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.

Activation of delta-opioid receptor contributes to the antinociceptive effect of oxycodone in mice.

Oxycodone has been used clinically for over 90 years. While it is known that it exhibits low affinity for the multiple opioid receptors, whether its pharmacological activities are due to oxycodone activation of the opioid receptor type or due to its active metabolite (oxymorphone) that exhibits high affinity for the mu-opioid receptors remains unresolved. Ross and Smith (1997) reported the antinociceptive effects of oxycodone (171nmol, i.c.v.) are induced by putative kappa-opioid receptors in SD rat while others have reported oxycodone activities are due to activation of mu- and/or delta-opioid receptors. In this study, using male mu-opioid receptor knock-out (MOR-KO) mice, we examined whether delta-opioid receptor was involved in oxycodone antinociception. Systemic subcutaneous (s.c.) administration of oxycodone (above 40mg/kg) could induce a small but significant antinociceptive effect in MOR-KO mice by the tail flick test. Delta-opioid receptor antagonist (naltrindole, 10mg/kg or 20mg/kg, i.p.) could block this effect. When oxycodone was injected directly into the brain of MOR-KO mice by intracerebroventricular (i.c.v.) route, oxycodone at doses of 50nmol or higher could induce similar level of antinociceptive responses to those observed in wild type mice at the same doses by i.c.v. Delta-opioid receptor antagonists (naltrindole at 10nmol or ICI 154,129 at 20µg) completely blocked the supraspinal antinociceptive effect of oxycodone in MOR-KO mice. Such oxycodone antinociceptive responses were probably not due to its active metabolites oxymorphone because (a) the relative low level of oxymorphone was found in the brain after systemically or centrally oxycodone injection using LC/MS/MS analysis; (b) oxymorphone at a dose that mimics the level detected in the mice brain did not show any significant antinocieption effect; (c) oxycodone exhibits equal potency as oxymorphone albeit being a partial agonist in regulating [Ca(2+)]I transients in a clonal cell line expressing high level of mu-opioid receptor. These data suggest that oxycodone by itself can activate both the mu- and delta-opioid receptors and that delta-opioid receptors may contribute to the central antinociceptive effect of oxycodone in mice.

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.

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.

Up-regulation of tau, a brain microtubule-associated protein, in lens cortical fractions of aged alphaA-, alphaB-, and alphaA/B-crystallin knockout mice.

PURPOSE: Alpha-crystallin is expressed at high levels in the lens in a complex of alphaA- and alphaB-crystallin subunits in 3:1 molar ratios, and is known to maintain the solubility of unpolymerized tubulin and enhance the resistance of microtubules to depolymerization, but its effect on proteins classically associated with microtubule stability (microtubule associated proteins) in the lens is unknown. In the present study we examined the expression of the brain microtubule associated protein tau in lenses of alpha-crystallin gene knockout mice. METHODS: Quantitative RT-PCR, immunoblotting, cryo-immunoelectron microscopic and immunohistochemical methods were used to characterize the expression of tau in the lenses of alphaA(-/-)-, alphaB(-/-)-, and alphaA/B(-/-)-crystallin mice. RESULTS: Immunoreactivity to tau, a 45-66 kDa brain microtubule associated protein that has been best characterized in neurons and neuronal pathologies, was uniquely upregulated in lens cortical fiber cells with aging and was associated with the microtubule fraction of alphaA(-/-)-, alphaB(-/-)-, and alphaA/B(-/-)-crystallin mouse lenses, but was undetectable in wild type lenses. Quantitative RT-PCR analysis further showed an upregulation of tau transcripts in alphaA(-/-)- and alphaA/B(-/-)-crystallin lenses. Brain microtubule fractions served as a positive control for tau in these experiments. An increase in phosphorylation of tau was detected in alphaA(-/-)- and alphaB(-/-)-crystallin brain proteins. CONCLUSIONS: Although tau aggregation and alphaB-crystallin expression have been shown to increase in neurodegenerative diseases, surprisingly tau expression increases in the alpha-crystallin knockout lenses, suggesting that alphaA- and alphaB-crystallins are potentially important regulators of tau expression in lens.

Alpha-crystallin expression affects microtubule assembly and prevents their aggregation.

The molecular chaperones alphaA- and alphaB-crystallins are important for cell survival and genomic stability and associate with the tubulin cytoskeleton. The mitotic spindle is abnormally assembled in a number of alphaA-/- and alphaB-/- lens epithelial cells. However, no report to date has studied the effect of alpha-crystallin expression on tubulin/microtubule assembly in lens epithelial cells. In the current work we tested the hypothesis that the absence of alphaA- and alphaB-crystallins alters microtubule assembly. Microtubules were reconstituted from freshly dissected explants of wild-type, alphaA-/-, alphaB-/-, and alpha(A/B) -/- (DKO) mouse lens epithelia and examined by electron microscopic and biochemical analyses. The wild-type microtubules were 4 mum long and approximately 25 nm wide and had a characteristic protofilament structure, but alphaB-/- microtubules were 2.5-fold longer. Microtubule-associated proteins (MAPs) extracted from microtubules by washing with salt included transketolase, alpha-enolase, and betaB2-crystallin. In DKO lens epithelial microtubules but not in wild-type, alphaA-/- or alphaB-/- microtubules, extraction of the MAPs gave very long (14-20 microm) "polyfilament" assemblies that were tightly bundled. Addition of exogenous alpha-crystallin (alphaA+ alphaB) was ineffective in preventing polyfilament formation. However, normal microtubule structure could be restored by including MAPs derived from wild-type lens epithelial cells during microtubule reconstitution. Intriguingly, these data suggest that alpha-crystallin may interact with MAPs to inhibit aggregation of microtubules in lens epithelial cells. Sedimentation analysis and 90 degrees light scattering measurements showed that alpha-crystallin suppressed tubulin assembly in vitro. Alpha-crystallin did not have a strong effect on the GTPase activity of purified tubulin. SDS-PAGE analysis showed that alpha-crystallin prevented heat-induced aggregation of tubulin, suggesting that alpha-crystallin may affect microtubule assembly by maintaining the pool of unassembled tubulin.

A comprehensive analysis of the expression of crystallins in mouse retina.

PURPOSE: Crystallins are expressed at high levels in lens fiber cells. Recent studies have revealed that several members of the alpha, beta, and gamma-crystallin family are also distributed in many non-lens tissues, though at lower levels. We observed that the use of retinal RNA as target for both custom I-Gene microarrays and Affymetrix GeneChips revealed significant expression of many crystallin genes. This prompted us to undertake a comprehensive investigation to delineate the baseline expression of crystallin genes in the adult mouse retina. METHODS: Quantitative RT-PCR was carried out using gene specific primers (derived from the mouse genomic sequence) for each crystallin gene. Immunofluorescence studies using frozen sections of the mouse retinas were performed with crystallin-specific antibodies. Retinal lysates were analyzed by immunoblotting using antibodies specific to alphaA and alphaB crystallins and those produced against total beta-crystallin and gamma-crystallin fractions of bovine lenses. RESULTS: Microarray analysis followed by quantitative RT-PCR revealed that mouse retinal cells express transcripts for 20 different members of the crystallin gene family; these are alphaA, alphaA-INS, alphaA-nov1, alphaB, betaA1, betaA2, betaA3, betaA4, betaB1, betaB2, betaB3, gammaA, gammaC, gammaD, gammaE, gammaF, gammaS, mu, zeta, and lambda-crystallin. The gene products of alphaA, alphaB, beta-, and gamma-crystallins are detected in the outer and inner nuclear layers of the retina by immunofluorescence analysis. In addition, alpha and beta-crystallins are detected in the photoreceptor inner segments. Retinal expression of these proteins was further confirmed by immunoblot analysis. Interestingly, our studies also showed a significant animal-to-animal variation in the expression level of some of the crystallins. CONCLUSIONS: Our results establish the expression of many crystallins in the adult mouse retina. Detection of crystallins in the retinal nuclear layers, though surprising, is consistent with their proposed role in cell survival and genomic stability. We suggest that crystallins play vital functions in protecting retinal neurons from damage by environmental and/or metabolic stress.

Identification of genes responsive to UV-A radiation in human lens epithelial cells using complementary DNA microarrays.

UV-A radiation produces cataract in animals, enhances photoaging of the lens and skin and increases the phototoxicity of drugs. However, the nature of genes that are activated or repressed after cellular exposure to UV-A radiation remains enigmatic. Because lens epithelial cells exposed to UV-A radiation undergo apoptosis 4 h after exposure to the stress, we sought to establish the change in gene expression in cells by UV-A radiation using gene expression profiling using complementary DNA microarrays containing about 12 000 genes. We identified 78 genes abnormally expressed in UV-A-irradiated cells (showing >2.5-fold change at P < 0.05). These genes are implicated in various biological processes, including signal transduction and nucleic acid binding, and genes encoding enzymes. A majority of the genes were downregulated. Our analysis revealed that the expression of genes for the transcription factors ATF-3 and Pilot increased four-fold, whereas the gene for the apoptosis regulator NAPOR-1 decreased five-fold. These changes were confirmed by real-time quantitative reverse transcriptase-polymerase chain reaction. The calpain large polypeptide 3 (CANP3) gene also increased nine-fold after UV-A radiation. In addition, peroxisomal biogenesis factor 7, glucocorticoid receptor-alpha and tumor-associated calcium signal transducer genes decreased three- to eight-fold. Western blot analysis further confirmed the increase in protein expression of ATF-3 and CANP3 and decreased expression of glucocorticoid receptor-alpha in the irradiated cells. Surprisingly, most of these genes had not been previously shown to be modulated by UV-A radiation. Our results show that human lens epithelial cells respond to a single dose of UV-A radiation by enhancing or suppressing functionally similar sets of genes, some of which have opposing functions, around the time at which apoptosis occurs. These studies support the intriguing concept that activation of competing pathways favoring either cell survival or death is a means to coordinate the response of cells to UV-A stress.

Mechanism of small heat shock protein function in vivo: a knock-in mouse model demonstrates that the R49C mutation in alpha A-crystallin enhances protein insolubility and cell death.

alphaA-crystallin (Cryaa/HSPB4) is a small heat shock protein and molecular chaperone that prevents nonspecific aggregation of denaturing proteins. Several point mutations in the alphaA-crystallin gene cause congenital human cataracts by unknown mechanisms. We took a novel approach to investigate the molecular mechanism of cataract formation in vivo by creating gene knock-in mice expressing the arginine 49 to cysteine mutation (R49C) in alphaA-crystallin (alphaA-R49C). This mutation has been linked with autosomal dominant hereditary cataracts in a four-generation Caucasian family. Homologous recombination in embryonic stem cells was performed using a plasmid containing the C to T transition in exon 1 of the cryaa gene. alphaA-R49C heterozygosity led to early cataracts characterized by nuclear opacities. Unexpectedly, alphaA-R49C homozygosity led to small eye phenotype and severe cataracts at birth. Wild type littermates did not show these abnormalities. Lens fiber cells of alphaA-R49C homozygous mice displayed an increase in cell death by apoptosis mediated by a 5-fold decrease in phosphorylated Bad, an anti-apoptotic protein, but an increase in Bcl-2 expression. However, proliferation measured by in vivo bromodeoxyuridine labeling did not decline. The alphaA-R49C heterozygous and homozygous knock-in lenses demonstrated an increase in insoluble alphaA-crystallin and alphaB-crystallin and a surprising increase in expression of cytoplasmic gamma-crystallin, whereas no changes in beta-crystallin were observed. Co-immunoprecipitation analysis showed increased interaction between alphaA-crystallin and lens substrate proteins in the heterozygous knock-in lenses. To our knowledge this is the first knock-in mouse model for a crystallin mutation causing hereditary human cataract and establishes that alphaA-R49C promotes protein insolubility and cell death in vivo.

The R116C mutation in alpha A-crystallin diminishes its protective ability against stress-induced lens epithelial cell apoptosis.

alphaA-crystallin is a small heat-shock protein expressed preferentially in the lens and is detected during the early stages of lens development. Recent work indicates that the expression of alphaA-crystallin enhances lens epithelial cell growth and resistance to stress conditions. Mutation of the arginine 116 residue to cysteine (R116C) in alphaA-crystallin has been associated with congenital cataracts in humans. However, the physiological consequences of this mutation have not been analyzed in lens epithelial cells. In the present study, we expressed wild type or R116C alphaA-crystallin in the human lens epithelial cell line HLE B-3. Immunofluorescence and confocal microscopy indicated that both wild type and R116C alphaA-crystallin were distributed mainly in the cytoplasm of lens epithelial cells. Size-exclusion chromatography indicated that the size of the alphaA-crystallin aggregate in lens epithelial cells increased from 500 to 600 kDa for the wild type protein to >2 MDa in the R116C mutant. When cells were exposed to physiological levels of UVA radiation, wild type alphaA-crystallin protected cells from apoptotic death as shown by annexin labeling and flow cytometric analysis, whereas the R116C mutant had a 4- to 10-fold lower protective ability. UVA-irradiated cells expressing the wild type protein had very low TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining, whereas cells expressing R116C mutant had a high level of TUNEL staining. F-actin was protected in UVA-treated cells expressing the wild type alphaA-crystallin but was either clumped around the apoptotic cells or was absent in apoptotic cells in cultures expressing the R116C mutant. Structural changes caused by the R116C mutation could be responsible for the reduced ability of the mutant to protect cells from stress. Our study shows that comparing the stress-induced apoptotic cell death is an effective way to compare the protective abilities of wild type and mutant alphaA-crystallin. We propose that the diminished protective ability of the R116C mutant in lens epithelial cells may contribute to the pathogenesis of cataract.

Cell kinetic status of mouse lens epithelial cells lacking alphaA- and alphaB-crystallin.

alphaA- and alphaB-crystallins are small heat shock proteins and molecular chaperones that are known to prevent non-specific aggregation of denaturing proteins. Recent work indicates that alphaA-/- lens epithelial cells grow at a slower rate than wild-type cells, and cultured alphaB-/- cells demonstrate increased hyperproliferation and genomic instability, suggesting that these proteins may exert a direct effect on the cell cycle kinetics, and influence cell proliferation. However, the cell cycle parameters of alphaA/alphaBKO (double knockout) cells have not been analyzed. Here we investigate the cell cycle kinetics of synchronized mouse lens epithelial cultures derived from wild-type and alphaA/alphaB double knockout (alphaA/alphaBKO) mice using BrdU labeling of proliferating cells, and flow cytometric analysis. We also provide data on the changing pattern of expression of HSP25, a small heat shock protein in alphaA/alphaBKO and wild-type cells during the cell cycle. Using serum starvation to synchronize cells in the quiescent G0 phase, and restimulation with serum followed by BrdU labeling and flow cytometry, the data indicated that as compared to wild-type cells, a <50% smaller fraction of the alphaA/alphaBKO cells entered the DNA synthetic S phase of the cell cycle. Furthermore, there was a delay in cell cycle transit through S phase in alphaA/alphaBKO cells, suggesting that although capable of entering S phase, the alphaA/alphaBKO cells are blocked in G1 phase, and are delayed in their cell cycle progression. Immunoblot analysis with antibodies to the small heat shock protein HSP25 indicated that although HSP25 increased in G1 phase of wild-type cells, and remained elevated on further progression through the cell cycle, HSP25 accumulation was delayed to S phase in alphaA/alphaBKO cells. These data can be interpreted to indicate that mouse lens epithelial cell progression through the cell cycle is significantly affected by expression of alphaA and alphaB-crystallin.

A comparative analysis of alphaA- and alphaB-crystallin expression during the cell cycle in primary mouse lens epithelial cultures.

AlphaA- and alphaB-crystallins are small heat shock proteins and molecular chaperones that prevent non-specific aggregation of denaturing proteins. Previous work in our laboratory has shown that lens epithelial cells derived from alphaA-/- mice exhibit slower growth, whereas alphaB-/- lens epithelial cells hyperproliferate at a higher rate in culture [Andley et al., J. Biol. Chem. 273 (1998) 31252; FASEB J. 15 (2001) 221]. Although both have been implicated in apoptosis and cell proliferation, direct analysis of their expression during the cell cycle has not been investigated. This study was undertaken to define the expression levels of alphaA and alphaB-crystallins during the cell cycle. Primary lens epithelial cell cultures derived from wild type mice were synchronized by serum starvation, and pulsed with bromodeoxyuridine (BrdU) at different times after re-stimulation with serum. Dual parameter flow cytometric studies with BrdU and propidium iodide (PI)-labeled cells were performed. Cells entered S phase 14 hr after serum re-stimulation. The duration of the S phase was 6 hr, and the total cell cycle transit time was between 24-27 hr. Enhanced expression of cyclin A, a protein essential for DNA synthesis was used as an additional marker to define the initiation of the S phase. Immunoblotting analysis demonstrated that the expression of alphaA and alphaB-crystallin was up to 10-fold higher in cells synchronized in G0 phase than in G1 phase. The levels of the proteins increased three-fold again as the cells entered the S phase and progressed to mitosis, but did not rise to the levels observed in G0 phase. This increase in expression of alphaA-crystallin resulted in part from enhanced synthesis during the S phase, as shown by an increase in [35S]methionine-labeling and immunoprecipitation of the radiolabeled alphaA-crystallin. The results were further confirmed by flow cytometric analysis using DNA content and alphaA-crystallin expression. The increase in alphaB-crystallin in S phase was paralleled by an increase in gene expression as shown by real-time RT-PCR analysis. These results demonstrate for the first time that in lens epithelial cells, alphaA and alphaB-crystallin levels are modulated during the cell cycle. Since the absence of alphaA and alphaB- crystallin in lens epithelial cells has been associated with disturbance of the tubulin cytoskeleton during mitosis, and with increased cell death or genomic instability, our results indicating that the alphaA- and alphaB-crystallin expression increases prior to mitosis are significant. The differential expression of these crystallins in the cell cycle may be important for optimal lens epithelial growth and lens transparency.