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The removal of intervening sequences from transcripts is catalyzed by the spliceosome, a multicomponent complex that assembles on the newly synthesized pre-mRNA. Pre-mRNA translation in the cytoplasm leads to the generation of aberrant proteins that are potentially harmful. Therefore, tight control to prevent undesired pre-mRNA export from the nucleus and its subsequent translation is an essential requirement for reliable gene expression. Here, we show that the natural product FR901464 ( 1 ) and its methylated derivative, spliceostatin A ( 2 ), inhibit in vitro splicing and promote pre-mRNA accumulation by binding to SF3b, a subcomplex of the U2 small nuclear ribonucleoprotein in the spliceosome. Importantly, treatment of cells with these compounds resulted in leakage of pre-mRNA to the cytoplasm, where it was translated. Knockdown of SF3b by small interfering RNA induced phenotypes similar to those seen with spliceostatin A treatment. Thus, the inhibition of pre-mRNA splicing during early steps involving SF3b allows unspliced mRNA leakage and translation.

Extracellular signal-regulated kinase (ERK), a serine/threonine protein kinase of the mitogen-activated protein kinase superfamily, is activated by various stimuli in inflammatory cells. We recently described FR180204 (5-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-amine), a novel selective ERK inhibitor. In this paper, we investigated the effect of FR180204 on collagen-induced arthritis (CIA) in DBA/1 mice, an animal model of rheumatoid arthritis (RA) mediated by type II collagen (CII)-reactive T cells and anti-CII antibodies. Preventive administration of FR180204 (100 mg/kg, i.p., b.i.d.) significantly ameliorated the clinical arthritis and body weight loss occurring in the CIA mice. Further, FR180204-treated mice showed a significant decrease in plasma anti-CII antibody levels (62%). FR180204 also attenuated delayed-type hypersensitivity in CII-immunized DBA/1 mice, an inflammatory response elicited by CII-reactive T cells, in a dose-dependent manner (52 and 62% inhibition at 32 and 100 mg/kg, respectively). Moreover, FR180204 inhibited in vitro CII-induced proliferation of lymph node cells prepared from CII-immunized mice, in which CII-specific T cells are known to undergo specific proliferation. In conclusion, our results suggest that ERK regulates both the cell-mediated and humoral immune responses in the development of CIA. ERK inhibitors may be useful as therapeutic reagents for the treatment of RA.

Enhancement of transgene expression is an important issue in human gene therapy. Here we describe a novel system for enhancing transgene expression by cointroduction of plasmid DNA with FR901228, a water-soluble histone deacetylase inhibitor. When a luciferase expression vector was cointroduced into cells with FR901228, luciferase gene expression was enhanced 50-fold in the mouse melanoma cell line B16-F1 and 5200-fold in NIH3T3 cells in comparison to cells without the drug. Luciferase gene expression enhancement was dependent on both drug dose and treatment time. Acetylated histones increased in accordance with drug dose, and the activation of gene expression occurred at the transcriptional level. The stimulation of luciferase gene expression by FR901228 was also observed in a B16-F1 clone stably expressing luciferase. Cointroduction of the luciferase plasmid with FR901228 into a B16-F1 tumor mass activated luciferase gene expression 3- to 4-fold. Thus, activation of transgene expression by FR901228 may serve as a new tool for gene therapy.

The novel gluconeogenesis inhibitor FR225654, isolated from the culture broth of Phoma sp. No. 00144, has an unique structure that consists of a highly oxygenated trans -decalin ring and a β-keto-enol, with a characteristic side chain. This compound selectively inhibited gluconeogenesis of rat primary hepatocytes and had hypoglycemic effects in several in vivo mouse models.

A novel gluconeogenesis inhibitor, FR225654 was isolated from the culture broth of Phoma sp. No. 00144. Spectroscopic analysis concluded that FR225654 has a highly oxygenated trans -decalin ring and β-keto-enol in its main part, and has a characteristic side chain possessing a conjugated carboxylic acid and tri-substituted olefin.

In the course of screening for a new anti-hyperlipidemic agent from microbial products, we found that FR177391, produced by Serratia liquefaciens No. 1821, alleviated the decrease in lipid droplet formation in differentiated 3T3-L1 adipocyte cells, induced by the addition of tumor necrosis factor-α. Structural elucidation by spectroscopic methods and X-ray crystallographic analysis of its propylamide derivative revealed that FR177391 was a chlorinated macrocyclic lactone.

Natural products with distinct biological activities are very promising molecular probes to dissect the novel pathways of biology. FR177391, a product of bacteria, was obtained as a natural compound possessing anti-hyperlipidemic effects. FR177391 enhances differentiation of mouse 3T3-L1 fibroblasts to adipocytes and reduces the circulating levels of triglyceride in C57BL/KsJ-db/db mice, a obese non-insulin-dependent diabetes mellitus animal model, although its mechanism of actions remained to be unknown. We report here that the target protein for FR177391 was identified to be protein phosphatase 2A (PP2A) by employing the method of affinity chromatography. FR177391 potently inhibited PP2A activity at nano molar concentration, and shared its binding pocket with a phosphatase inhibitor, okadaic acid. In addition to the phenotypic alterations, the enhancement for phosphorylation of extracellular signal-regulated kinase (ERK) protein was observed in the FR177391-treated 3T3-L1 cells. These results suggest that prolonged activation of ERK protein due to inhibition of its dephosphorylation by PP2A plays an important role in adipocyte maturation and regulation of the blood revels of lipids.

FR225654, a novel gluconeogenesis inhibitor, was isolated from the culture broth of Phoma sp. No. 00144 and purified by adsorptive resin and reverse-phase column chromatography. This compound is a potent inhibitor of gluconeogenesis and is a promising candidate of anti-diabetic agent.

FR177391 produced by Serratia liquefaciens No. 1821 enhances differentiation of mouse 3T3-L1 fibroblasts to adipocytes and reduces the circulating levels of triglyceride in C57BL/KsJ-db/bd mice, an obese non-insulin-dependent diabetes mellitus animal model, although its mechanism of actions remained to be unknown. Its active derivative, 20-hydroxy FR177391, and its inactive derivative, 3-hydroxy FR177391 were produced by microbial conversion of FR177391, and biotin-labeled FR177391 was synthesized from 20-hydroxy FR177391 as an active affinity ligand to identify target molecules of FR177391 by chemical genetic approaches.

In the course of screening for a new anti-hyperlipidemic agent from microbial products, we found that FR177391, produced by Serratia liquefaciens No. 1821, alleviated the decrease in lipid droplet formation in differentiated 3T3-L1 adipocyte cells, induced by the addition of tumor necrosis factor-α. Structural elucidation by spectroscopic methods and X-ray crystallographic analysis of its propylamide derivative revealed that FR177391 was a chlorinated macrocyclic lactone.