There is increasing evidence that the retinoic acid receptor-related orphan receptor a (RORa) plays an important role in the regulation of metabolic pathways, particularly of fatty acid and cholesterol metabolism; however, the role of RORa in the regulation of hepatic lipogenesis has not been studied. Here, we report that RORa attenuates hepatic steatosis, probably via activation of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) and repression of the liver X receptor a (LXRa). First, RORa and its activator, cholesterol sulfate (CS), induced phosphorylation of AMPK, which was accompanied by the activation of serine-threonine kinase liver kinase B1 (LKB1). Second, the activation of RORa, either by transient transfection or CS treatment, decreased the TO901317-induced transcriptional expression of LXRa and its downstream target genes, such as the sterol regulatory element binding protein-1 (SREBP-1) and fatty acid synthase. RORa interacted physically with LXRa and inhibited the LXRa response element in the promoter of LXRa, indicating that RORa interrupts the autoregulatory activation loop of LXRa. Third, infection with adenovirus encoding RORa suppressed the lipid accumulation that had been induced by a free-fatty-acid mixture in cultured cells. Furthermore, we observed that the level of expression of the RORa protein was decreased in the liver of mice that were fed a high-fat diet. Restoration of RORa via tail-vein injection of adenovirus (Ad)-RORa decreased the high-fat-diet-induced hepatic steatosis. Finally, we synthesized thiourea derivatives that activated RORa, thereby inducing activation of AMPK and repression of LXRa. These compounds decreased hepatic triglyceride levels and lipid droplets in the high-fat-diet-fed mice. Conclusion: We found that RORa induced activation of AMPK and inhibition of the lipogenic function of LXRa, which may be key phenomena that provide the beneficial effects of RORa against hepatic steatosis. (HEPATOLOGY 2012;55:1379-1388 A n increasing number of populations in the world suffer from fatty liver, which is a disease defined as hepatic fat accumulation greater than 5% of the liver wet weight. The major causes of fatty liver are obesity, diabetes, hyperlipidemia, drugs, and metabolic disorders.1 Although this relativelyAbbreviations: ACC, acetyl-CoA carboxylase; Ad-RORa, adenovirus-RORa; AICAR, aminoimidazole carboxamide ribonucleotide; AKT2, v-akt murine thymoma viral oncogene homolog 1; AMPK, adenosine monophosphate (AMP)-activated protein kinase; ATP, adenosine triphosphate; BODIPY, borondipyrromethene; CA-AMPK, constitutively active AMPK; ChIP, chromatin immunoprecipitation; CS, cholesterol sulfate; Cyp7b1, oxysterol 7a-hydroxylase; DBD, DNA binding domain; FA, fatty acid; FAS, fatty acid synthase; FFA, free fatty acid; HFD, high-fat diet; LBD, ligand binding domain; LKB1, serine-threonine kinase liver kinase B1; LXRa, liver X receptor a; LXRE, LXR response element; NADH, reduced nicotinamide adenine dinucleotide; p, phosphorylated; RORa, retinoic acid rec...
Thirty-two C(5)-C(5a) exomethylene-modified bicyclomycin derivatives were prepared to determine the effect of structural modification of this unit on bicyclomycin (1) function. The compounds were grouped into three categories: the C(5)-unsaturated bicyclomycins, the C(5a)-substituted C(5)-C(5a)-dihydrobicyclomycin derivatives, and the C(5)-modified norbicyclomycins. An efficient three-step procedure was developed to synthesize C(5a)-substituted C(5),C(5a)-dihydrobicyclomycins. Bicyclomycin was converted to bicyclomycin C(2'),C(3')-acetonide (36) and then treated with a nucleophile in 50% aqueous methanol ("pH" 10.5) to give the C(5a)-substituted C(5),C(5a)-dihydrobicyclomycin C(2'),C(3')-acetonide. Removal of the acetonide group (trifluoroacetic acid in 50% aqueous methanol) in the final step provided the desired bicyclomycin derivative. All the compounds were evaluated using the rho-dependent ATPase assay and their antimicrobial activities determined using a filter disc assay. Most of the compounds were also tested in the transcription termination assay. We observed that many of the C(5)-unsaturated bicyclomycins effectively inhibited ATP hydrolysis at 400 &mgr;M and inhibited the production of rho-dependent transcripts at 100 &mgr;M. The biochemical activities of C(5a)-bicyclomycincarboxylic acid (5), methyl C(5a)-bicyclomycincarboxylate (6), ethyl C(5a)-bicyclomycincarboxylate (7), and bicyclomycin C(5)-norketone O-methyloxime (11) were all similar to 1. Compounds 6, 7, and 11 exhibited diminished antibiotic activity compared to 1, and 5 displayed no detectable activity. Several C(5a)-substituted C(5),C(5a)-dihydrobicyclomycins showed significant inhibition of rho-dependent ATPase and transcription termination activities. The inhibitory properties of C(5),C(5a)-dihydrobicyclomycin C(5a)-methyl sulfide (18), C(5),C(5a)-dihydrobicyclomycin C(5a)-phenyl sulfide (23), and C(5)-C(5a)-dihydrobicyclomycin-5,5a-diol (31) approached those of 1. Compounds 18, 23, and 31 did not exhibit antibiotic activity. Two of the four C(5)-modified norbicyclomycin adducts showed moderate inhibitory activities in the ATPase assay, and none showed significant antibiotic activity. Our findings showed that the C(5)-C(5a) exomethylene unit retention in 1 was not essential for inhibition of in vitro rho activity. The structure-activity relationship data indicated that bicyclomycins that contained a small unsaturated C(5) unit or C(5),C(5a)-dihydrobicyclomycins that possessed a small, nonpolar C(5a) substituent effectively inhibited rho function in in vitro biochemical assays. We concluded that the C(5)-C(5a) unit in 1 was not a critical structural element necessary for drug binding to rho and that irreversible, inactivating units placed at this site would permit the bicyclomycin derivative to bind efficiently to rho.
Twelve bicyclomycin derivatives were synthesized to determine the effect of modification of the [4.2.2] bicyclic unit in bicyclomycin (1) on drug function. Few bicyclomycin derivatives have been described in which the [4.2.2] ring system has been modified. The compounds evaluated were divided into two categories: the two N-methyl-modified bicyclomycins (2, 3) and the ten C(6)-substituted bicyclomycins (4-13). Substituents introduced at the C(6) site included alkoxy, thioalkoxy, thiophenoxy, anilino, and hydrogen. A procedure was developed to synthesize select C(6)-substituted bicyclomycins. Bicyclomycin was first converted to bicyclomycin C(2'),C(3')-acetonide (16) and then treated with methanesulfonyl chloride to give in situ the corresponding C(6) mesylate 17. Treatment of 17 with the appropriate nucleophile followed by removal of the C(2'),C(3')-acetonide group gave the desired C(6)-substituted bicyclomycin. The chemical properties of C(6) O-methylbicyclomycin (4) were examined. Treatment of THF-H(2)O mixtures of 4 with excess EtSH maintained at "pH" 8.0-9.0 led to no detectable reaction, while at more basic "pH" values 4 underwent stereospecific conversion to the bis-spiro derivative 33 and no appreciable EtSH addition to the C(5)-C(5a) exomethylene unit. These results were compared to the reactivity of 1 with EtSH. The stability (pH 7.4, 37 degrees C) of C(6)-substituted bicyclomycins 4, 6, and 10-13 in aqueous solutions were examined. We observed that most of these compounds (4, 6, 10-12) underwent near complete change (>75%) within 200 h. The [4.2.2] bicyclic-modified bicyclomycins were evaluated in the rho-dependent ATPase assay and their antimicrobial activities determined using a filter disc assay. Most of the compounds were also tested in the transcription termination assay. We observed that all structural modifications conducted within the [4.2.2] bicyclic unit led to a loss of rho-dependent ATPase (I(50) > 400 &mgr;M) and to transcription termination (I(50) > 100 &mgr;M) inhibitory activities, as well as a loss of antimicrobial activity (MIC > 32 mg/mL). Only N(10)-methylbicyclomycin (2) displayed moderate inhibitory activities in these assays. These findings indicated that the [4.2.2] bicyclic unit played an important role in the antibiotic-rho recognition process. Potential factors that govern this interaction are briefly discussed. We concluded that placement of an irreversible inactivating unit at the N- and O-sites within the [4.2.2] bicyclic unit in 1 would likely prohibit the bicyclomycin derivative from efficiently binding to rho.
The commercial antibiotic bicyclomycin (1) has been shown to target the essential transcriptional termination factor rho in Escherichia coli. Little is known, however, about the bicyclomycin binding site in rho. A recent structure−activity relationship study permitted us to design modified bicyclomycins that may irreversibly inactivate rho. The four compounds selected were C(5a)-(4-azidoanilino)dihydrobicyclomycin (3), C(5a)-(3-formylanilino)dihydrobicyclomycin (4), C(5)−norbicyclomycin C(5)-O-(4-azidobenzoate) (5), and C(5)-norbicyclomycin C(5)-O-(3-formylbenzoate) (6). In each of these compounds the inactivating unit was placed at the C(5)−C(5a) site in bicyclomycin. In compounds 3 and 5 an aryl azide moiety was used as a photoaffinity label whereas in 4 and 6 an aryl aldehyde group was employed as a reductive amination probe. The synthesis and spectral properties of 3−6 are described. Chemical studies demonstrated that 3 and 4 were stable in D2O and CD3OD (room temperature, 7 d), while 5 and 6 underwent significant change within 1 d. Biochemical investigations showed that 3 and 4 retained appreciable inhibitory activities in rho-dependent ATPase and transcription termination assays. In the ATPase assay, I 50 values for 1, 3, and 4 were 60, 135, and 70 μM, respectively. Correspondingly, the I 50 values for 5 and 6 were >400 and 225 μM, respectively. In the transcription termination assay, compounds 1, 3, and 4 all prevented (≥97%) the production of rho-dependent transcripts at 40 μM, whereas little (≤15%) inhibition of transcription termination was observed for 5 and 6 at this concentration. Antimicrobial evaluation of 3−6 showed that none of the four compounds exhibited antibiotic activity at 32 mg/mL or less against W3350 E. coli. The combined chemical and biochemical studies led to our further evaluation of 3 and 4. Photochemical irradiation (254 nm) of 3 in the presence of rho led to a 29−32% loss of rho ATPase activity. Attempts to confirm the irreversible adduction of 3 to rho by electrospray mass spectrometry were unsuccessful. No higher molecular weight adducts were detected. Incubation of rho with 4 at room temperature (4 h) followed by the addition of NaBH4 led to significant losses (>62%) of rho ATPase activity. Analyses of the 4−rho modified adduct showed appreciable levels of adduction (∼40%). Mass spectrometric analyses indicated a molecular weight for the adduct of approximately 47 410, consistent with a modification of a rho lysine residue by 4. Compound 4 was selected for additional studies.
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