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.

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.

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.

Reduced survival of lens epithelial cells in the alphaA-crystallin-knockout mouse.

alphaA-Crystallin (alphaA) is a molecular chaperone expressed preferentially in the lens. alphaA transcripts are first detected during the early stages of lens development and its synthesis continues as the lens grows throughout life. alphaA(-/-) mouse lenses are smaller than controls, and lens epithelial cells derived from these mice have diminished growth in culture. In the current work, we tested the hypothesis thatalphaA prevents cell death at a specific stage of the cell cycle in vivo. Seven-day-old 129Sv (wild-type) and alphaA(-/-) mice were injected with 5-bromo-2'-deoxyuridine (BrdU) to label newly synthesized DNA in proliferating cells. To follow the fate of the labeled cells, wholemounts of the capsule epithelial explants were made at successive times after the BrdU pulse, and the labeling index was determined. Immunofluorescence and confocal microscopy showed that both wild-type and alphaA(-/-) cells had a 3-hour labeling index of 4.5% in the central region of the wholemount, indicating that the number of cells in S phase was the same. Twenty-four hours after the pulse, individual cells labeled with BrdU had divided and BrdU-labeled cells were detected in pairs. The 24-hour labeling index in the wild-type lens was 8.6%, but in the alphaA(-/-) lens it was significantly lower, suggesting that some of the cells failed to divide and/or that the daughter cells died during mitosis. TUNEL labeling was rarely detected in the wild-type lens, but was significant and always detected in pairs in the alphaA(-/-) wholemounts. Dual labeling with TUNEL and BrdU also suggested that the labeled cells were dying in pairs in the alphaA(-/-) lens epithelium. Immunolabeling of wholemounts with beta-tubulin antibodies indicated that the anaphase spindle in a significant proportion of alphaA(-/-) cells was not well organized. Examination of the cellular distribution of alphaA in cultured lens epithelial cells showed that it was concentrated in the intercellular microtubules of cells undergoing cytokinesis. These data suggest that alphaA expression in vivo protects against cell death during mitosis in the lens epithelium, and the smaller size of the alphaA(-/-) lens may be due to a decrease in the net production of epithelial cells.

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.

Hyperproliferation and p53 status of lens epithelial cells derived from alphaB-crystallin knockout mice.

alphaB-Crystallin, a major protein of lens fiber cells, is a stress-induced chaperone expressed at low levels in the lens epithelium and numerous other tissues, and its expression is enhanced in certain pathological conditions. However, the function of alphaB in these tissues is not known. Lenses of alphaB-/- mice develop degeneration of specific skeletal muscles but do not develop cataracts. Recent work in our laboratory indicates that primary cultures of alphaB-/- lens epithelial cells demonstrate genomic instability and undergo hyperproliferation at a frequency 4 orders of magnitude greater than that predicted by spontaneous immortalization of rodent cells. We now demonstrate that the hyperproliferative alphaB-/- lens epithelial cells undergo phenotypic changes that include the appearance of the p53 protein as shown by immunoblot analysis. Sequence analysis showed a lack of mutations in the p53 coding region of hyperproliferative alphaB-/- cells. However, the reentry of hyperproliferative alphaB-/- cells into S phase and mitosis after DNA damage by gamma-irradiation were consistent with impaired p53 checkpoint function in these cells. The results demonstrate that expression of functionally impaired p53 is one of the factors that promote immortalization of lens epithelial cells derived from alphaB-/- mice. Fluorescence in situ hybridization using probes prepared from centromere-specific mouse P1 clones of chromosomes 1 and 9 demonstrated that the hyperproliferative alphaB-/- cells were 30% diploid and 70% tetraploid, whereas wild type cells were 83% diploid. Further evidence of genomic instability was obtained when the hyperproliferative alphaB-/- cells were labeled with anti-beta-tubulin antibodies. Examination of the hyperproliferative alphaB-/- mitotic profiles revealed the presence of cells that failed to round up for mitosis, or arrested in cytokinesis, and binucleated cells in which nuclear division had occurred without cell division. These results suggest that the stress protein and molecular chaperone alphaB-crystallin protects cells from acquiring impaired p53 protein and genomic instability.