below) will help elucidate the mechanism of action of ML350 and future improved analogs. New OPRK1 antagonists possessing novel scaffolds and improved selectivity will serve as useful pharmacological tools to better understand the OPRK-dynorphin system.

4.3. Planned Future Studies

Additional medicinal chemistry to improve potency, selectivity, and pharmacokinetic properties is in progress. Working with collaborators, we plan to test ML350 and improved analogs in: 1) a stress-induced migraine model assay that measures cutaneous (tactile) allodynia in rats as a measurable translational endpoint for headache-related pain [5], 2) an alcohol withdrawal assay that measures working memory performance and anxiety-like behavior in rats after withdrawal from alcohol [6], and 3) a cocaine reward assay that measures the effect on drug-seeking behavior in a cocaine addiction model. A CNS-penetrant single oral daily dosed picomolar OPRK antagonist with 3 logs selectivity of OPRM1 and OPRD1 for titration of OPRK1 tone in depression and psychosis is the desired goal.

5. References

1.
Kuzmin AV, Gerrits MA, Van Ree JM. Kappa-opioid receptor blockade with nor-binaltorphimine modulates cocaine self-administration in drug-naive rats. Eur J Pharmacol. 1998;358(3):197–202. [PubMed: 9822884]
2.
Mague SD, et al. Antidepressant-like effects of kappa-opioid receptor antagonists in the forced swim test in rats. J Pharmacol Exp Ther. 2003;305(1):323–30. [PubMed: 12649385]
3.
Jewett DC, et al. The kappa-opioid antagonist GNTI reduces U50,488-, DAMGO-, and deprivation-induced feeding, but not butorphanol- and neuropeptide Y-induced feeding in rats. Brain Res. 2001;909(1-2):75–80. [PubMed: 11478923]
4.
Roth BL, et al. Salvinorin A: a potent naturally occurring nonnitrogenous kappa opioid selective agonist. Proc Natl Acad Sci U S A. 2002;99(18):11934–9. [PMC free article: PMC129372] [PubMed: 12192085]
5.
De Felice M, et al. Triptan-induced enhancement of neuronal nitric oxide synthase in trigeminal ganglion dural afferents underlies increased responsiveness to potential migraine triggers. Brain. 2010;133(Pt 8):2475–88. [PMC free article: PMC3139937] [PubMed: 20627971]
6.
George O, et al. Recruitment of medial prefrontal cortex neurons during alcohol withdrawal predicts cognitive impairment and excessive alcohol drinking. Proc Natl Acad Sci U S A. 2012;109(44):18156–61. [PMC free article: PMC3497825] [PubMed: 23071333]
7.
Belcheva MM, et al. Mu and kappa opioid receptors activate ERK/MAPK via different protein kinase C isoforms and secondary messengers in astrocytes. J Biol Chem. 2005;280(30):27662–9. [PMC free article: PMC1400585] [PubMed: 15944153]
8.
Bruchas MR, Chavkin C. Kinase cascades and ligand-directed signaling at the kappa opioid receptor. Psychopharmacology (Berl) 2010;210(2):137–47. [PMC free article: PMC3671863] [PubMed: 20401607]
9.
Bruchas MR, et al. Stress-induced p38 mitogen-activated protein kinase activation mediates kappa-opioid-dependent dysphoria. J Neurosci. 2007;27(43):11614–23. [PMC free article: PMC2481272] [PubMed: 17959804]
10.
Bruchas MR, et al. Kappa opioid receptor activation of p38 MAPK is GRK3- and arrestin-dependent in neurons and astrocytes. J Biol Chem. 2006;281(26):18081–9. [PMC free article: PMC2096730] [PubMed: 16648139]
11.
Bruchas MR, et al. Selective p38alpha MAPK deletion in serotonergic neurons produces stress resilience in models of depression and addiction. Neuron. 2011;71(3):498–511. [PMC free article: PMC3155685] [PubMed: 21835346]
12.
Childers SR, et al. Opiate receptor binding affected differentially by opiates and opioid peptides. Eur J Pharmacol. 1979;55(1):11–8. [PubMed: 220062]
13.
Hahn JW, et al. Mu and kappa opioids modulate mouse embryonic stem cell-derived neural progenitor differentiation via MAP kinases. J Neurochem. 2010;112(6):1431–41. [PMC free article: PMC2856797] [PubMed: 19895666]
14.
Walwyn WM, Miotto KA, Evans CJ. Opioid pharmaceuticals and addiction: the issues, and research directions seeking solutions. Drug Alcohol Depend. 2010;108(3):156–65. [PMC free article: PMC3072810] [PubMed: 20188495]
15.
Xu M, et al. Sciatic nerve ligation-induced proliferation of spinal cord astrocytes is mediated by kappa opioid activation of p38 mitogen-activated protein kinase. J Neurosci. 2007;27(10):2570–81. [PMC free article: PMC2104780] [PubMed: 17344394]
16.
Melief EJ, et al. Ligand-directed c-Jun N-terminal kinase activation disrupts opioid receptor signaling. Proc Natl Acad Sci U S A. 2010;107(25):11608–13. [PMC free article: PMC2895055] [PubMed: 20534436]
17.
Melief EJ, et al. Duration of action of a broad range of selective kappa-opioid receptor antagonists is positively correlated with c-Jun N-terminal kinase-1 activation. Mol Pharmacol. 2011;80(5):920–9. [PMC free article: PMC3198912] [PubMed: 21832171]
18.
Finley MJ, et al. Transcriptional regulation of the major HIV-1 coreceptor, CXCR4, by the kappa opioid receptor. J Leukoc Biol. 2011;90(1):111–21. [PMC free article: PMC3114596] [PubMed: 21447649]
19.
Bruchas MR, Xu M, Chavkin C. Repeated swim stress induces kappa opioid-mediated activation of extracellular signal-regulated kinase 1/2. Neuroreport. 2008;19(14):1417–22. [PMC free article: PMC2641011] [PubMed: 18766023]
20.
McLennan GP, et al. Kappa opioids promote the proliferation of astrocytes via Gbetagamma and beta-arrestin 2-dependent MAPK-mediated pathways. J Neurochem. 2008;107(6):1753–65. [PMC free article: PMC2606093] [PubMed: 19014370]
21.
Potter DN, et al. Repeated exposure to the kappa-opioid receptor agonist salvinorin A modulates extracellular signal-regulated kinase and reward sensitivity. Biol Psychiatry. 2011;70(8):744–53. [PMC free article: PMC3186866] [PubMed: 21757186]
22.
Oderda G. Challenges in the management of acute postsurgical pain. Pharmacotherapy. 2012;32(9 Suppl):6S–11S. [PubMed: 22956493]
23.
Kieffer BL, et al. The delta-opioid receptor: isolation of a cDNA by expression cloning and pharmacological characterization. Proc Natl Acad Sci U S A. 1992;89(24):12048–52. [PMC free article: PMC50695] [PubMed: 1334555]
24.
Mansson E, Bare L, Yang D. Isolation of a human kappa opioid receptor cDNA from placenta. Biochem Biophys Res Commun. 1994;202(3):1431–7. [PubMed: 8060324]
25.
Wang JB, et al. mu opiate receptor: cDNA cloning and expression. Proc Natl Acad Sci U S A. 1993;90(21):10230–4. [PMC free article: PMC47748] [PubMed: 8234282]
26.
Xu M, et al. Neuropathic pain activates the endogenous kappa opioid system in mouse spinal cord and induces opioid receptor tolerance. J Neurosci. 2004;24(19):4576–84. [PMC free article: PMC2376823] [PubMed: 15140929]
27.
Chavkin C, James IF, Goldstein A. Dynorphin is a specific endogenous ligand of the kappa opioid receptor. Science. 1982;215(4531):413–5. [PubMed: 6120570]
28.
Muschamp JW, Van't Veer A, Carlezon WA Jr. Tracking down the molecular substrates of stress: new roles for p38alpha MAPK and kappa-opioid receptors. Neuron. 2011;71(3):383–5. [PMC free article: PMC3155977] [PubMed: 21835335]
29.
Wagner R, et al. Spinal dynorphin immunoreactivity increases bilaterally in a neuropathic pain model. Brain Res. 1993;629(2):323–6. [PubMed: 7906604]
30.
Wang Z, et al. Pronociceptive actions of dynorphin maintain chronic neuropathic pain. J Neurosci. 2001;21(5):1779–86. [PubMed: 11222667]
31.
Nakazawa T, et al. Spinal kappa receptor-mediated analgesia of E-2078, a systemically active dynorphin analog, in mice. J Pharmacol Exp Ther. 1991;256(1):76–81. [PubMed: 1671100]
32.
Kolesnikov Y, et al. Peripheral kappa 1-opioid receptor-mediated analgesia in mice. Eur J Pharmacol. 1996;310(2-3):141–3. [PubMed: 8884210]
33.
Obara I, et al. Antagonists of the kappa-opioid receptor enhance allodynia in rats and mice after sciatic nerve ligation. Br J Pharmacol. 2003;140(3):538–46. [PMC free article: PMC1574046] [PubMed: 12970097]
34.
Aldrich JV, McLaughlin JP. Peptide kappa opioid receptor ligands: potential for drug development. AAPS J. 2009;11(2):312–22. [PMC free article: PMC2691465] [PubMed: 19430912]
35.
WHO. Mental health: a new understanding, new hope. The world health report. 2001.
36.
Tejeda HA, Shippenberg TS, Henriksson R. The dynorphin/kappa-opioid receptor system and its role in psychiatric disorders. Cell Mol Life Sci. 2012;69(6):857–96. [PubMed: 22002579]
37.
Yoo JH, Kitchen I, Bailey A. The endogenous opioid system in cocaine addiction: what lessons have opioid peptide and receptor knockout mice taught us? Br J Pharmacol. 2012;166(7):1993–2014. [PMC free article: PMC3402766] [PubMed: 22428846]
38.
Nestler EJ, Aghajanian GK. Molecular and cellular basis of addiction. Science. 1997;278(5335):58–63. [PubMed: 9311927]
39.
Sivam SP. Cocaine selectively increases striatonigral dynorphin levels by a dopaminergic mechanism. J Pharmacol Exp Ther. 1989;250(3):818–24. [PubMed: 2476548]
40.
Bailey A, et al. Downregulation of kappa-opioid receptors in basolateral amygdala and septum of rats withdrawn for 14 days from an escalating dose “binge” cocaine administration paradigm. Synapse. 2007;61(10):820–6. [PubMed: 17621646]
41.
Collins SL, et al. Chronic cocaine increases kappa-opioid receptor density: lack of effect by selective dopamine uptake inhibitors. Synapse. 2002;45(3):153–8. [PubMed: 12112394]
42.
Hurd YL, Herkenham M. Molecular alterations in the neostriatum of human cocaine addicts. Synapse. 1993;13(4):357–69. [PubMed: 7683144]
43.
Mash DC, et al. Dopamine transport function is elevated in cocaine users. J Neurochem. 2002;81(2):292–300. [PubMed: 12064476]
44.
Rosin A, et al. Downregulation of kappa opioid receptor mRNA levels by chronic ethanol and repetitive cocaine in rat ventral tegmentum and nucleus accumbens. Neurosci Lett. 1999;275(1):1–4. [PubMed: 10554970]
45.
Schroeder JA, Niculescu M, Unterwald EM. Cocaine alters mu but not delta or kappa opioid receptor-stimulated in situ [35S]GTPgammaS binding in rat brain. Synapse. 2003;47(1):26–32. [PubMed: 12422370]
46.
Spangler R, et al. Regulation of kappa opioid receptor mRNA in the rat brain by “binge' pattern cocaine administration and correlation with preprodynorphin mRNA. Brain Res Mol Brain Res. 1996;38(1):71–6. [PubMed: 8737669]
47.
Spangler R, et al. Prodynorphin, proenkephalin and kappa opioid receptor mRNA responses to acute “binge” cocaine. Brain Res Mol Brain Res. 1997;44(1):139–42. [PubMed: 9030708]
48.
Staley JK, et al. Kappa2 opioid receptors in limbic areas of the human brain are upregulated by cocaine in fatal overdose victims. J Neurosci. 1997;17(21):8225–33. [PubMed: 9334398]
49.
Turchan J, et al. Effects of repeated psychostimulant administration on the prodynorphin system activity and kappa opioid receptor density in the rat brain. Neuroscience. 1998;85(4):1051–9. [PubMed: 9681945]
50.
Unterwald EM, Kreek MJ, Cuntapay M. The frequency of cocaine administration impacts cocaine-induced receptor alterations. Brain Res. 2001;900(1):103–9. [PubMed: 11325352]
51.
McLaughlin JP, Marton-Popovici M, Chavkin C. Kappa opioid receptor antagonism and prodynorphin gene disruption block stress-induced behavioral responses. J Neurosci. 2003;23(13):5674–83. [PMC free article: PMC2104777] [PubMed: 12843270]
52.
Shippenberg TS, Zapata A, Chefer VI. Dynorphin and the pathophysiology of drug addiction. Pharmacol Ther. 2007;116(2):306–21. [PMC free article: PMC2939016] [PubMed: 17868902]
53.
Zimmer A, et al. Absence of delta -9-tetrahydrocannabinol dysphoric effects in dynorphin-deficient mice. J Neurosci. 2001;21(23):9499–505. [PubMed: 11717384]
54.
Gawin FH. Cocaine addiction: psychology and neurophysiology. Science. 1991;251(5001):1580–6. [PubMed: 2011738]
55.
Carey AN, et al. Reinstatement of cocaine place-conditioning prevented by the peptide kappa-opioid receptor antagonist arodyn. Eur J Pharmacol. 2007;569(1-2):84–9. [PMC free article: PMC1994084] [PubMed: 17568579]
56.
Chefer VI, et al. Endogenous kappa-opioid receptor systems regulate mesoaccumbal dopamine dynamics and vulnerability to cocaine. J Neurosci. 2005;25(20):5029–37. [PMC free article: PMC1405843] [PubMed: 15901784]
57.
Di Chiara G, Imperato A. Opposite effects of mu and kappa opiate agonists on dopamine release in the nucleus accumbens and in the dorsal caudate of freely moving rats. J Pharmacol Exp Ther. 1988;244(3):1067–80. [PubMed: 2855239]
58.
McLaughlin JP, et al. Prior activation of kappa opioid receptors by U50,488 mimics repeated forced swim stress to potentiate cocaine place preference conditioning. Neuropsychopharmacology. 2006;31(4):787–94. [PMC free article: PMC2096772] [PubMed: 16123754]
59.
Mori T, et al. Effects of a newly synthesized kappa-opioid receptor agonist, TRK-820, on the discriminative stimulus and rewarding effects of cocaine in rats. Psychopharmacology (Berl) 2002;161(1):17–22. [PubMed: 11967626]
60.
Redila VA, Chavkin C. Stress-induced reinstatement of cocaine seeking is mediated by the kappa opioid system. Psychopharmacology (Berl) 2008;200(1):59–70. [PMC free article: PMC2680147] [PubMed: 18575850]
61.
Simonin F, et al. Disruption of the kappa-opioid receptor gene in mice enhances sensitivity to chemical visceral pain, impairs pharmacological actions of the selective kappa-agonist U-50,488H and attenuates morphine withdrawal. EMBO J. 1998;17(4):886–97. [PMC free article: PMC1170438] [PubMed: 9463367]
62.
Zhang Y, et al. Effect of the kappa opioid agonist R-84760 on cocaine-induced increases in striatal dopamine levels and cocaine-induced place preference in C57BL/6J mice. Psychopharmacology (Berl) 2004;173(1-2):146–52. [PubMed: 14712342]
63.
Jackson KJ, et al. Effect of the selective kappa-opioid receptor antagonist JDTic on nicotine antinociception, reward, and withdrawal in the mouse. Psychopharmacology (Berl) 2010;210(2):285–94. [PMC free article: PMC2866121] [PubMed: 20232057]
64.
Matsuzawa S, et al. Different roles of mu-, delta- and kappa-opioid receptors in ethanol-associated place preference in rats exposed to conditioned fear stress. Eur J Pharmacol. 1999;368(1):9–16. [PubMed: 10096764]
65.
Sperling RE, et al. Endogenous kappa-opioid mediation of stress-induced potentiation of ethanol-conditioned place preference and self-administration. Psychopharmacology (Berl) 2010;210(2):199–209. [PubMed: 20401606]
66.
Glick SD, et al. Kappa opioid inhibition of morphine and cocaine self-administration in rats. Brain Res. 1995;681(1-2):147–52. [PubMed: 7552272]
67.
Nylander I, Vlaskovska M, Terenius L. The effects of morphine treatment and morphine withdrawal on the dynorphin and enkephalin systems in Sprague-Dawley rats. Psychopharmacology (Berl) 1995;118(4):391–400. [PubMed: 7568625]
68.
Henriksen G, Willoch F. Imaging of opioid receptors in the central nervous system. Brain. 2008;131(Pt 5):1171–96. [PMC free article: PMC2367693] [PubMed: 18048446]
69.
Shippenberg TS. The dynorphin/kappa opioid receptor system: a new target for the treatment of addiction and affective disorders? Neuropsychopharmacology. 2009;34(1):247. [PubMed: 19079072]
70.
Schwarzer C. 30 years of dynorphins--new insights on their functions in neuropsychiatric diseases. Pharmacol Ther. 2009;123(3):353–70. [PMC free article: PMC2872771] [PubMed: 19481570]
71.
Bortolato M, Solbrig MV. The price of seizure control: dynorphins in interictal and postictal psychosis. Psychiatry Res. 2007;151(1-2):139–43. [PubMed: 17395273]
72.
Sheffler DJ, Roth BL. Salvinorin A: the “magic mint” hallucinogen finds a molecular target in the kappa opioid receptor. Trends Pharmacol Sci. 2003;24(3):107–9. [PubMed: 12628350]
73.
Bortolato M, et al. Kappa opioid receptor activation disrupts prepulse inhibition of the acoustic startle in rats. Biol Psychiatry. 2005;57(12):1550–8. [PubMed: 15953492]
74.
Bruchas MR, Land BB, Chavkin C. The dynorphin/kappa opioid system as a modulator of stress-induced and pro-addictive behaviors. Brain Res. 2010;1314:44–55. [PMC free article: PMC2819621] [PubMed: 19716811]
75.
Knoll AT, Carlezon WA Jr. Dynorphin, stress, and depression. Brain Res. 2010;1314:56–73. [PMC free article: PMC2819644] [PubMed: 19782055]
76.
Carlezon WA Jr, et al. Depressive-like effects of the kappa-opioid receptor agonist salvinorin A on behavior and neurochemistry in rats. J Pharmacol Exp Ther. 2006;316(1):440–7. [PubMed: 16223871]
77.
Land BB, et al. The dysphoric component of stress is encoded by activation of the dynorphin kappa-opioid system. J Neurosci. 2008;28(2):407–14. [PMC free article: PMC2612708] [PubMed: 18184783]
78.
Knoll AT, et al. Anxiolytic-like effects of kappa-opioid receptor antagonists in models of unlearned and learned fear in rats. J Pharmacol Exp Ther. 2007;323(3):838–45. [PubMed: 17823306]
79.
Pliakas AM, et al. Altered responsiveness to cocaine and increased immobility in the forced swim test associated with elevated cAMP response element-binding protein expression in nucleus accumbens. J Neurosci. 2001;21(18):7397–403. [PMC free article: PMC4205577] [PubMed: 11549750]
80.
McLaughlin JP, et al. Social defeat stress-induced behavioral responses are mediated by the endogenous kappa opioid system. Neuropsychopharmacology. 2006;31(6):1241–8. [PMC free article: PMC2096774] [PubMed: 16123746]
81.
Willner P. The validity of animal models of depression. Psychopharmacology (Berl) 1984;83(1):1–16. [PubMed: 6429692]
82.
Porsolt RD, et al. Behavioural despair in rats: a new model sensitive to antidepressant treatments. Eur J Pharmacol. 1978;47(4):379–91. [PubMed: 204499]
83.
Newton SS, et al. Inhibition of cAMP response element-binding protein or dynorphin in the nucleus accumbens produces an antidepressant-like effect. J Neurosci. 2002;22(24):10883–90. [PubMed: 12486182]
84.
Shirayama Y, et al. Stress increases dynorphin immunoreactivity in limbic brain regions and dynorphin antagonism produces antidepressant-like effects. J Neurochem. 2004;90(5):1258–68. [PubMed: 15312181]
85.
Artaiz I, Zazpe A, Del Rio J. Characterization of serotonergic mechanisms involved in the behavioural inhibition induced by 5-hydroxytryptophan in a modified light-dark test in mice. Behav Pharmacol. 1998;9(2):103–12. [PubMed: 10065930]
86.
Bagdy G, et al. Anxiety-like effects induced by acute fluoxetine, sertraline or m-CPP treatment are reversed by pretreatment with the 5-HT2C receptor antagonist SB-242084 but not the 5-HT1A receptor antagonist WAY-100635. Int J Neuropsychopharmacol. 2001;4(4):399–408. [PubMed: 11806866]
87.
Drapier D, et al. Effects of acute fluoxetine, paroxetine and desipramine on rats tested on the elevated plus-maze. Behav Brain Res. 2007;176(2):202–9. [PubMed: 17095104]
88.
Carlezon WA Jr, et al. Kappa-opioid ligands in the study and treatment of mood disorders. Pharmacol Ther. 2009;123(3):334–43. [PMC free article: PMC2740476] [PubMed: 19497337]
89.
Munro TA, et al. Long-acting kappa opioid antagonists nor-BNI, GNTI and JDTic: pharmacokinetics in mice and lipophilicity. BMC Pharmacol. 2012;12:5. [PMC free article: PMC3411462] [PubMed: 22642416]
90.
Takemori AE, Portoghese PS. Selective naltrexone-derived opioid receptor antagonists. Annu Rev Pharmacol Toxicol. 1992;32:239–69. [PubMed: 1318671]
91.
Stevens WC Jr, et al. Potent and selective indolomorphinan antagonists of the kappa-opioid receptor. J Med Chem. 2000;43(14):2759–69. [PubMed: 10893314]
92.
Thomas JB, et al. Identification of the first trans-(3R,4R)- dimethyl-4-(3-hydroxyphenyl)piperidine derivative to possess highly potent and selective opioid kappa receptor antagonist activity. J Med Chem. 2001;44(17):2687–90. [PubMed: 11495579]
93.
Endoh T, et al. Nor-binaltorphimine: a potent and selective kappa-opioid receptor antagonist with long-lasting activity in vivo. Arch Int Pharmacodyn Ther. 1992;316:30–42. [PubMed: 1326932]
94.
Horan P, et al. Extremely long-lasting antagonistic actions of nor-binaltorphimine (nor-BNI) in the mouse tail-flick test. J Pharmacol Exp Ther. 1992;260(3):1237–43. [PubMed: 1312164]
95.
Metcalf MD, Coop A. Kappa opioid antagonists: past successes and future prospects. AAPS J. 2005;7(3):E704–22. [PMC free article: PMC2751273] [PubMed: 16353947]
96.
Beguin C, Cohen BM. Medicinal Chemistry of Kappa Opioid Receptor Antagonists. In: Dean RL, Bilsky EJ, Negus SS, editors. Opiate Receptors and Antagonists: from Bench to Clinic. Humana Press; New York: 2009. pp. 99–118.
97.
Peters MF, et al. Identification of short-acting kappa-opioid receptor antagonists with anxiolytic-like activity. Eur J Pharmacol. 2011;661(1-3):27–34. [PubMed: 21539838]
98.
Brugel TA, et al. Discovery of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides as selective antagonists of the kappa opioid receptor. Part 1. Bioorg Med Chem Lett. 2010;20(19):5847–52. [PubMed: 20727752]
99.
Verhoest PR, et al. Design and discovery of a selective small molecule kappa opioid antagonist (2-methyl-N-((2′-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine, PF-4455242) J Med Chem. 2011;54(16):5868–77. [PubMed: 21744827]
100.
Mitch CH, et al. Discovery of aminobenzyloxyarylamides as kappa opioid receptor selective antagonists: application to preclinical development of a kappa opioid receptor antagonist receptor occupancy tracer. J Med Chem. 2011;54(23):8000–12. [PubMed: 21958337]
101.
Li X, et al. Characterization of dasatinib and its structural analogs as CYP3A4 mechanism-based inactivators and the proposed bioactivation pathways. Drug Metab Dispos. 2009;37(6):1242–50. [PMC free article: PMC3202349] [PubMed: 19282395]
102.
Jones RM, Portoghese PS. 5′-Guanidinonaltrindole, a highly selective and potent kappa-opioid receptor antagonist. Eur J Pharmacol. 2000;396(1):49–52. [PubMed: 10822054]
103.
Grimwood S, et al. Pharmacological characterization of 2-methyl-N-((2′-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine (PF-04455242), a high-affinity antagonist selective for kappa-opioid receptors. J Pharmacol Exp Ther. 2011;339(2):555–66. [PubMed: 21821697]

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