Substrate-induced conformational changes and dynamics of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase-2.

O-Glycan biosynthesis is initiated by the transfer of N-acetylgalactosamine (GalNAc) from a nucleotide sugar donor (UDP-GalNAc) to Ser/Thr residues of an acceptor substrate. The detailed transfer mechanism, catalyzed by the UDP-GalNAc polypeptide:N-acetyl-alpha-galactosaminyltransferases (ppGalNAcTs), remains unclear despite structural information available for several isoforms in complex with substrates at various stages along the catalytic pathway. We used all-atom molecular dynamics simulations with explicit solvent and counterions to study the conformational dynamics of ppGalNAcT-2 in several enzymatic states along the catalytic pathway. ppGalNAcT-2 is simulated both in the presence and in the absence of substrates and reaction products to examine the role of conformational changes in ligand binding. In multiple 40-ns-long simulations of more than 600 ns total run time, we studied systems ranging from 45,000 to 95,000 atoms. Our simulations accurately identified dynamically active regions of the protein, as previously revealed by the X-ray structures, and permitted a detailed, atomistic description of the conformational changes of loops near the active site and the characterization of the ensemble of structures adopted by the transferase complex on the transition pathway between the ligand-bound and ligand-free states. In particular, the conformational transition of a functional loop adjacent to the active site from closed (active) to open (inactive) is correlated with the rotameric state of the conserved residue W331. Analysis of water dynamics in the active site revealed that internal water molecules have an important role in enhancing the enzyme flexibility. We also found evidence that charged side chains in the active site rearrange during site opening to facilitate ligand binding. Our results are consistent with the single-displacement transfer mechanism previously proposed for ppGalNAcTs based on X-ray structures and mutagenesis data and provide new evidence for possible functional roles of certain amino acids conserved across several isoforms.

Multi-targeted antifolates aimed at avoiding drug resistance form covalent closed inhibitory complexes with human and Escherichia coli thymidylate synthases.

Crystal structures of four pyrrolo(2,3-d)pyrimidine-based antifolate compounds, developed as inhibitors of thymidylate synthase (TS) in a strategy to circumvent drug-resistance, have been determined in complexes with their in vivo target, human thymidylate synthase, and with the structurally best-characterized Escherichia coli enzyme, to resolutions of 2.2-3.0 A. The 2.9 A crystal structure of a complex of human TS with one of the inhibitors, the multi-targeted antifolate LY231514, demonstrates that this compound induces a "closed" enzyme conformation and leads to formation of a covalent bond between enzyme and substrate. This structure is one of the first liganded human TS structures, and its solution was aided by mutation to facilitate crystallization. Structures of three other pyrrolo(2,3-d)pyrimidine-based antifolates in complex with Escherichia coli TS confirm the orientation of this class of inhibitors in the active site. Specific interactions between the polyglutamyl moiety and a positively charged groove on the enzyme surface explain the marked increase in affinity of the pyrrolo(2,3-d)pyrimidine inhibitors once they are polyglutamylated, as mediated in vivo by the cellular enzyme folyl polyglutamate synthetase.

Predicting and harnessing protein flexibility in the design of species-specific inhibitors of thymidylate synthase.

BACKGROUND: Protein plasticity in response to ligand binding abrogates the notion of a rigid receptor site. Thus, computational docking alone misses important prospective drug design leads. Bacterial-specific inhibitors of an essential enzyme, thymidylate synthase (TS), were developed using a combination of computer-based screening followed by in-parallel synthetic elaboration and enzyme assay [Tondi et al. (1999) Chem. Biol. 6, 319-331]. Specificity was achieved through protein plasticity and despite the very high sequence conservation of the enzyme between species. RESULTS: The most potent of the inhibitors synthesized, N,O-didansyl-L-tyrosine (DDT), binds to Lactobacillus casei TS (LcTS) with 35-fold higher affinity and to Escherichia coli TS (EcTS) with 24-fold higher affinity than to human TS (hTS). To reveal the molecular basis for this specificity, we have determined the crystal structure of EcTS complexed with DDT and 2'-deoxyuridine-5'-monophosphate (dUMP). The 2.0 A structure shows that DDT binds to EcTS in a conformation not predicted by molecular docking studies and substantially differently than other TS inhibitors. Binding of DDT is accompanied by large rearrangements of the protein both near and distal to the enzyme's active site with movement of C alpha carbons up to 6 A relative to other ternary complexes. This protein plasticity results in novel interactions with DDT including the formation of hydrogen bonds and van der Waals interactions to residues conserved in bacterial TS but not hTS and which are hypothesized to account for DDT's specificity. The conformation DDT adopts when bound to EcTS explains the activity of several other LcTS inhibitors synthesized in-parallel with DDT suggesting that DDT binds to the two enzymes in similar orientations. CONCLUSIONS: Dramatic protein rearrangements involving both main and side chain atoms play an important role in the recognition of DDT by EcTS and highlight the importance of incorporating protein plasticity in drug design. The crystal structure of the EcTS/dUMP/DDT complex is a model system to develop more selective TS inhibitors aimed at pathogenic bacterial species. The crystal structure also suggests a general formula for identifying regions of TS and other enzymes that may be treated as flexible to aid in computational methods of drug discovery.

Phase I clinical trials of MRX-115. A new ultrasound contrast agent.

RATIONALE AND OBJECTIVES: Stabilized microbubbles are under development as contrast agents for medical ultrasound. The authors report the results of Phase I clinical trials of a new ultrasound contrast agent based on lipidencapsulated perfluorocarbon gas microbubbles. METHODS: Lipids encapsulating perfluoropropane gas (Aerosomes MRX-115, ImaRx Pharmaceutical Corp., Tucson, AZ) were evaluated in Phase I clinical trials. Two separate studies were performed. The first was a single escalating-dose study (n = 30 subjects), and the second was a multiple-dose study (n = 18 subjects) with rechallenge in several subjects (n = 4) after 21 days. Echocardiographic examinations were performed before and after contrast agent for each test drug administration for both studies, with the exception of the rechallenge group. Doses tested in the single-dose study ranged from 0.005 mL/kg to 0.100 mL/kg body weight. In the multiple-dose study, five doses of 0.005 mL/ kg to 0.030 mL/kg (0.025-0.150 mL/kg total dose) were evaluated. Studies were single-masked, placebo-controlled, and safety assessment and adverse events were monitored. RESULTS: All doses in both studies were well tolerated with no treatment-related changes in safety measures for either study. Left ventricular cavity and myocardial enhancement were seen with all doses of MRX-115. CONCLUSIONS: MRX-115 is a promising new intravascular ultrasound contrast agent that was safe and well tolerated at the doses evaluated in these studies.

Partial purification and substrate specificity of heparan sulfate alpha-N-acetylglucosaminyltransferase I: synthesis, NMR spectroscopic characterization and in vitro assays of two aryl tetrasaccharides.

Studies of heparan sulfate biosynthesis on beta-D-xylosides have led to the hypothesis that heparan sulfate alpha-N-acetylglucosaminyltransferase I (alpha-GlcNAc-TI) recognizes structures at the reducing end of the proteoglycan linkage tetrasaccharide. We report here the in vivo and in vitro testing of this hypothesis using four synthetic substrates, benzyl- and 2-naphthalenemethanyl-beta-D-xylosides, and two proteoglycan linkage tetrasaccharides containing benzyl alcohol or naphthalmethanol aglycones, viz., GlcAbeta(1 --> 3)Gal beta(1 --> 3)Gal beta(1 --> 4)Xyl beta-O-Bn (BNT) and GlcAbeta(1 --> 3)Gal beta(1 --> 3)Gal beta(1 --> 4)Xyl beta-O-NM (NMT). The aryl tetrasaccharides were chemically synthesized and the 1H and 13C resonances were assigned by two-dimensional NMR spectroscopy. The inter-residue spatial constraints, determined by the 2D NOESY data, revealed essentially identical conformations for the interglycosidic linkages and Xyl-O-CH2Ar linkages in both compounds. Interestingly, the aromatic rings in both tetrasaccharides undergo rapid internal rotation across the CH2-Ar bond. These tetrasaccharides were used to assay heparan sulfate alpha-GlcNAc-TI from homogenates of wild-type CHO cells. alpha-GlcNAc-TI was also purified approximately 900-fold from rat liver and assayed with BNT and NMT. At nearly all concentrations tested, alpha-GlcNAc-TI activity from both CHO cell homogenates and rat liver was greater with the NMT. When fed to CHO cells, benzyl-beta-D-xyloside primed heparan sulfate poorly relative to 2-naphthalenemethanyl-beta-D-xyloside. Thus, the in vitro enzyme activity is consistent with the in vivo priming data that suggests that alpha-GlcNAc-TI can directly recognize structure at the reducing end of the linkage tetrasaccharide. These studies provide an in vivo basis for the possible role of core protein sequences in the biosynthesis of specific glycosaminoglycans.

Electron beams and ion composition measured at Io and in its torus.

Intense, magnetic field-aligned, bidirectional, energetic (>15 kiloelectron volts) electron beams were discovered by the Galileo energetic particles detector during the flyby of Io. These beams can carry sufficient energy flux into Jupiter's atmosphere to produce a visible aurora at the footprint of the magnetic flux tube connecting Io to Jupiter. Composition measurements through the torus showed that the spatial distributions of protons, oxygen, and sulfur are different, with sulfur being the dominant energetic (> approximately 10 kiloelectron volts per nucleon) ion at closest approach.

Disaccharide uptake and priming in animal cells: inhibition of sialyl Lewis X by acetylated Gal beta 1-->4GlcNAc beta-O-naphthalenemethanol.

Inhibitors of glycosylation provide a tool for studying the biology of glycoconjugates. One class of inhibitors consists of glycosides that block glycoconjugate synthesis by acting as primers of free oligosaccharide chains. A typical primer contains one sugar linked to a hydrophobic aglycone. In this report, we describe a way to use disaccharides as primers. Chinese hamster ovary cells readily take up glycosides containing a pentose linked to naphthol, but they take up hexosides less efficiently and disaccharides not at all. Linking phenanthrol to a hexose improves its uptake dramatically but has no effect on disaccharides. To circumvent this problem, analogs of Xyl beta 1-->6Gal beta-O-2-naphthol were tested as primers of glycosaminoglycan chains. The unmodified disaccharide did not prime, but methylated derivatives had activity in the order Xyl beta 1-->6Gal(Me)3-beta-O-2-naphthol > Xyl beta 1-->6Gal (Me)2 beta-O-2-naphthol >> Xyl beta 1-->6Gal(Me)beta-O-2-naphthol. Acetylated Xyl beta 1-->6Gal beta-O-2-naphthol also primed glycosaminoglycans efficiently, suggesting that the terminal xylose residue was exposed by removing the acetyl groups. The general utility of using acetyl groups to create disaccharide primers was shown by the priming of oligosaccharides on peracetylated Gal beta 1-->4GlcNAc beta-O-naphthalenemethanol. This disaccharide inhibited sialyl Lewis X expression on HL-60 cells.

Heparan sulfate primed on beta-D-xylosides restores binding of basic fibroblast growth factor.

Heparan sulfate proteoglycans (HSPG) are obligatory for receptor binding and mitogenic activity of basic fibroblast growth factor (bFGF). Mutant Chinese hamster ovary cells (pgsA-745) deficient in xylosyltransferase are unable to initiate glycosaminoglycan synthesis and hence can not bind bFGF to low- and high-affinity cell surface receptors. Exposure of pgsA-745 cells to beta-D-xylopyranosides containing hydrophobic aglycones resulted in restoration of bFGF binding in a manner similar to that induced by soluble heparin or by heparan sulfate (HS) normally associated with cell surfaces. Restoration of bF-GF binding correlated with the ability of the beta-D-xylosides to prime the synthesis of heparan sulfate. Thus, both heparan sulfate synthesis and bFGF receptor binding were induced by low concentrations (10-30 microM) of estradiol-beta-D-xyloside and naphthyl-beta-D-xyloside, but not by cis/trans-decahydro-2-naphthyl-beta-D-xyloside, which at low concentration primes mainly chondroitin sulfate. The obligatory involvement of xyloside-primed heparan sulfate in restoration of bFGF-receptor binding was also demonstrated by its sensitivity to heparinase treatment and by the lack of restoration activity in CHO cell mutants that lack enzymatic activities required to form the repeating disaccharide unit characteristic of heparan sulfate. Xyloside-primed heparan sulfate binds to the cell surface. Restoration of bFGF receptor binding was induced by both soluble and cell bound xyloside-primed heparan sulfate and was abolished in cells that were exposed to 0.5-1.0 M NaCl prior to the bFGF binding reaction. These results indicate that heparan sulfate chains produced on xyloside primers behave like heparan sulfate chains attached to cellular core proteins in terms of affinity for bFGF and ability to function as low-affinity sites in a dual receptor mechanism characteristic of bFGF and other heparin-binding growth promoting factors.

Two N-acetylglucosaminyltransferases catalyze the biosynthesis of heparan sulfate.

We report that two N-acetylglucosaminyltransferases catalyze the biosynthesis of heparan sulfate in Chinese hamster ovary cells. The first enzyme initiates heparan sulfate biosynthesis and can be measured by the transfer of GlcNAc from UDP-GlcNAc to GlcUA beta 1-3Gal beta 1-O-naphthalenemethanol. The second enzyme catalyzes the polymerization of heparan sulfate and can be measured by the transfer of GlcNAc from UDP-GlcNAc to the nonreducing terminal GlcUA present in oligosaccharide fragments prepared from the Escherichia coli K5 capsular polysaccharide, N-acetylheparosan. Kinetic characterization of the initiating GlcNAc-transferase (alpha-GlcNAc-TI) indicates an apparent Km for UDP-GlcNAc of 36 +/- 4 microM. The apparent Km for UDP-GlcNAc of the polymerizing GlcNAc-transferase (alpha-GlcNAc-TII) is 230 +/- 30 microM. Both enzymes have broad pH optima and require a divalent cation for activity. alpha-GlcNAc-TI can use both Mn2+ and Ca2+, while alpha-GlcNAc-TII will use only Mn2+. Chinese hamster ovary cells deficient in the synthesis of heparan sulfate and lacking alpha-GlcNAc-TII activity and S49 Thy 1-a lymphoma cells deficient in alpha GlcNAc addition to phosphatidylinositol have wild-type alpha-GlcNAc-TI activity. Thus, distinct alpha-GlcNAc-transferases catalyze the initiation and polymerization of heparan sulfate.

The glycosylation of phosphoglucomutase is modulated by carbon source and heat shock in Saccharomyces cerevisiae.

Phosphoglucomutase is the acceptor for UDP-glucose: glycoprotein glucose-1-phosphotransferase and contains Glc in a phosphodiester linkage to O-linked Man. In this study, we have characterized the glycosylation of phosphoglucomutase by Saccharomyces cerevisiae in response to heat shock and growth in media containing carbon sources other than Glc. Phosphoglucomutase synthesized under these conditions is underglucosylated relative to that synthesized during logarithmic growth in Glc. The underglucosylation results in increased UDP-glucose:glycoprotein glucose-1-phosphotransferase acceptor activity in in vitro assays and a newly appearing less negatively charged form of phosphoglucomutase resolvable by anion exchange chromatography. Utilizing a yeast strain in which phosphoglucomutase is overexpressed via a multicopy plasmid, metabolic labeling of the enzyme with [35S]Met and [3H]Man increased in response to heat shock, whereas [3H]Glc labeling decreased. The glucosylation state of phosphoglucomutase was also compared in cells grown in media containing various carbon sources and was found to be lowest in cells utilizing Gal as the sole carbon source compared with Glc or lactate. In mammalian cells, the glucosylation of phosphoglucomutase has been shown to be sensitive to changes in cytoplasmic Ca2+ and to correlate with a change in its membrane association. The change in phosphoglucomutase's oligosaccharide in Saccharomyces cerevisiae may be important to alterations in its distribution under conditions of nutrient deprivation or metabolic stress.

Biosynthesis of heparan sulfate on beta-D-xylosides depends on aglycone structure.

We have reported that 3-estradiol-beta-D-xyloside primes heparan sulfate synthesis in Chinese hamster ovary cells and that the proportion of heparan sulfate made rises with increasing concentration of xyloside (Lugemwa, F.N. and Esko, J.D. (1991) J. Biol. Chem. 266, 6674-6677). Using estradiol as a guide, we varied the structure of the aglycone and showed that beta-D-xylosides containing two fused aromatic rings efficiently prime heparan sulfate. Thus, 2-naphthol-beta-D-xyloside primed heparan sulfate at low dose (< or = 10 microM) and the proportion of heparan sulfate increased with concentration (up to 50% of total glycosaminoglycan). Various ring additions and heterocyclic ring substitutions altered the efficiency of heparan sulfate priming, but had no effect on the overall level of glycosaminoglycan synthesis. Replacement of the bridging oxygen with sulfur (2-naphthalenethiol-beta-D-xyloside) increased the efficiency of heparan sulfate priming. Priming of heparan sulfate correlated with hydrophobicity of the xyloside, but several exceptions suggested that the chemical structure of the aglycone played an equally important role. Interestingly, the heparan sulfate chains generated on 2-naphthol-beta-D-xyloside showed a 2-fold decrease in the proportion of disaccharides containing 6-O-sulfate groups and a striking diminution in non-sulfated iduronic acid containing disaccharides compared to the chains attached to cellular proteoglycans. Thus, both the type of glycosaminoglycan made on a xyloside and its fine structure depends on the aglycone.

Phosphoglucomutase in Saccharomyces cerevisiae is a cytoplasmic glycoprotein and the acceptor for a Glc-phosphotransferase.

UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor in vertebrates and Paramecium tetraurelia is a cytoplasmic 62-kDa glycoprotein. To determine if the yeast Saccharomyces cerevisiae also possesses Glc-phosphotransferase activity, a crude cellular lysate was incubated with [beta-32P]UDP-Glc and analyzed. A phosphoglycoprotein having an apparent molecular mass of 62 kDa (pgp62) was found to be the predominant labeled macromolecule. Reconstitution experiments determined that both a soluble and membrane fraction were required for labeling, and suggested that the Glc-phosphotransferase is membrane-associated while pgp62 is cytoplasmic. The reaction is evolutionarily conserved to the extent that rat liver Glc-phosphotransferase was capable of recognizing the yeast acceptor and vice versa. The yeast 62-kDa acceptor was purified, and partial amino acid sequences showed a high level of identity with rabbit muscle phosphoglucomutase. Subsequently, both yeast and rabbit muscle phosphoglucomutase were found to be acceptors in the Glc-phosphotransferase reaction. The label was found on a tryptic peptide distinct from that containing the enzyme's active site serine. When phosphoglucomutase was overexpressed, an increase was seen in Glc-phosphotransferase acceptor activity and in specific metabolic labeling of the acceptor by glucose and mannose.

Preliminary evaluation of iron phytate (inositol hexaphosphate) as a gastrointestinal MR contrast agent.

A simple, effective, safe, and well-tolerated contrast agent is needed as a bowel marker for magnetic resonance (MR) imaging. The authors tested a variety of foodstuffs admixed with ferric iron as potential gastrointestinal MR contrast agents. Phytate (inositol hexaphosphate) more than doubled the relaxivity of solutions of ferric iron. Because of the improved relaxivity of iron phytate, the concentration of iron could be reduced substantially relative to free ferric iron (eg, ferric chloride or ferric ammonium citrate). Imaging studies were performed in five volunteers to determine the optimal dose of iron phytate and in five additional volunteers to test its effectiveness. A 200 mg/L concentration of ferric iron with phytate functions as an effective gastrointestinal MR contrast agent for T1-weighted abdominal MR imaging, significantly improving bowel contrast (P < .01). Blood studies after contrast agent administration showed no appreciable increase in serum iron. Compared with standard chelate complexes that decrease the relaxivity of a given paramagnetic ion, phytate not only decreases the absorption of the iron but increases its relaxivity.

Status of liposomes as MR contrast agents.

Recent work on the development of liposomal magnetic resonance (MR) contrast agents has yielded structures with higher overall relaxivity than that of other nanoparticles of similar diameter. Liposomes incorporating membrane-bound complexes of manganase ("memsomes") produce greater hepatic enhancement per micromole of metal ion than either ferrite particles or paramagnetic chelates. Memsomes also hold promise for targeting of sites outside the liver. Work is in progress to take these agents into clinical trials.

Cellulose as a gastrointestinal US contrast agent.

Ultrasound (US) imaging of the abdomen often is compromised by artifacts due to adjacent bowel gas. In an attempt to decrease gas artifacts and improve US image quality, the authors evaluated the use of cellulose preparations as gastrointestinal US contrast agents. Optimal homogeneity and reflectivity were evaluated in phantom solutions, and two suitable agents were selected for clinical trial. Ten volunteers underwent abdominal US imaging before and after contrast agent administration on three separate occasions. The volunteers drank 800 mL of freshly degassed water and two different gastrointestinal US contrast agents. US images obtained before and after administration of contrast material were evaluated by five radiologists and scored for bowel marking, visualization of abdominal anatomy, and image degradation by bowel gas. Compared with water, the orally administered US contrast agents improved visualization of bowel and abdominal anatomy, with diminished gas artifact.

Nitrogen-filled liposomes as a vascular US contrast agent: preliminary evaluation.

Liposomes with a mean diameter of 1-2 microns were made to entrap nitrogen gas and tested as an ultrasound (US) contrast agent. The gas-filled liposomes, or Aerosomes (ImaRx Pharmaceutical, Tucson) were tested in vitro for size, stability, reflectivity, and acoustic characterization, and were tested in vivo for acute toxicity in mice and for cardiac imaging in rabbits after intravenous injection. Aerosomes have much greater reflectivity and higher attenuation than do standard liposomes and retain their acoustic properties after storage in aqueous media for several months. The interpolated median lethal dose of Aerosomes is approximately 2.5 mmol of lipid per kilogram, and the imaging dose is under 5 mumol of lipid per kilogram, yielding a potential therapeutic index of over 500 to 1. Postcontrast US images showed sustained enhancement of all four cardiac chambers as well as enhancement in the aorta, vena cava, and hepatic veins. Aerosomes hold promise as a contrast agent for cardiac and blood-pool imaging. Further work is in progress to characterize and develop this novel US contrast agent.

Clearance of liposomal gadolinium: in vivo decomplexation.

The contrast agents gadolinium-DTPA (diethylenetriaminepentaacetic acid), Gd-DOTA (tetraazacyclododecanetetraacetic acid), and Gd-HP-DO3A (1,4,7-tris[carboxymethyl]-10-[2' hydroxypropyl]-1,4,7,10-tetraazacyclododecane) are used in humans as extracellular contrast agents. Although free Gd+ ion is toxic, the intact Gd3+ complexes are rapidly excreted and are relatively nontoxic. Decomplexation with release of free gadolinium is a relevant clinical concern in patients with altered renal clearance. Blood pool contrast agents currently under development may have longer clearance half-lives and be more prone to decomplexation. The present study was designed to evaluate the clearance of liposomally encapsulated Gd3+ complexes (DTPA, DOTA, and HP-DO3A). The macrocyclic compounds had more rapid and complete clearance than DTPA (P less than .05). Parallel studies with carbon-14 and Gd-153-labeled complexes showed significant differences (P less than .05) in the amount of these isotopes retained in the heart, kidney, lungs, and spleen, providing strong supportive evidence for in vivo decomplexation.

Energetic particles at venus: galileo results.

At Venus the Energetic Particles Detector (EPD) on the Galileo spacecraft measured the differential energy spectra and angular distributions of ions >22 kiloelectron volts (keV) and electrons > 15 keV in energy. The only time particles were observed by EPD was in a series of episodic events [0546 to 0638 universal time (UT)] near closest approach (0559:03 UT). Angular distributions were highly anisotropic, ordered by the magnetic field, and showed ions arriving from the hemisphere containing Venus and its bow shock. The spectra showed a power law form with intensities observed into the 120- to 280-keV range. Comparisons with model bow shock calculations show that these energetic ions are associated with the venusian foreshock-bow shock region. Shock-drift acceleration in the venusian bow shock seems the most likely process responsible for the observed ions.