Cyclin-dependent kinase 8 mediates chemotherapy-induced tumor-promoting paracrine activities.

Conventional chemotherapy not only kills tumor cells but also changes gene expression in treatment-damaged tissues, inducing production of multiple tumor-supporting secreted factors. This secretory phenotype was found here to be mediated in part by a damage-inducible cell-cycle inhibitor p21 (CDKN1A). We developed small-molecule compounds that inhibit damage-induced transcription downstream of p21. These compounds were identified as selective inhibitors of a transcription-regulating kinase CDK8 and its isoform CDK19. Remarkably, p21 was found to bind to CDK8 and stimulate its kinase activity. p21 and CDK8 also cooperate in the formation of internucleolar bodies, where both proteins accumulate. A CDK8 inhibitor suppresses damage-induced tumor-promoting paracrine activities of tumor cells and normal fibroblasts and reverses the increase in tumor engraftment and serum mitogenic activity in mice pretreated with a chemotherapeutic drug. The inhibitor also increases the efficacy of chemotherapy against xenografts formed by tumor cell/fibroblast mixtures. Microarray data analysis revealed striking correlations between CDK8 expression and poor survival in breast and ovarian cancers. CDK8 inhibition offers a promising approach to increasing the efficacy of cancer chemotherapy.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 9: Synthesis, characterization and molecular modeling of pyridinyl isosteres of N-BPE-8-CAC (1), a high affinity ligand for opioid receptors.

Derivatives of the lead compound N-BPE-8-CAC (1) where each CH of the biphenyl group was individually replaced by N were prepared in hopes of identifying high affinity ligands with improved aqueous solubility. Compared to 1, binding affinities of the five possible pyridinyl derivatives for the µ opioid receptor were between threefold lower to fivefold higher with the Ki of the most potent compound being 0.064 nM. Docking of 8-CAC (2) into the unliganded binding site of the mouse µ opioid receptor (pdb: 4DKL) revealed that 8-CAC and ß-FNA (from 4DKL) make nearly identical interactions with the receptor. However, for 1 and the new pyridinyl derivatives 4-8, binding is not tolerated in the 8-CAC binding mode due to the steric constraints of the large N-substituents. Either an alternative binding mode or rearrangement of the protein to accommodate these modifications may account for their high binding affinity.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 8. High affinity ligands for opioid receptors in the picomolar Ki range: oxygenated N-(2-[1,1'-biphenyl]-4-ylethyl) analogues of 8-CAC.

N-[2-(4'-methoxy[1,1'-biphenyl]-4-yl)ethyl]-8-CAC (1) is a high affinity (K(i)=0.084 nM) ligand for the µ opioid receptor and served as the lead compound for this study. Analogues of 1 were made in hopes of identifying an SAR within a series of oxygenated (distal) phenyl derivatives. A number of new analogues were made having single-digit pM affinity for the µ receptor. The most potent was the 3',4'-methylenedioxy analogue 18 (K(i)=1.6 pM).

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 7: syntheses and opioid receptor properties of cyclic variants of cyclazocine.

A series of 7,8- and 8,9-fused triazole and imidazole analogues of cyclazocine have been made and characterized in opioid receptor binding and [(35)S]GTPgammaS assays. Target compounds were designed to explore the SAR surrounding our lead molecule for this study, namely the 8,9-fused pyrrolo analogue 2 of cyclazocine. Compared to 2, many of the new compounds in this study displayed very high affinity for opioid receptors.

Syntheses and opioid receptor binding properties of carboxamido-substituted opioids.

A series of 15 novel opioid derivatives were made where the prototypic phenolic-OH group of traditional opioids was replaced by a carboxamido (CONH(2)) group. For 2,6-methano-3-benzazocines and morphinans similar or, in a few instances, enhanced affinity for mu, delta and kappa opioid receptors was observed when the OH-->CONH(2) switch was applied. For 4,5alpha-epoxymorphinans, binding affinities for the corresponding carboxamide derivatives were much lower than the OH partner consistent with our pharmacophore hypothesis concerning carboxamide bioactive conformation. The active metabolite of tramadol and its carboxamide counterpart had comparable affinities for the three receptors.

Syntheses of novel high affinity ligands for opioid receptors.

A series of novel high affinity opioid receptor ligands have been made whereby the phenolic-OH group of nalbuphine, naltrexone methiodide, 6-desoxonaltrexone, hydromorphone and naltrindole was replaced by a carboxamido group and the furan ring was opened to the corresponding 4-OH derivatives. These furan ring 'open' derivatives display very high affinity for mu and kappa receptors and much less affinity for delta. The observation that these target compounds have much higher receptor affinity than the corresponding ring 'closed' carboxamides significantly strengthens our underlying pharmacophore hypothesis concerning the bioactive conformation of the carboxamide group.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 6: Opioid receptor binding properties of cyclic variants of 8-carboxamidocyclazocine.

A series of 7,8- and 8,9-fused pyrimidinone, aminopyrimidine and pyridone derivatives of 8-carboxamidocyclazocine (8-CAC) have been prepared and evaluated in opioid receptor binding assays. Targets were designed to corroborate a pharmacophore hypothesis regarding the bioactive conformation of the carboxamide of 8-CAC. In addition to the results from this study strongly supporting this pharmacophore hypothesis, a number of novel compounds with high affinity to opioid receptors have been identified.

CC12, a high-affinity ligand for [3H]cimetidine binding, is an improgan antagonist.

Improgan, a chemical congener of cimetidine, is a highly effective non-opioid analgesic when injected into the CNS. Despite extensive characterization, neither the improgan receptor, nor a pharmacological antagonist of improgan has been previously described. Presently, the specific binding of [(3)H]cimetidine (3HCIM) in brain fractions was used to discover 4(5)-((4-iodobenzyl)thiomethyl)-1H-imidazole, which behaved in vivo as the first improgan antagonist. The synthesis and pharmacological properties of this drug (named CC12) are described herein. In rats, CC12 (50-500nmol, i.c.v.) produced dose-dependent inhibition of improgan (200-400nmol) antinociception on the tail flick and hot plate tests. When given alone to rats, CC12 had no effects on nociceptive latencies, or on other observable behavioral or motor functions. Maximal inhibitory effects of CC12 (500nmol) were fully surmounted with a large i.c.v. dose of improgan (800nmol), demonstrating competitive antagonism. In mice, CC12 (200-400nmol, i.c.v.) behaved as a partial agonist, producing incomplete improgan antagonism, but also limited antinociception when given alone. Radioligand binding, receptor autoradiography, and electrophysiology experiments showed that CC12's antagonist properties are not explained by activity at 25 sites relevant to analgesia, including known receptors for cannabinoids, opioids or histamine. The use of CC12 as an improgan antagonist will facilitate the characterization of improgan analgesia. Furthermore, because CC12 was also found presently to inhibit opioid and cannabinoid antinociception, it is suggested that this drug modifies a biochemical mechanism shared by several classes of analgesics. Elucidation of this mechanism will enhance understanding of the biochemistry of pain relief.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. 5. Opioid receptor binding properties of N-((4'-phenyl)-phenethyl) analogues of 8-CAC.

A series of aryl-containing N-monosubstituted analogues of the lead compound 8-[N-((4'-phenyl)-phenethyl)]-carboxamidocyclazocine were synthesized and evaluated to probe a putative hydrophobic binding pocket of opioid receptors. Very high binding affinity to the mu opioid receptor was achieved though the N-(2-(4'-methoxybiphenyl-4-yl)ethyl) analogue of 8-CAC. High binding affinity to mu and very high binding affinity to kappa opioid receptors was observed for the N-(3-bromophenethyl) analogue of 8-CAC. High binding affinity to all three opioid receptors were observed for the N-(2-naphthylethyl) analogue of 8-CAC.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. 4. Opioid receptor binding properties of 8-[N-(4'-phenyl)-phenethyl)carboxamido] analogues of cyclazocine and ethylketocycalzocine.

The synthesis and evaluation of a series of aryl-containing N-monosubstituted analogues of the lead compound 8-carboxamidocyclazocine were performed to probe a putative hydrophobic binding pocket of opioid receptors. High binding affinity to mu, kappa, and delta opioid receptors was observed for the 8-[N-(4'-phenyl)-phenethyl)carboxamido] analogue.

Antinociceptive, brain-penetrating derivatives related to improgan, a non-opioid analgesic.

The antinociceptive profile of selected histamine H(2) and histamine H(3) receptor antagonists led to the discovery of improgan, a non-brain-penetrating analgesic agent which does not act on known histamine receptors. Because no chemical congener of improgan has yet been discovered which has both antinociceptive and brain-penetrating properties, the present study investigated the antinociceptive effects of a series of chemical compounds related to zolantidine, a brain-penetrating histamine H(2) receptor antagonist. The drugs studied presently contain the piperidinomethylphenoxy (PMPO) moiety, hypothesized to introduce brain-penetrating characteristics. Following intracerebroventricular (i.c.v.) dosing in rats, six of eight drugs produced dose- and time-related antinociception on both the tail flick and hot plate tests over a nearly eight-fold range of potencies. Ataxia and other motor side effects were observed after high doses of these drugs, but two of the compounds (SKF94674 and loxtidine) produced maximal antinociception at doses which were completely devoid of these motor effects. Consistent with the hypothesis that PMPO-containing drugs are brain-penetrating analgesics, SKF94674 and another derivative (JB-9322) showed dose-dependent antinociceptive activity 15 to 30 min after systemic dosing in mice, but these effects were accompanied by seizures and death beginning 45 min after dosing. Other drugs showed a similar pattern of antinociceptive and toxic effects. In addition, loxtidine produced seizures without antinociception, whereas zolantidine produced neither effect after systemic dosing in mice. Although several of the drugs tested have histamine H(2) receptor antagonist activity, neither the antinociception nor the toxicity was correlated with histamine H(2) receptor activity. The present results are the first to demonstrate the existence of brain-penetrating antinociceptive agents chemically related to zolantidine and improgan, but further studies are needed to understand the mechanisms of both the pain relief and toxicity produced by these agents.

10-Ketomorphinan and 3-substituted-3-desoxymorphinan analogues as mixed kappa and micro opioid ligands: synthesis and biological evaluation of their binding affinity at opioid receptors.

A series of 10-ketomorphinan analogues were synthesized, and their binding affinity at all three opioid receptors was investigated. In most cases, high affinity at micro and kappa receptors, and lower affinity at delta receptor was observed, resulting in good selectivity for micro and kappa receptors. A wide range of substituents can be accommodated on the nitrogen position. The N-(S)-tetrahydrofurfuryl analogue 11 displayed the highest affinity at all three receptors. The N-cyclobutylmethyl analogue 13 gave both high affinity and selectivity at kappa receptor, and N-2-phenylethyl analogue 18 exhibited good affinity and selectivity at micro receptor. Further modifications of the 3-substituent indicated that one H-bond donor was an essential requirement for good affinity at micro and kappa receptors. Similar modifications were investigated at the 3-OH group of morphinans: levorphanol (2a), cyclorphan (2b), and MCL-101 (2c) lacking the 10-keto group. The 3-amino bioisosteric analogues (40 and 41) displayed reasonably good affinity at micro and kappa receptors. The 3-carboxamido replacement (compounds 46-48) in the morphinan subseries resulted in similar affinities comparable to their corresponding 3-OH congeners. The high affinity of these carboxamido analogues, along with their greater lipophilicity and metabolic stability, make them promising candidates for further pharmacological investigation.

Syntheses and opioid receptor binding affinities of 8-amino-2,6-methano-3-benzazocines.

8-Amino-2,6-methano-3-benzazocine derivatives have been made using Pd-catalyzed amination procedures, and their affinities for opioid receptors were assessed. The 8-amino group was hypothesized to be a replacement for the prototypic 8-OH substituent for 2,6-methano-3-benzazocines and related opiates. This OH group is generally required for binding yet is implicated in unfavorable pharmacokinetic characteristics such as low oral bioavailability and rapid clearance via O-glucuronidation. The core structures in which the 8-OH group was replaced were cyclazocine and its enantiomers, ethylketocyclazocine and its enantiomers, ketocyclazocine, and Mr2034. Many new analogues had high affinity for opioid receptors with several in the subnanomolar range. Highest affinity was seen in analogues with secondary 8-(hetero)arylamino appendages. Binding to opioid receptors was enantioselective with the (2R,6R,11R)-configuration preferred and high selectivity for mu and kappa over delta opioid receptors was observed within the series. Several derivatives were shown to have intrinsic opioid-receptor-mediated activity in [(35)S]GTPgammaS assays.

Effects of the mixed-action kappa/mu opioid agonist 8-carboxamidocyclazocine on cocaine- and food-maintained responding in rhesus monkeys.

The present study evaluated the effects of 8-carboxamidocyclazocine (8-CAC), a novel mixed-action kappa/mu agonist with a long duration of action, on food- and cocaine-maintained responding in rhesus monkeys to assess the potential utility of 8-CAC as a medication for the treatment of cocaine dependence. The effects of acute and chronic (10 days) 8-CAC were examined in rhesus monkeys responding under a multiple schedule for both cocaine and food reinforcement. Acute 8-CAC (0.032-0.56 mg/kg, i.m.) dose-dependently eliminated cocaine-maintained responding in all three monkeys. However, doses of 8-CAC that decreased cocaine self-administration typically also decreased food-maintained responding, and 8-CAC-induced decreases in cocaine self-administration diminished during chronic 8-CAC treatment. These results confirm that 8-CAC acutely decreases cocaine self-administration. However, non-selective effects of 8-CAC on food-maintained responding and tolerance to 8-CAC effects on cocaine self-administration may limit its potential for the treatment of cocaine dependence.

Synthesis and pharmacology of 8-amino-3-[(tetrahydro-2-furanyl)methyl] benzomorphan.

AIM: To design and synthesize new chiral 8-(substituted) amino-analogues of 3-[(tetrahydro-2-furanyl)methyl] benzomorphans, to expand knowledge of the structure-activity relationship (SAR) for 8-aminobenzomorphan. METHODS: Target compounds were synthesized from the 8-triflate of the optically active 3-[(tetrahydro-2-furanyl)methyl]-2,6-methano-benzomorphans using Pd-catalyzed aminations. Opioid receptor binding experiments were performed to evaluate their biological activities. RESULTS: Both 8-amino and 8-phenylamino analogues showed lower binding affinity for mu, delta and kappa receptors than corresponding 8-hydroxy-3-[(tetrahydro-2-furanyl)methyl]-2,6-methano-benzomorphan in vitro. CONCLUSION: The relative poor binding affinity of the target compounds did not warrant conducting the in vivo studies to determine if they have the profile(kappa agonist/mu antagonist) that will be potentially useful in the treatment of drug addiction. Further study is in progress.

Antinociceptive activity of CC44, a biotinylated improgan congener.

Improgan, a non-opioid, antinociceptive drug, activates descending analgesic circuits following brain administration, but the improgan receptor remains unidentified. Since biotinylation of drugs can enhance drug potency or facilitate discovery of new drug targets, a biotinylated congener of improgan (CC44) and several related compounds were synthesized and tested for antinociceptive activity. In rats and mice, intracerebroventricular (i.c.v.) administration of CC44 produced dose-dependent reductions in thermal nociceptive (tail flick and hot plate) responses, with 5-fold greater potency than improgan. CC44 also robustly attenuated mechanical (tail pinch) nociception in normal rats and mechanical allodynia in a spinal nerve ligation model of neuropathic pain. Similar to the effects of improgan, CC44 antinociception was reversed by the GABAA agonist muscimol (consistent with activation of analgesic circuits), and was resistant to the opioid antagonist naltrexone (implying a non-opioid mechanism). Also like improgan, CC44 produced thermal antinociception when microinjected into the rostral ventromedial medulla (RVM). Unlike improgan, CC44 (i.c.v.) produced antinociception which was resistant to antagonism by the cannabinoid CB1 antagonist/inverse agonist rimonabant. CC44 was inactive in mice following systemic administration, indicating that CC44 does not penetrate the brain. Preliminary findings with other CC44 congeners suggest that the heteroaromatic nucleus (imidazole), but not the biotin moiety, is required for CC44's antinociceptive activity. These findings demonstrate that CC44 is a potent analgesic compound with many improgan-like characteristics. Since powerful techniques are available to characterize and identify the binding partners for biotin-containing ligands, CC44 may be useful in searching for new receptors for analgesic drugs.

Cytochrome P450 2C24: Expression, Tissue Distribution, High-Throughput Assay, and Pharmacological Inhibition.

Cytochrome P450 (CYP)-mediated epoxidation of arachidonic acid (AA) contributes to important biological functions, including the pain-relieving responses produced by analgesic drugs. However, the relevant epoxygenase(s) remain unidentified. Presently, we describe the tissue distribution, high-throughput assay, and pharmacological characteristics of the rat epoxygenase CYP2C24. Following cloning from male rat liver, recombinant baculovirus containing the C-terminal His-tagged cDNA was constructed and used to express the protein in Spodoptera frugiperda (Sf9) cells. Enzymatic activity was detected with membranes, NADPH regenerating system and CYP reductase, and optimized for high throughput screening by use of the Vivid Blue© BOMCC fluorescence substrate. Quantitative real-time PCR identified CYP2C24 m-RNA in liver, kidney, heart, lung, gonad and brain. Screening of CYP2C24 activity against a panel of inhibitors showed a very strong correlation with activity against the human homologue CYP2C19. In agreement with recent findings on CYP2C19, the epoxygenase blockers PPOH and MS-PPOH inhibited CYP2C24 only weakly, confirming that these drugs are not universal epoxygenase inhibitors. Finally, comparisons of the CYP2C24 inhibitor profile with anti-analgesic activity suggests that this isoform does not contribute to brain analgesic drug action. The present methods and pharmacological data will aid in study of the biological significance of this CYP isoform.

Brain P450 epoxygenase activity is required for the antinociceptive effects of improgan, a nonopioid analgesic.

The search for the mechanism of action of improgan (a nonopioid analgesic) led to the recent discovery of CC12, a compound that blocks improgan antinociception. Because CC12 is a cytochrome P450 inhibitor, and brain P450 mechanisms were recently shown to be required in opioid analgesic signaling, pharmacological and transgenic studies were performed in rodents to test the hypothesis that improgan antinociception requires brain P450 epoxygenase activity. Intracerebroventricular (i.c.v.) administration of the P450 inhibitors miconazole and fluconazole, and the arachidonic acid (AA) epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) potently inhibited improgan antinociception in rats at doses that were inactive alone. MW06-25, a new P450 inhibitor that combines chemical features of CC12 and miconazole, also potently blocked improgan antinociception. Although miconazole and CC12 were weakly active at opioid and histamine H(3) receptors, MW06-25 showed no activity at these sites, yet retained potent P450-inhibiting properties. The P450 hypothesis was also tested in Cpr(low) mice, a viable knock-in model with dramatically reduced brain P450 activity. Improgan (145 nmol, i.c.v.) antinociception was reduced by 37% to 59% in Cpr(low) mice, as compared with control mice. Moreover, CC12 pretreatment (200 nmol, i.c.v.) abolished improgan action (70% to 91%) in control mice, but had no significant effect in Cpr(low) mice. Thus, improgan's activation of bulbospinal nonopioid analgesic circuits requires brain P450 epoxygenase activity. A model is proposed in which (1) improgan activates an unknown receptor to trigger downstream P450 activity, and (2) brainstem epoxygenase activity is a point of convergence for opioid and nonopioid analgesic signaling.

Opioids activate brain analgesic circuits through cytochrome P450/epoxygenase signaling.

To assess the importance of brain cytochrome P450 (P450) activity in mu opioid analgesic action, we generated a mutant mouse with brain neuron-specific reductions in P450 activity; these mice showed highly attenuated morphine antinociception compared with controls. Pharmacological inhibition of brain P450 arachidonate epoxygenases also blocked morphine antinociception in mice and rats. Our findings indicate that a neuronal P450 epoxygenase mediates the pain-relieving properties of morphine.

Synthesis and opioid receptor binding properties of a highly potent 4-hydroxy analogue of naltrexone.

Very high affinity for opioid receptors (e.g., K(i)=0.052nM for mu) has been observed in the rationally designed naltrexone analogue 5. SAR and physical data supports the hypothesis that the 4-OH group of 5 stabilizes the 3-carboxamido group in the putative bioactive conformation.