Selective and antagonist-dependent µ-opioid receptor activation by the combination of 2-{[2-(6-chloro-3,4-dihydro-1(2H)-quinolinyl)-2-oxoethyl]sulfanyl}-5-phenyl-4,6-(1H,5H)-pyrimidinedione and naloxone/naltrexone.

We identified, via high-throughput screening using a FLIPR® calcium assay, compound 1, which incorporated a dihydroquinolinyl-2-oxoethylsulfanyl-(1H,5H)-pyrimidinedione core and activated the µ-opioid receptor (MOR) in the presence of naloxone or naltrexone. A structure-activity relationship study of the analogs of 1 led to the design of compound 21, which activated MOR in the presence of naloxone with an EC50 of 3.3 ± 0.2 µM. MOR activation by the compound 21-antagonist pair was antagonist-dependent. Compound 21 did not affect the potency of the orthosteric agonist, morphine, toward MOR, indicating that it affected the function of MOR antagonists rather than that of the agonists. Computer modeling of the compound 21-MOR-naloxone complex revealed major interactions between compound 21 and MOR, including hydrogen bonding with Ser196, π-π stacking with Tyr149, and sulfur-aromatic interaction with Trp192. This study may pave the way for developing agents capable of safe and effective MOR modulation.

1-(2,4-Dibromophenyl)-3,6,6-trimethyl-1,5,6,7-tetrahydro-4H-indazol-4-one: A Novel Opioid Receptor Agonist with Less Accompanying Gastrointestinal Dysfunction than Morphine.

BACKGROUND: The authors investigated the pharmacology and signaling pathways of the opioid receptors modulated by compound 1, 1-(2,4-dibromophenyl)-3,6,6-trimethyl-1,5,6,7-tetrahydro-4H-indazol-4-one. METHODS: In vitro studies of compound 1 were assessed by using a radioligand-binding assay (n = 3), a cyclic adenosine monophosphate assay (n = 3), a ß-arrestin assay (n = 3), an internalization assay (n = 3), and an immunohistochemistry (n = 8). In vivo studies of compound 1 were characterized using a tail-flick test (n = 5 to 6), tail-clip test (n = 7), von Frey hair test (n = 5), and charcoal meal test (n = 5). RESULTS: Compound 1 elicited robust effects in µ-opioid (mean ± SD; binding affinity: 15 ± 2 nM; cyclic adenosine monophosphate assay: 24 ± 6 nM), δ-opioid (82 ± 7 nM; 1.9 ± 0.1 µM), and κ-opioid (76 ± 9 nM; 1.4 ± 0.5 µM) receptor-expressing cells. Compound 1 acts as a full agonist of ß-arrestin-2 recruitment in µ-opioid (1.1 ± 0.3 µM) and δ-opioid (9.7 ± 1.9 µM) receptor-expressing cells. Compound 1 caused less gastrointestinal dysfunction (charcoal meal test: morphine: 82 ± 5%; compound 1: 42 ± 5%) as well as better antinociception in mechanical pain hypersensitivity (tail-clip test: morphine: 10 ± 3 s; compound 1: 19 ± 1 s) and in cancer-induced pain (von Frey hair test: morphine: 0.1 ± 0.1 g; compound 1: 0.3 ± 0.1 g) than morphine at equi-antinociceptive doses. CONCLUSIONS: Compound 1 produced antinociception with less gastrointestinal dysfunction than morphine.

Morphine drives internal ribosome entry site-mediated hnRNP K translation in neurons through opioid receptor-dependent signaling.

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) binds to the promoter region of mu-opioid receptor (MOR) to regulate its transcriptional activity. How hnRNP K contributes to the analgesic effects of morphine, however, is largely unknown. We provide evidence that morphine increases hnRNP K protein expression via MOR activation in rat primary cortical neurons and HEK-293 cells expressing MORs, without increasing mRNA levels. Using the bicistronic reporter assay, we examined whether morphine-mediated accumulation of hnRNP K resulted from translational control. We identified potential internal ribosome entry site elements located in the 5' untranslated regions of hnRNP K transcripts that were regulated by morphine. This finding suggests that internal translation contributes to the morphine-induced accumulation of hnRNP K protein in regions of the central nervous system correlated with nociceptive and antinociceptive modulatory systems in mice. Finally, we found that down-regulation of hnRNP K mediated by siRNA attenuated morphine-induced hyperpolarization of membrane potential in AtT20 cells. Silencing hnRNP K expression in the spinal cord increased nociceptive sensitivity in wild-type mice, but not in MOR-knockout mice. Thus, our findings identify the role of translational control of hnRNP K in morphine-induced analgesia through activation of MOR.

The beneficial effects of leptin on REM sleep deprivation-induced cognitive deficits in mice.

Leptin, a 167 amino acid peptide, is synthesized predominantly in the adipose tissues and plays a key role in the regulation of food intake and body weight. Recent studies indicate that leptin receptor is expressed with high levels in many brain regions that may regulate synaptic plasticity. Here we show that deprivation of rapid eye movement (REMD) sleep resulted in impairment of both cue and contextual fear memory. In parallel, surface expression of GluR1 was reduced in the amygdala. Intraperitoneal injection of leptin to the REMD mice rescued memory impairment and reversed surface GluR1 reduction. Using whole-cell recording to evaluate the synaptic function of the thalamus-lateral amygdala (LA) pathway, we found a decrease in frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) concomitant with reduced AMPA/NMDA ratios in the REMD mice. By contrast, paired-pulse facilitation (PPF) was increased. The effects of REMD on mEPSCs and AMPA/NMDA ratio could be reversed by leptin treatment, whereas on PPR it could not. Phosphatase and tensin homolog (PTEN), a dual protein/lipid phosphatase, down-regulates the effect of the PI-3 kinase pathway. Fear conditioning increased whereas REMD led to a decrease in the phosphorylated states of PTEN, Akt, and glycogen synthase kinase-3ß (GSK3ß), and the effects of REMD were reversed by leptin. These results suggest that both pre- and postsynaptic functions of the thalamus-LA pathway were altered by fear conditioning and REMD in opposite directions. Leptin treatment reversed REMD-induced memory deficits primarily by a postsynaptic action by restoring surface expression of GluR1 without affecting PPR.

Discovery of a mu-opioid receptor modulator that in combination with morphinan antagonists induces analgesia.

Morphinan antagonists, which block opioid effects at mu-opioid receptors, have been studied for their analgesic potential. Previous studies have suggested that these antagonists elicit analgesia with fewer adverse effects in the presence of the mutant mu-opioid receptor (MOR; S196A). However, introducing a mutant receptor for medical applications represents significant challenges. We hypothesize that binding a chemical compound to the MOR may elicit a comparable effect to the S196A mutation. Through high-throughput screening and structure-activity relationship studies, we identified a modulator, 4-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)-3-methylbenzoic acid (BPRMU191), which confers agonistic properties to small-molecule morphinan antagonists, which induce G protein-dependent MOR activation. Co-application of BPRMU191 and morphinan antagonists resulted in MOR-dependent analgesia with diminished side effects, including gastrointestinal dysfunction, antinociceptive tolerance, and physical and psychological dependence. Combining BPRMU191 and morphinan antagonists could serve as a potential therapeutic strategy for severe pain with reduced adverse effects and provide an avenue for studying G protein-coupled receptor modulation.

DBPR116, a Prodrug of BPRMU191, in Combination with Naltrexone as a Safer Opioid Analgesic Than Morphine via Peripheral Administration.

The development of opioid analgesics with reduced adverse effects is an unmet need. In a previous study, we discovered a unique combination of BPRMU191 and morphinan antagonists that produced potent antinociception with reduced adverse effects after central administration (intrathecal or intracerebroventricular). BPRMU191/naltrexone exhibits notable in vitro and in vivo pharmacological properties. However, the poor blood-brain barrier penetrative ability of BPRMU191 restricts its clinical application. In this study, we utilized a prodrug strategy to deliver sufficient brain concentrations of BPRMU191 and selected compound 2 (DBPR116) with the best physicochemical and pharmacological properties among other in vivo active prodrugs. The in vivo pharmacological studies of compound 2/naltrexone, including thermally stimulated pain, cancer pain, constipation, sedation, psychological dependence, heart rate, and respiratory frequency measurements, demonstrated that it was a safer opioid analgesic than morphine in pain control.

A dual nociceptin and mu opioid receptor agonist exhibited robust antinociceptive effect with decreased side effects.

The compelling demand of a consummate analgesic medication without addiction is rising due to the clinical mistreatment. Additionally, the series of severe untoward effects usually deterred the utilization while coping with serious pain. As a possible turning point, we revealed that compound 14 is a dual agonist of mu opioid receptor (MOR) and nociceptin-orphanin FQ opioid peptide (NOP) receptor in this study. More importantly, compound 14 achieves pain relieving at very small doses, meanwhile, reduces several unwanted side effects such as constipation, reward, tolerance and withdrawal effects. Here, we evaluated the antinociception and side effects of this novel compound from wild type and humanized mice to further develop a safer prescription analgesic drug.

Benzo[b]thiophene-2-carboxamides as novel opioid receptor agonists with potent analgesic effect and reduced constipation.

Currently, there is a significant unmet need for novel analgesics with fewer side effects. In this study, we carried out structural modification of a hit compound previously identified in an artificial-intelligence (AI) virtual screening and discovered the potent analgesic, benzo[b]thiophene-2-carboxamide analog (compound 25) with new structural scaffold. We investigated the signaling pathways of opioid receptors mediated by compound 25, and found this racemic compound activated mu-opioid receptor through the cyclic adenosine monophosphate (cAMP) and ß-arrestin-2-mediated pathways with strong potency and efficacy, and accompanying nociceptin-orphanin FQ opioid peptide and delta-opioid receptors through the cAMP pathway with weak potencies. Compound 25 elicited potent antinociception in thermal-stimulated pain (ED50 value of 127.1 ± 34.65 µg/kg) and inflammatory-induced allodynia models with less gastrointestinal transit inhibition and antinociceptive tolerance than morphine. Overall, this study revealed a novel analgesic with reduced risks of side effects.

Comparative study between deep learning and QSAR classifications for TNBC inhibitors and novel GPCR agonist discovery.

Machine learning is a well-known approach for virtual screening. Recently, deep learning, a machine learning algorithm in artificial neural networks, has been applied to the advancement of precision medicine and drug discovery. In this study, we performed comparative studies between deep neural networks (DNN) and other ligand-based virtual screening (LBVS) methods to demonstrate that DNN and random forest (RF) were superior in hit prediction efficiency. By using DNN, several triple-negative breast cancer (TNBC) inhibitors were identified as potent hits from a screening of an in-house database of 165,000 compounds. In broadening the application of this method, we harnessed the predictive properties of trained model in the discovery of G protein-coupled receptor (GPCR) agonist, by which computational structure-based design of molecules could be greatly hindered by lack of structural information. Notably, a potent (~ 500 nM) mu-opioid receptor (MOR) agonist was identified as a hit from a small-size training set of 63 compounds. Our results show that DNN could be an efficient module in hit prediction and provide experimental evidence that machine learning could identify potent hits in silico from a limited training set.

Morphine produces potent antinociception, sedation, and hypothermia in humanized mice expressing human mu-opioid receptor splice variants.

Morphine is a strong painkiller acting through mu-opioid receptor (MOR). Full-length 7-transmembrane (TM) variants of MOR share similar amino acid sequences of TM domains in rodents and humans; however, interspecies differences in N- and C-terminal amino acid sequences of MOR splice variants dramatically affect the downstream signaling. Thus, it is essential to develop a mouse model that expresses human MOR splice variants for opioid pharmacological studies. We generated 2 lines of fully humanized MOR mice (hMOR; mMOR mice), line #1 and #2. The novel murine model having human OPRM1 genes and human-specific variants was examined by reverse-transcription polymerase chain reaction and the MinION nanopore sequencing. The differences in the regional distribution of MOR between wild-type and humanized MOR mice brains were detected by RNAscope and radioligand binding assay. hMOR; mMOR mice were characterized in vivo using a tail-flick, charcoal meal, open field, tail suspension, naloxone precipitation tests, and rectal temperature measurement. The data indicated that wild-type and humanized MOR mice exhibited different pharmacology of morphine, including antinociception, tolerance, sedation, and withdrawal syndromes, suggesting the presence of species difference between mouse and human MORs. Therefore, hMOR; mMOR mice could serve as a novel mouse model for pharmacogenetic studies of opioids.

BPR1M97, a dual mu opioid receptor/nociceptin-orphanin FQ peptide receptor agonist, produces potent antinociceptive effects with safer properties than morphine.

There is unmet need to design an analgesic with fewer side effects for severe pain management. Although traditional opioids are the most effective painkillers, they are accompanied by severe adverse responses, such as respiratory depression, constipation symptoms, tolerance, withdrawal, and addiction. We indicated BPR1M97 as a dual mu opioid receptor (MOP)/nociceptin-orphanin FQ peptide (NOP) receptor full agonist and investigated the pharmacology of BPR1M97 in multiple animal models. In vitro studies on BPR1M97 were assessed using cyclic-adenosine monophosphate production, ß-arrestin, internalization, and membrane potential assays. In vivo studies were characterized using the tail-flick, tail-clip, lung functional, heart functional, acetone drop, von Frey hair, charcoal meal, glass bead, locomotor activity, conditioned place preference (CPP) and naloxone precipitation tests. BPR1M97 elicited full agonist properties for all cell-based assays tested in MOP-expressing cells. However, it acted as a G protein-biased agonist for NOP. BPR1M97 initiated faster antinociceptive effects at 10 min after subcutaneous injection and elicited better analgesia in cancer-induced pain than morphine. Unlike morphine, BPR1M97 caused less respiratory, cardiovascular, and gastrointestinal dysfunction. In addition, BPR1M97 decreased global activity and induced less withdrawal jumping precipitated by naloxone. Thus, BPR1M97 could serve as a novel small molecule dual receptor agonist for antinociception with fewer side effects than morphine. This article is part of the Special Issue entitled 'New Vistas in Opioid Pharmacology'.

Convallatoxin enhance the ligand-induced mu-opioid receptor endocytosis and attenuate morphine antinociceptive tolerance in mice.

Morphine is a unique opioid analgesic that activates the mu-opioid receptor (MOR) without efficiently promoting its endocytosis that may underlie side effects. Our objective was to discover a novel enhancer of ligand-induced MOR endocytosis and determine its effects on analgesia, tolerance and dependence. We used high-throughput screening to identify convallatoxin as an enhancer of ligand-induced MOR endocytosis with high potency and efficacy. Treatment of cells with convallatoxin enhanced morphine-induced MOR endocytosis through an adaptor protein 2 (AP2)/clathrin-dependent mechanism, attenuated morphine-induced phosphorylation of MOR, and diminished desensitization of membrane hyperpolarization. Furthermore, co-treatment with chronic convallatoxin reduced morphine tolerance in animal models of acute thermal pain and chronic inflammatory pain. Acute convallatoxin administration reversed morphine tolerance and dependence in morphine-tolerant mice. These findings suggest convallatoxin are potentially therapeutic for morphine side effects and open a new avenue to study MOR trafficking.

The in vivo antinociceptive and µ-opioid receptor activating effects of the combination of N-phenyl-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amines and naloxone.

Morphine is widely used for the treatment of severe pain. This analgesic effect is mediated principally by the activation of µ-opioid receptors (MOR). However, prolonged activation of MOR also results in tolerance, dependence, addiction, constipation, nausea, sedation, and respiratory depression. To address this problem, we sought alternative ways to activate MOR - either by use of novel ligands, or via a novel activation mechanism. To this end, a series of compounds were screened using a sensitive CHO-K1/MOR/Gα15 cell-based FLIPR® calcium high-throughput screening (HTS) assay, and the bithiazole compound 5a was identified as being able activate MOR in combination with naloxone. Structural modifications of 5a resulted in the discovery of lead compound 5j, which could effectively activate MOR in combination with the MOR antagonist naloxone or naltrexone. In vivo, naloxone in combination with 100 mg/kg of compound 5j elicited antinociception in a mouse tail-flick model with an ED50 of 17.5 ±â€¯4 mg/kg. These results strongly suggest that the mechanism by which the 5j/naloxone combination activates MOR is worthy of further study, as its discovery has the potential to yield an entirely novel class of analgesics.

Discovery, structure-activity relationship studies, and anti-nociceptive effects of N-(1,2,3,4-tetrahydro-1-isoquinolinylmethyl)benzamides as novel opioid receptor agonists.

µ-Opioid receptor (MOR) agonists are analgesics used clinically for the treatment of moderate to severe pain, but their use is associated with severe adverse effects such as respiratory depression, constipation, tolerance, dependence, and rewarding effects. In this study, we identified N-({2-[(4-bromo-2-trifluoromethoxyphenyl)sulfonyl]-1,2,3,4-tetrahydro-1-isoquinolinyl}methyl)cyclohexanecarboxamide (1) as a novel opioid receptor agonist by high-throughput screening. Structural modifications made to 1 to improve potency and blood-brain-barrier (BBB) penetration resulted in compounds 45 and 46. Compound 45 was a potent MOR/KOR (κ-opioid receptor) agonist, and compound 46 was a potent MOR and medium KOR agonist. Both 45 and 46 demonstrated a significant anti-nociceptive effect in a tail-flick test performed in wild type (WT) B6 mice. The ED50 value of 46 was 1.059 mg/kg, and the brain concentrations of 45 and 46 were 7424 and 11696 ng/g, respectively. Accordingly, compounds 45 and 46 are proposed for lead optimization and in vivo disease-related pain studies.

Protein kinase mζ is necessary for cocaine-induced synaptic potentiation in the ventral tegmental area.

BACKGROUND: Much progress has been made in our understanding of brain regions and specific receptors that are involved in the action of cocaine addiction. Although long-term modifications of mesolimbic reward circuit following cocaine exposure are responsible for cocaine-addicted behaviors, the underlying molecular mechanism at the cellular level is still obscure. Here, we investigated the possible participation of protein kinase Mζ (PKMζ) in synaptic potentiation following cocaine exposure. METHODS: Spontaneous and evoked synaptic activity of glutamate synapse in saline- and cocaine-treated rats were examined by preparing acute brain slices and performing whole-cell voltage-clamp recordings from individual dopamine neurons in the ventral tegmental area (VTA). We also assessed the role of PKMζ on the behavioral responses by cocaine conditioned place preference. RESULTS: Chelerythrine, an inhibitor of PKMζ, reversed the cocaine-induced facilitation of spontaneous synaptic transmission in the VTA. PKMζ inhibition by chelerythrine or myristoylated ζ inhibitory peptide significantly attenuated the cocaine exposure-induced enhancement of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor/N-methyl-D-aspartate receptor ratio. Myristoylated ζ inhibitory peptide had no effect on spike timing-dependent long-term potentiation in rats previously injected with saline but remarkably restored spike timing-dependent long-term potentiation in VTA dopamine neurons in slices prepared from rats that received single or multiple cocaine exposure. Western blot analyses showed that both single and five consecutive cocaine injections induced a significant increase in PKMζ level. Furthermore, intracranial infusion of myristoylated ζ inhibitory peptide in the VTA disrupted cocaine conditioned place preference. CONCLUSIONS: Our results suggest that persistent activity of PKMζ is a requisite for cocaine-induced enhancement of synaptic plasticity in the VTA and cocaine conditioned place preference.