A selective detection of lysophosphatidylcholine in dried blood spots for diagnosis of adrenoleukodystrophy by LC-MS/MS.

X-linked adrenoleukodystrophy (X-ALD) is a rare inherited metabolic disorder characterized by an impaired beta-oxidation of very long chain fatty acids in the peroxisomes. Recent studies have suggested that 1-hexacosanoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso-PC 26:0) can be a sensitive biomarker for X-ALD. Although approximately 10-fold increase in the concentration of Lyso-PC 26:0 in DBSs from X-ALD-affected individuals were reported, whether the carriers might be distinguished from the healthy controls remained unclear. To address this question, we have validated previously developed LC-MS/MS-based analytical procedures using QC DBS. We found that the recovery of Lyso-PC 26:0 from the QC DBSs was 73.6 ± 0.3% when 2 µM of Lyso-PC 26:0 was spiked into the blood. Based on this result, the amounts of Lyso-PC 26:0 in the controls and ALD-affected individuals were 0.090 ± 0.004 (n = 11) and 1.078 ± 0.217 (n = 4) pmol/DBS, respectively. Interestingly, the concentration of Lyso-PC 26:0 in the carriers were 0.548 ± 0.095 pmol/DBS (n = 3), indicating that the carriers and the healthy controls can be distinguished. These results suggest that LC-MS/MS-based technique can be used for the detection of asymptomatic carriers and X-ALD-affected subjects in the newborn screening.

Generation of robust vascular networks from cardiovascular blast populations derived from human induced pluripotent stem cells in vivo and ex vivo organ culture system.

Vascular network formation is a key therapeutic event in regenerative medicine because it is essential for mitigating or ameliorating ischemic conditions implicated in various diseases and repair of tissues and organs. In this study, we induced human induced pluripotent stem cells (hiPSCs) to differentiate into heterogeneous cell populations which have abilities to form vascular vessel-like structures by recapitulating the embryonic process of vasculogenesis in vitro. These cell populations, named cardiovascular blast populations (CBPs) in this report, primarily consisted of CD31(+) and CD90(+) cells. By using cell-sheet technology, we observed that CBP with CD31(+) cells to CD90(+) cells in the ratio of 1:1.5 could reproducibly form robust vascular networks in vivo and ex vivo organ culture system. To our knowledge, this is the first report demonstrating the generation of vascular network from hiPSCs in ex vivo organ culture system that correlates closely with in vivo results. Our results suggest that CBP provides a promising approach for studying vasculogenesis and subsequently can be used in regenerative medicine.

Renoprotective properties of pirfenidone in subtotally nephrectomized rats.

Renal fibrosis is the final common pathway of chronic kidney disease, and its progression predicts the degree of renal dysfunction. We investigated the renoprotective properties of pirfenidone in a remnant kidney model of chronic renal failure to determine its pharmacological potency compared to enalapril. Five-sixths nephrectomized rats were fed diet containing pirfenidone (approximately 700mg/kg/day) for 8weeks. Pirfenidone steadily inhibited the progression of proteinuria, but not to a significant degree. Pirfenidone prevented the elevation of plasma creatinine and blood urea nitrogen. At the end of the experiment, pirfenidone had reduced systolic blood pressure by means of its renoprotective effect. In a histological study, pirfenidone improved interstitial fibrosis in the renal cortex. These effects were supported by the suppression of the expression of TGF-beta and fibronectin in the mRNA of the kidney. In contrast, pirfenidone had little effect on the expression of alpha-smooth muscle actin, which is one of the proteins responsible for epithelial-mesenchymal transition. This property was confirmed by the TGF-beta-induced transdifferentiation observed in cultured normal rat kidney tubular epithelial NRK52E cells. These results suggest that pirfenidone improves the progression of chronic renal failure via its antifibrotic action, although pirfenidone has less effective TGF-beta-induced epithelial to mesenchymal transdifferentiation.

Anti-herpes virus activity of polyether antibiotic CP-44161 in vivo.

In the previous study, we discovered a polyether antibiotic CP-44161, which was reported earlier as an anticoccidal agent, as an anti-varicella zoster virus compound. In this study, we demonstrated that CP-44161 had a very strong and broad anti-herpes virus activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in vitro. To determine the antiviral activity of CP-44161 in vivo, we examined its effect on the cutaneous HSV-2 infection model in Balb/c mice. CP-44161 showed inhibitory effect on lesion development as well as acyclovir (ACV) when the treatment was started from day 3. Meanwhile, in case the start of treatment was delayed until day 4, when ACV was no longer effective, the effectiveness of CP-44161 still remained. In this model, CP-44161 also showed inhibitory effect on the proliferation of HSV-2 DNA in dorsal root ganglia. This is the first article to report that polyether antibiotics can be effective on viral infection in vivo.

Inhibition of splicing and nuclear retention of pre-mRNA by spliceostatin A in fission yeast.

Nuclear retention of pre-mRNAs is tightly regulated by several security mechanisms that prevent pre-mRNA export into the cytoplasm. Recently, spliceostatin A, a methylated derivative of a potent antitumor microbial metabolite FR901464, was found to cause pre-mRNA accumulation and translation in mammalian cells. Here we report that spliceostatin A also inhibits splicing and nuclear retention of pre-mRNA in a fission yeast strain that lacks the multidrug resistance protein Pmd1. As observed in mammalian cells, spliceostatin A is bound to components of the SF3b complex in the spliceosome. Furthermore, overexpression of nup211, a homolog of Saccharomyces cerevisiae MLP1, suppresses translation of pre-mRNAs accumulated by spliceostatin A. These results suggest that the SF3b complex has a conserved role in pre-mRNA retention, which is independent of the Mlp1 function.

Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of pre-mRNA.

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.

Role of a cysteine residue in the active site of ERK and the MAPKK family.

Kinases of mitogen-activated protein kinase (MAPK) cascades, including extracellular signal-regulated protein kinase (ERK), represent likely targets for pharmacological intervention in proliferative diseases. Here, we report that FR148083 inhibits ERK2 enzyme activity and TGFbeta-induced AP-1-dependent luciferase expression with respective IC50 values of 0.08 and 0.05 microM. FR265083 (1'-2' dihydro form) and FR263574 (1'-2' and 7'-8' tetrahydro form) exhibited 5.5-fold less and no activity, respectively, indicating that both the alpha,beta-unsaturated ketone and the conformation of the lactone ring contribute to this inhibitory activity. The X-ray crystal structure of the ERK2/FR148083 complex revealed that the compound binds to the ATP binding site of ERK2, involving a covalent bond to Sgamma of ERK2 Cys166, hydrogen bonds with the backbone NH of Met108, Nzeta of Lys114, backbone C=O of Ser153, Ndelta2 of Asn154, and hydrophobic interactions with the side chains of Ile31, Val39, Ala52, and Leu156. The covalent bond motif in the ERK2/FR148083 complex assures that the inhibitor has high activity for ERK2 and no activity for other MAPKs such as JNK1 and p38MAPKalpha/beta/gamma/delta which have leucine residues at the site corresponding to Cys166 in ERK2. On the other hand, MEK1 and MKK7, kinases of the MAPKK family which also can be inhibited by FR148083, contain a cysteine residue corresponding to Cys166 of ERK2. The covalent binding to the common cysteine residue in the ATP-binding site is therefore likely to play a crucial role in the inhibitory activity for these MAP kinases. These findings on the molecular recognition mechanisms of FR148083 for kinases with Cys166 should provide a novel strategy for the pharmacological intervention of MAPK cascades.

FR180204, a novel and selective inhibitor of extracellular signal-regulated kinase, ameliorates collagen-induced arthritis in mice.

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.

The novel gluconeogenesis inhibitor FR225654 that originates from Phoma sp. no. 00144. I. Taxonomy, fermentation, isolation and physico-chemical properties.

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.

The novel gluconeogenesis inhibitor FR225654 that originates from Phoma sp. no. 00144. II. Biological activities.

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 beta-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.

The novel gluconeogenesis inhibitor FR225654 that originates from Phoma sp. no. 00144. III. Structure determination.

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 beta-keto-enol in its main part, and has a characteristic side chain possessing a conjugated carboxylic acid and tri-substituted olefin.

Identification of a selective ERK inhibitor and structural determination of the inhibitor-ERK2 complex.

Selective inhibition of extracellular signal-regulated kinase (ERK) represents a potential approach for the treatment of cancer and other diseases; however, no selective inhibitors are currently available. Here, we describe an ERK-selective inhibitor, FR180204, and determine the structural basis of its selectivity. FR180204 inhibited the kinase activity of ERK1 and ERK2, with K(i) values 0.31 and 0.14microM, respectively. Lineweaver-Burk analysis of the binding interaction revealed that FR180204 acted as competitive inhibitor of ATP. In mink lung epithelial Mv1Lu cells, FR180204 inhibited TGFbeta-induced luciferase-expression. X-ray crystal structure analysis of the human ERK2/FR180204 complex revealed that Q105, D106, L156, and C166, which form the ATP-binding pocket on ERK, play important roles in the drug/protein interaction. These results suggest that FR180204 is an ERK-selective and cell-permeable inhibitor, and could be useful for elucidating the roles of ERK as well as for drug development.

A putative role for histone deacetylase in the differentiation of human erythroid cells.

Histone acetylation controls the expression of specific genes in eukaryotic cells. We investigated the role of histone deacetylases (HDACs) in the differentiation of human erythroid cells, using pharmacological approaches. When CD36+ erythroid precursor cells, generated from CD34+ cells with stem cell factor, flt-3 ligand, thrombopoietin, interleukin-3, interleukin-6, and erythropoietin, were cultured with an HDAC inhibitor FK228 (depsipeptide) at a specified dose in the presence of erythropoietin, their differentiation was inhibited, as determined by the expression of CD45 and glycophorin A. Addition of the same dose of FK228 to cultures did not affect the growth of CD36+ cells. Regardless of the presence or absence of FK228, cultured CD36+ cells displayed similar proliferation kinetics. Analysis of acetylated histones revealed that FK228 upregulated the acetylation status of histones H3 and H4 in CD36+ cells. The inhibition of CD36+ cell differentiation was restored by removal of FK228 from the culture, indicating that the modification of CD36+ cell differentiation by FK228 is reversible. Furthermore, interference with histone deacetylation by FK228 inhibited the generation of CD36+ erythroid cells from CD34+ hematopoietic progenitor cells. Our results indicate the possible involvement of HDACs in human erythropoiesis, especially the regulation of erythroid cell differentiation.

Signal therapy of breast cancers by the HDAC inhibitor FK228 that blocks the activation of PAK1 and abrogates the tamoxifen-resistance.

PAK1, a Rac/CDC42-dependent Ser/Thr kinase, is required for both neurofibromatosis (NF) and RAS transformation in vivo. FK228, a histone deacetylase (HDAC) inhibitor, activates a very specific set of genes such as the tumor suppressor WAF1, an inhibitor of cyclin-dependent kinases (CDKs), and suppresses the growth of these tumors. In addition, this drug downregulates cyclin D1, which is upregulated by RAS through PAK1, in breast cancers. In this study, we demonstrate that FK228 at 0.1-1 nM significantly reduces the kinase activity of PAK1 in these cells, without affecting the protein level of PAK1. Interestingly, estrogen receptor (ER) and PAK1 mutually activate each other in breast cancers. Here we provide an evidence suggesting that breast cancers require PAK1 for their estrogen-dependent growth. Moreover, the treatment with FK228 strongly inhibits the estrogen-dependent growth of human breast cancers (both tamoxifen-sensitive and resistant cell lines) in vivo, suggesting that FK228 and other anti-PAK1 drugs would be useful for the treatment of breast cancers which become resistant to currently used estrogen antagonists such as tamoxifen.

Signal therapy of NF1-deficient tumor xenograft in mice by the anti-PAK1 drug FK228.

PAK1, a Rac/CDC42-dependent Ser/Thr kinase, is required for the malignant growth of RAS transformants as well as both NF1-deficient and NF2-deficient cancer cells. FK228, a histone deacetylase (HDAC) inhibitor, suppresses the growth of more than 70% of human cancers in vivo including RAS transformants, breast cancers and prostate cancers by activating a set of genes including the tumor suppressors gelsolin and p21(WAF1), that block upstream and downstream of PAK1, respectively. Here we demonstrate that (1) the anti-PAK1 drug FK228 (0.1 nM) completely blocks the growth of both NF1-deficient and NF2-deficient cancer cells in vitro, and that (2) FK228 (2.5 mg/kg, i.p., twice a week) causes the complete regression of an NF1-deficient human malignant peripheral nerve sheath tumor (MPNST) xenograft in nude mice. This is the very first case where a chemical drug in clinical trials for cancers has ever worked so effectively on neurofibromatosis (experimental neurofibromas) in vivo.

Structure of a high-resolution crystal form of human triosephosphate isomerase: improvement of crystals using the gel-tube method.

Crystals of human triosephosphate isomerase with two crystal morphologies were obtained using the normal vapour-diffusion technique with identical crystallization conditions. One had a disordered plate shape and the crystals were hollow (crystal form 1). As a result, this form was very fragile, diffracted to 2.8 A resolution and had similar crystallographic parameters to those of the structure 1hti in the Protein Data Bank. The other had a fine needle shape (crystal form 2) and was formed more abundantly than crystal form 1, but was unsuitable for structure analysis. Since the normal vapour-diffusion method could not control the crystal morphology, gel-tube methods, both on earth and under microgravity, were applied for crystallization in order to control and improve the crystal quality. Whereas crystal form 1 was only slightly improved using this method, crystal form 2 was greatly improved and diffracted to 2.2 A resolution. Crystal form 2 contained a homodimer in the asymmetric unit, which was biologically essential. Its overall structure was similar to that of 1hti except for the flexible loop, which was located at the active centre Lys13.

FR177391, a new anti-hyperlipidemic agent from Serratia. I. Taxonomy, fermentation, isolation, physico-chemical properties, structure elucidation and biological activities.

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-alpha. Structural elucidation by spectroscopic methods and X-ray crystallographic analysis of its propylamide derivative revealed that FR177391 was a chlorinated macrocyclic lactone.

FR177391, a new anti-hyperlipidemic agent from Serratia. IV. Target identification and validation by chemical genetic approaches.

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.