Synthesis of covalently immobilized phospholipid analogues

Cyclic 1-O-acyl-2-O-alkyl-glycero-3-phosphotriesters and 1-O-acyl-2-O-alkyl-glycero-3-bromoethylphosphate with a free acyl moiety in position 1 of the glycerol backbone were synthesized. These phospholipid intermediates were covalently bound to AH-Sepharose via the carbodiimide method. After immobilization the corresponding phosphatidylethanolamine analogues were obtained by acid hydrolysis of the cyclic phosphotriesters and by direct amination of the bromoethylphosphate. Thus, in a short, stepwise synthesis including minimum use of protecting groups, a variety of immobilized phospholipid analogues are available as affinity adsorbents for the purification of enzymes related to phospholipid metabolism.     

Protein farnesyltransferase isoprenoid substrate discrimination is dependent on isoprene double bonds and branched methyl groups

Farnesylation is a posttranslational lipid modification in which a 15-carbon farnesyl isoprenoid is linked via a thioether bond to specific cysteine residues of proteins in a reaction catalyzed by protein farnesyltransferase (FTase). We synthesized the benzyloxyisoprenyl pyrophosphate (BnPP) series of transferable farnesyl pyrophosphate (FPP) analogues (1a-e) to test the length dependence of the isoprenoid substrate on the FTase-catalyzed transfer of lipid to protein substrate. Kinetic analyses show that pyrophosphates 1a-e and geranyl pyrophosphate (GPP) transfer with a lower efficiency than FPP whereas geranylgeranyl pyrophosphate (GGPP) does not transfer at all. While a correlation was found between K(m) and analogue hydrophobicity and length, there was no correlation between k(cat) and these properties. Potential binding geometries of FPP, GPP, GGPP, and analogues 1a-e were examined by modeling the molecules into the active site of the FTase crystal structure. We found that analogue 1d displaces approximately the same volume of the active site as does FPP, whereas GPP and analogues 1a-c occupy lesser volumes and 1e occupies a slightly larger volume. Modeling also indicated that GGPP adopts a different conformation than the farnesyl chain of FPP, partially occluding the space occupied by the Ca(1)a(2)X peptide in the ternary X-ray crystal structure. Within the confines of the FTase pocket, the double bonds and branched methyl groups of the geranylgeranyl chain significantly restrict the number of possible conformations relative to the more flexible lipid chain of analogues 1a-e. The modeling results also provide a molecular explanation for the observation that an aromatic ring is a good isostere for the terminal isoprene of FPP.     

Synthesis, cannabinoid receptor activity, and enzymatic stability of reversed amide derivatives of arachidonoyl ethanolamide

Retroanandamide (2f) and its 10 analogues (1a-e, 2a-e) were synthesized and evaluated for the cannabinoid receptor activation by a [35S]GTPgammaS binding assay using rat cerebellar membranes, and Chinese hamster ovary cell membranes expressing human CB2 receptors. The primary goal of the study was to develop cannabinoid receptor agonists having improved enzymatic stability compared to endogenous N-arachidonoyl ethanolamide (AEA). Furthermore, by reversing the amide bond of AEA, the formation of arachidonic acid would be prevented. Finally, an effect of the carbonyl carbon position on the cannabinoid receptor activity was explored by synthesizing retroanandamide analogues having different chain lengths (1a-e, C19; 2a-f, C20). All the synthesized compounds, except 2c, behaved as partial agonists for the both cannabinoid receptors. In rat brain homogenate, the reversed amides possessed significantly higher stability against FAAH induced degradation than AEA. Therefore, the reversed amide analogues of AEA may serve as enzymatically stable structural basis for the drug design based on the endogenous cannabinoids.     

Anti-tumor and proapoptotic effect of novel synthetic benzophenone analogues in Ehrlich ascites tumor cells

A series of substituted benzophenone analogues, (2-aroyl-4-methylphenoxy)acetamides 4a-e, have been synthesized via three-step synthesis sequence beginning with the 2-hydroxybenzophenones 1a-e in excellent yield. 1a-e on reaction with ethyl chloroacetate afford ethyl (2-aroyl-4-methylphenoxy)acetates 2a-e which on alkaline hydrolysis afforded (2-aroyl-4-methylphenoxy)ethanoic acid 3a-e. Compounds 3a-e on condensation with p-chloroaniline furnished benzophenone analogues 4a-e. In the present report, we investigated the anti-tumor and proapoptotic effect of benzophenones in Ehrlich ascites tumor (EAT) cells. Treatment of benzophenones in vivo resulted in inhibition of proliferation of EAT cells and ascites formation. Further, we demonstrate that the induction of apoptosis in EAT cells is mediated through activation of caspase-3. These results suggest a further possible clinical application of these synthetic compounds as potent anti-tumor and proapoptotic compounds.     

Specificity of antibodies to sea anemone toxin III and immunogenicity of the pharmacological site of anemone and scorpion toxins

Toxin III (ATX III) of the sea anemone (Anemonia sulcata) is a polypeptide containing 27 amino acid residues. It has no sequence similarity with other toxins (ATX I and II) from the same species, or with scorpion toxins, although they apparently act in a similar manner by prolonging action potentials. The specificity of ATX III antibodies was characterized using ATX III, ATX I, native and chemically modified ATX II, and scorpion alpha-toxins. The results obtained suggest that a region of ATX III, partially or totally overlapping the pharmacological site shared with ATX I and ATX II, is immunogenic. It includes a guanidino and at least two carboxylate groups. The corresponding region is not immunogenic in ATX I and ATX II. Anti-(ATX III) antibodies recognize the similar regions of ATX I and ATX II and apparently do not recognize scorpion toxins.     

Comparative HPLC enantioseparation of ferrocenylalcohols on two cellulose-based chiral stationary phases

The direct HPLC enantiomeric separation of several ferrocenylalcohols on the commercially available Chiralcel OD and Chiralcel OJ columns has been evaluated in normal-phase mode. Almost all the compounds were resolved on one or both chiral stationary phases (CSPs) with separation factor (alpha) ranging from 1.06 to 2.88 while the resolution (R(s)) varied from 0.63 to 12.70 In the separation of the alpha-ferrocenylalcohols 1a-e and the phenyl analogues 2a-e, which were all resolved except 1c, a similar trend in the retention behavior for the two series of alcohols was evidenced and the selectivity was roughly complementary on the two investigated CSP. For three ferrocenylacohols, chosen as model compounds, the influence of the mobile phase composition and temperature on the enantioseparation were investigated and additional information on the chiral recognition mechanism were deduced from the chromatographic behavior of their acetylderivatives.     

An integrative approach to gain insights into the cellular function of human ataxin-2

Spinocerebellar ataxia type 2 (SCA2) is a hereditary neurodegenerative disorder caused by a trinucleotide expansion in the SCA2 gene, encoding a polyglutamine stretch in the gene product ataxin-2 (ATX2), whose cellular function is unknown. However, ATX2 interacts with A2BP1, a protein containing an RNA-recognition motif, and the existence of an interaction motif for the C-terminal domain of the poly(A)-binding protein (PABC) as well as an Lsm (Like Sm) domain in ATX2 suggest that ATX2 like its yeast homolog Pbp1 might be involved in RNA metabolism. Here, we show that, similar to Pbp1, ATX2 suppresses the petite (pet-) phenotype of Deltamrs2 yeast strains lacking mitochondrial group II introns. This finding points to a close functional relationship between the two homologs. To gain insight into potential functions of ATX2, we also generated a comprehensive protein interaction network for Pbp1 from publicly available databases, which implicates Pbp1 in diverse RNA-processing pathways. The functional relationship of ATX2 and Pbp1 is further corroborated by the experimental confirmation of the predicted interaction of ATX2 with the cytoplasmic poly(A)-binding protein 1 (PABP) using yeast-2-hybrid analysis as well as co-immunoprecipitation experiments. Immunofluorescence studies revealed that ATX2 and PABP co-localize in mammalian cells, remarkably, even under conditions in which PABP accumulates in distinct cytoplasmic foci representing sites of mRNA triage.     

In the search for new anticancer drugs XII. Synthesis and biological evaluation of spin labeled nitrosoureas

The spin labeled nitrosourea 1-(2-chloroethyl)-3-(1-oxyl-2,2,6,6- tetramethyl-piperidinyl)-1-nitrosourea (SLCNU, 4) and its analogues 5-7 were synthesized either by a regio-selective method or by a conventional route via the nitrosation of the spin labeled intermediates (11a-e). Nitrosation of the ureas 11a-e with dinitrogen tetraoxide resulted in better yields than those obtained with sodium nitrite. The nitrosoureas 4-8 were tested for their anticancer activity against the lymphocytic leukemia P388 in mice. Thus, either at the equal molar dose or at the dose of equal toxicity level, the SLCNU (4) was found to be more active than the clinically used CCNU (1). Unlike CCNU (1) whose LD50 is 56 mg/kg, the SLCNU (4) possesses a low toxicity (LD50 123 mg/kg). Therefore, SLCNU (4) is a promising new entry into the nitrosourea class of anticancer drugs.     

The role of the thymus in the maintenance of natural killer cells in vivo

This report describes a model for investigating the role of the thymus in regulating natural killer (NK) cell activity in vivo. Evidence is presented that the thymus can regulate NK cells, and that at least some NK cells can develop without thymic help. Marrow from thymectomized rats depleted of circulating T cells by thoracic duct cannulation was transplanted into rats without a thymus (1 degree ATX.BM). These 1 degree ATX.BM rats had NK cell levels above controls 3 months after reconstitution but markedly depressed NK cell levels by 9 months. When 1 degree ATX.BM marrow was used to reconstitute rats with or without a thymus, those without a thymus (2 degrees ATX.BM) exhibited low NK cell levels after 3 months, and a similar result was obtained when 2 degrees ATX.BM marrow was used to reconstitute 3 degrees ATX.BM rats. The low NK cell levels in 2 degrees and 3 degrees ATX.BM rats were due to a deficiency in spontaneously cytotoxic NK cells, as they had normal numbers of interferon-responsive pre-NK cells. Spleen cells from 2 degrees and 3 degrees ATX.BM rats produced less interferon than control spleen cells when cultured with P815 tumor cells in vitro. However, 2 degrees and 3 degrees ATX.BM rats had higher numbers of large granular lymphocytes than controls despite their low NK cell levels. In marked contrast to 2 degrees and 3 degrees ATX.BM rats, spleen cells from 4 degrees ATX.BM rats had higher levels of cytotoxicity and a higher frequency of both spontaneously cytotoxic and pre-NK cells than controls. The 4 degrees ATX.BM rats also had the highest frequency of large granular lymphocytes in the spleen.     

Synthesis of analogues of a potent antitumor saponin OSW-1

A series of side chain analogues (5a-e), a 22-glycosylated isomer (10), and 16beta-O-l-arabinosyl (13a) or 16beta-O-d-xylosyl (13b) analogues of OSW-1 were synthesized. All analogues were found to be less cytotoxic against breast and endometrial cancer cell lines than the natural product.     

Synthesis and evaluation of 6-hydroxy-7-methoxy-4-chromanone- and chroman-2-carboxamides as antioxidants

A series of 6-hydroxy-7-methoxy-4-chromanone- (2a-e) and chroman-2-carboxamides (3a-e) were synthesized and their antioxidant activities were evaluated. While compounds 2a-e were less active, compounds 3a-e exhibited more potent inhibition of lipid peroxidation initiated by Fe(2+) and ascorbic acid in rat brain homogenates. Among them, N-arylsubstituted-chroman-2-carboxamides (3d and 3e) exhibited 25-40 times more potent inhibition than trolox (1). The DPPH radical scavenging activity of compound 3d was comparable to that of trolox.     

Structure-activity relationships study at the 3'-N position of paclitaxel. Part 2: synthesis and biological evaluation of 3'-N-thiourea- and 3'-N-thiocarbamate-bearing paclitaxel analogues

The syntheses and preliminary biological evaluation of 3'-N-thiocarbamate- and 3'-N-thiourea-bearing paclitaxel analogues, 4a-f and 5a-e, are described. 3'-N-thiocarbamates 4a-e were found to be more potent than paclitaxel in both the tubulin polymerization assay and the in vitro cytotoxicity assay. Several derivatives of this class such as 4c, 4d, and 4e also exhibited some in vivo activity.     

The highly similar Arabidopsis homologs of trithorax ATX1 and ATX2 encode proteins with divergent biochemical functions

Gene duplication followed by functional specialization is a potent force in the evolution of biological diversity. A comparative study of two highly conserved duplicated genes, ARABIDOPSIS TRITHORAX-LIKE PROTEIN1 (ATX1) and ATX2, revealed features of both partial redundancy and of functional divergence. Although structurally similar, their regulatory sequences have diverged, resulting in distinct temporal and spatial patterns of expression of the ATX1 and ATX2 genes. We found that ATX2 methylates only a limited fraction of nucleosomes and that ATX1 and ATX2 influence the expression of largely nonoverlapping gene sets. Even when coregulating shared targets, ATX1 and ATX2 may employ different mechanisms. Most remarkable is the divergence of their biochemical activities: both proteins methylate K4 of histone H3, but while ATX1 trimethylates it, ATX2 dimethylates it. ATX2 and ATX1 provide an example of separated K4 di from K4 trimethyltransferase activity.     

Suppression of oxidative damage by Saccharomyces cerevisiae ATX2, which encodes a manganese-trafficking protein that localizes to Golgi-like vesicles.

Oxygen toxicity in Saccharomyces cerevisiae lacking the copper/zinc superoxide dismutase (SOD1) can be suppressed by overexpression of the S. cerevisiae ATX2 gene. Multiple copies of ATX2 were found to reverse the aerobic auxotrophies of sod1(delta) mutants for lysine and methionine and also to enhance the resistance of these yeast strains to paraquat and atmospheric levels of oxygen. ATX2 encodes a novel 34.4-kDa polypeptide with a number of potential membrane-spanning domains. Our studies indicate that Atx2p localizes to the membrane of a vesicular compartment in yeast cells reminiscent of the Golgi apparatus. With indirect immunofluorescence microscopy, Atx2p exhibited a punctate pattern of staining typical of the Golgi apparatus, and upon subcellular fractionation, Atx2p colocalized with a biochemical marker for the yeast Golgi apparatus. We demonstrate here that this vesicle protein normally functions in the homeostasis of manganese ions and that this role in metal metabolism is necessary for the ATX1 suppression of SOD1 deficiency. First, overexpression of ATX2 caused cells to accumulate increased levels of manganese. Second, a deletion in ATX2 caused a decrease in the apparent available level of intracellular manganese and caused sod1(delta) mutants to become dependent upon exogenous manganese for aerobic growth. Third, ATX2 was incapable of suppressing oxidative damage in cells depleted of manganese ions or lacking the plasma membrane transporter for manganese. The effect of ATX2 overexpression on manganese accumulation and oxygen resistance is similar to what we have previously reported for mutations in PMR1, which encodes a manganese-trafficking protein that also resides in a vesicular compartment. Our studies are consistent with a model in which Atx2p and Pmr1p work in opposite directions to control manganese homeostasis.     

Identification and biochemical characterization of a novel autotaxin isoform, ATXδ, with a four-amino acid deletion

Autotaxin (ATX) is lysophospholipase D, which converts lysophospholipids such as lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a bioactive lipid mediator with multiple biological roles. ATX is present in high concentrations in various biological fluids and is responsible for LPA production in these fluids. The plasma ATX level is altered in some patho-physiological conditions. Three splicing isoforms of ATX have been reported so far (ATXα, β and γ). In this study, we identified and characterized ATXδ, a novel alternative splice variant of ATX, which has a four-amino acid deletion in the L2 linker region of ATXβ. ATXδ was found to be the second major isoform following ATXβ and fully active. ATXβ and ATXδ showed similar divalent cation sensitivity and cell motility-stimulating activity. ATXβ and ATXδ are present in wide range of organism from fish to mammals. Among them, only ATXδ was found in Gallus gallus and Xenopus laevis, suggesting the indispensable role of the isoform. ATXδ was expressed in various human tissues with different expression patterns from that of ATXβ. These results show that ATXδ is a second major ATX isoform sharing similar biochemical characters with the major isoform, ATXβ, and is a potential biomarker.     

Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line.

Glycoglycerolipids constitute a family of glycolipids with apparently very restricted expression in human tissues. They have previously been detected only in the testis and the nervous system. In the present study, two glycoglycerolipids were isolated from the HT29 human colon carcinoma cell line. The glycoglycerolipids were structurally characterized as a monogalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl)-sn-glycerol) and a digalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl(1-4)alpha-galactosyl)-sn-glycerol) using NMR and mass spectrometry. This digalactosylglycerolipid has not previously been structurally characterized. When HT29 cells were allowed to differentiate into more enterocyte-like cells by culture in glucose-free medium, expression of both of these glycoglycerolipids was greatly diminished. The presence of glycoglycerolipids in a human colon carcinoma cell line indicates that expression of this family of glycolipids may not be as restricted as previously thought. Instead this class of glycolipids may serve as differentiation antigens in various normal tissues and in tumor development. The Galalpha1-4Gal epitope was previously identified as a receptor for bacterial adhesins and toxins. The finding that this epitope is also linked to a glycerolipid moiety opens up new possible roles for this carbohydrate receptor in intracellular signaling.     

Autotaxin regulates vascular development via multiple lysophosphatidic acid (LPA) receptors in zebrafish

Autotaxin (ATX) is a multifunctional ecto-type phosphodiesterase that converts lysophospholipids, such as lysophosphatidylcholine, to lysophosphatidic acid (LPA) by its lysophospholipase D activity. LPA is a lipid mediator with diverse biological functions, most of which are mediated by G protein-coupled receptors specific to LPA (LPA1-6). Recent studies on ATX knock-out mice revealed that ATX has an essential role in embryonic blood vessel formation. However, the underlying molecular mechanisms remain to be solved. A data base search revealed that ATX and LPA receptors are conserved in wide range of vertebrates from fishes to mammals. Here we analyzed zebrafish ATX (zATX) and LPA receptors both biochemically and functionally. zATX, like mammalian ATX, showed lysophospholipase D activity to produce LPA. In addition, all zebrafish LPA receptors except for LPA5a and LPA5b were found to respond to LPA. Knockdown of zATX in zebrafish embryos by injecting morpholino antisense oligonucleotides (MOs) specific to zATX caused abnormal blood vessel formation, which has not been observed in other morphant embryos or mutants with vascular defects reported previously. In ATX morphant embryos, the segmental arteries sprouted normally from the dorsal aorta but stalled in midcourse, resulting in aberrant vascular connection around the horizontal myoseptum. Similar vascular defects were not observed in embryos in which each single LPA receptor was attenuated by using MOs. Interestingly, similar vascular defects were observed when both LPA1 and LPA4 functions were attenuated by using MOs and/or a selective LPA receptor antagonist, Ki16425. These results demonstrate that the ATX-LPA-LPAR axis is a critical regulator of embryonic vascular development that is conserved in vertebrates.     

Synthesis and anti-histaminic evaluation of N-phenyl-(alkyl)-5-(dialkylamino)methyl-2-amino-2-oxazolines

New N-phenyl(alkyl)-5-(dialkylamino)methyl-2-amino-2-oxazolines, 5a-e, have been synthesized from the corresponding 3-phenyl(alkyl)carbamoyl-2-iminooxazolidines 2. A two-stage hydrolysis reaction led finally to the corresponding ring-opened N-phenyl(alkyl)-N'-[1-(3-(dialkylamino)-propan-2-ol)]ureas 4. The oxazoline ring was regenerated through an intramolecular nucleophilic substitution involving an halogen atom introduced by the reaction of thionyl chloride on 4. Pharmacological properties of 5a-e were evaluated on histaminic and adrenergic receptors in guinea-pig trachea and rat aorta. Compounds 5b and 5e showed a selective anti-histaminic effect on guinea-pig airways, but a significant response was obtained for a concentration >10(-6) M. No pharmacological activity was obtained with oxazoline 5c whereas oxazolines 5a and 5d seemed to present a non-selective effect on the contractile mechanism of the smooth muscle cell.     

Type I Interferons Function as Autocrine and Paracrine Factors to Induce Autotaxin in Response to TLR Activation

Lysophosphatidic acid (LPA) is an important phospholipid mediator in inflammation and immunity. However, the mechanism of LPA regulation during inflammatory response is largely unknown. Autotaxin (ATX) is the key enzyme to produce extracellular LPA from lysophosphatidylcholine (LPC). In this study, we found that ATX was induced in monocytic THP-1 cells by TLR4 ligand lipopolysaccharide (LPS), TLR9 ligand CpG oligonucleotide, and TLR3 ligand poly(I:C), respectively. The ATX induction by TLR ligand was abolished by the neutralizing antibody against IFN-β or the knockdown of IFNAR1, indicating that type I IFN autocrine loop is responsible for the ATX induction upon TLR activation. Both IFN-β and IFN-α were able to induce ATX expression via the JAK-STAT and PI3K-AKT pathways but with different time-dependent manners. The ATX induction by IFN-β was dramatically enhanced by IFN-γ, which had no significant effect on ATX expression alone, suggesting a synergy effect between type I and type II IFNs in ATX induction. Extracellular LPA levels were significantly increased when THP-1 cells were treated with IFN-α/β or TLR ligands. In addition, the type I IFN-mediated ATX induction was identified in human monocyte-derived dendritic cells (moDCs) stimulated with LPS or poly(I:C), and IFN-α/β could induce ATX expression in human peripheral blood mononuclear cells (PBMCs) and monocytes isolated form blood samples. These results suggest that, in response to TLR activation, ATX is induced through a type I INF autocrine-paracrine loop to enhance LPA generation.     

Synthesis and biological evaluation of 2- and 7-substituted 9-deazaadenosine analogues

A series of 2-halogen and 7-alkyl substituted analogues of 9-deazaadenosine and 2'-deoxy-9-deazaadenosine was synthesized by new efficient methodology involving transformation of corresponding 9-deazaguanosine and 2'-deoxyguanosine, which in turn were synthesized by direct C-glycosylation of 1-benzyl-9-deazaguanine with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose and methyl 2-deoxy-3,5-di-O-(p-toluoyl)-D-ribofuranoside, respectively. Deoxychlorination of C6 and diazotization/chloroor fluoro-dediazoniation of the sugar-protected 9-deazaguanosine, followed by selective ammonolysis at C6 and deprotection of the sugar moiety, gave 2-chloro- and 2-fluoro-9-deazaadenosine (6 and 9). Substitution of the 7-position of the dihalogen-intermediate with alkyl groups, followed by ammonolysis and deprotection, provided 2-chloro-7-alkyl-9-deazaadenosines (13a-e) and 2-fluoro-7-benzyl-9-deazaadenosine (13f). Catalytic hydrogenation of 13a-e gave 7-alkyl-9-deazaadenosines 14a-e. Similarly, 2-chloro-2'-deoxy-9-deazaadenosine (21), 2-chloro-2'-deoxy-7-methyl-9-deazaadenosine (25), 2'-deoxy-9-deazaadenosine (22), and 2'-deoxy-7-methyl-9-deazaadenosine (26) were prepared from sugar-protected 2'-deoxy-9-deazaguanosine. Among these compounds, 7-benzyl-9-deazaadenosine (14b) showed the most potent cytotoxic activity, with IC50 values of 0.07, 0.1, 0.2 and 1.5 microM, while both 7-methyl-9-deazaadenosine (14a) and 2-fluoro-9-deazaadenosine (9) also demonstrated significant cytotoxic activity with IC50 values of 0.4, 0.7, 0.3, and 1.5 microM, and 1.5, 0.9, 0.3, and 5 microM against L 1210 leukemia, P388 leukemia, CCRF-CEM lymphoblastic leukemia, and B16F10 melanoma cells, respectively.