The tunicamycin derivative TunR2 exhibits potent antibiotic properties with low toxicity in an in vivo Mycobacterium marinum-zebrafish TB infection model.

Tunicamycins (TUN) are well-defined, Streptomyces-derived natural products that inhibit protein N-glycosylation in eukaryotes, and by a conserved mechanism also block bacterial cell wall biosynthesis. TUN inhibits the polyprenylphosphate-N-acetyl-hexosamine-1-phospho-transferases (PNPT), an essential family of enzymes found in both bacteria and eukaryotes. We have previously published the development of chemically modified TUN, called TunR1 and TunR2, that have considerably reduced activity on eukaryotes but that retain the potent antibacterial properties. A mechanism for this reduced toxicity has also been reported. TunR1 and TunR2 have been tested against mammalian cell lines in culture and against live insect cells but, until now, no in vivo evaluation has been undertaken for vertebrates. In the current work, TUN, TunR1, and TunR2 are investigated for their relative toxicity and antimycobacterial activity in zebrafish using a well-established Mycobacterium marinum (M. marinum) infection system, a model for studying human Mycobacterium tuberculosis infections. We also report the relative ability to activate the unfolded protein response (UPR), the known mechanism for the eukaryotic toxicity observed with TUN treatment. Importantly, TunR1 and TunR2 retained their antimicrobial properties, as evidenced by a reduction in M. marinum bacterial burden, compared to DMSO-treated zebrafish. In summary, findings from this study highlight the characteristics of recently developed TUN derivatives, mainly TunR2, and its potential for use as a novel anti-bacterial agent for veterinary and potential medical purposes.

Synergistic enhancement of beta-lactam antibiotics by modified tunicamycin analogs TunR1 and TunR2.

The ß-lactams are the most widely used group of antibiotics in human health and agriculture, but this is under threat due to the persistent rise of pathogenic resistance. Several compounds, including tunicamycin (TUN), can enhance the antibacterial activity of the ß-lactams to the extent of overcoming resistance, but the mammalian toxicity of TUN has precluded its use in this role. Selective hydrogenation of TUN produces modified compounds (TunR1 and TunR2), which retain the enhancement of ß-lactams while having much lower mammalian toxicity. Here we show that TunR1 and TunR2 enhance the antibacterial activity of multiple ß-lactam family members, including penems, cephems, and third-generation penicillins, to a similar extent as does the native TUN. Eleven of the ß-lactams tested were enhanced from 2 to >256-fold against Bacillus subtilis, with comparable results against a penicillin G-resistant strain. The most significant enhancements were obtained with third-generation aminothiazolidyl cephems, including cefotaxime, ceftazidime, and cefquinome. These results support the potential of low toxicity tunicamycin analogs (TunR1 and TunR2) as clinically valid, synergistic enhancers for a broad group of ß-lactam antibiotics.

Bisubstrate analogues as glycosyltransferase inhibitors.

Oligosaccharides in glycoconjugates such as glycoproteins and glycolipids play important roles in a variety of biological functions. Since glycosyltransferases are responsible for the biosynthesis of these oligosaccharides, inhibitors of glycosyltransferases are targets for drug discovery. Bisubstrate analogues, in which donor and acceptor analogue are covalently attached to each other, offer donor's high affinity and acceptor's high selectivity. In this review, we describe the design and synthesis of bisubstrate analogues of glycosyltransferases as well as their inhibitory potency hoping to inform the development of potent and selective inhibitors.

Synthesis of complex nucleoside antibiotics.

Herbicidin B and fully prtected tunicaminyluracil, which were undecose nucleoside antibiotics, were synthesized using a samarium diiodide (SmI2) mediated aldol reaction with the use of alpha-phenylthioketone as an enolate. The characteristics of the SmI2-mediated aldol reaction are that the enolate can be regioselectively generated and the aldol reaction proceeds under near neutral condition. This reaction is proved to be a powerful reaction for the synthesis of complex nucleoside antibiotics. The synthesis of caprazol, the core structure of caprazamycins, was conducted by the strategy including beta-selective ribosylation without using a neighboring group participation and the construction of a diazepanone by a modified reductive amination. Our synthetic route would provide a range of key analogues with partial structures to define the pharmacophore, which can be a lead for the development of more effective anti-bacterial agents.

Synthesis of tunicaminyluracil derivatives.

A tunicaminyluracil derivative, which is a key component of the tunicamycin nucleoside antibiotics, was synthesized using a samarium diiodide (SmI2) mediated aldol reaction and intramolecular Pummerer reaction as the key steps. The alpha-phenylthio ketone 11, the precursor of the samarium enolate, was prepared from D-galactose. Treatment of 11 with SmI2 at -40 degrees C resulted in complete conversion to the corresponding samarium enolate, and subsequent addition of uridine 5'-aldehyde 12 afforded the desired aldol products 13a,b. Compound 13a was converted to the sulfoxide 15 by a sequential diastereoselective reduction of the ketone and an oxidation with mCPBA. Activation of 15 with Tf2O provided the desired cyclized compound 17. In this reaction, the aldol product 13a was also obtained as a consequence of a competitive intramolecular version of DMSO-oxidation via a 7-membered ring intermediate. Compound 18 or 19 are ready for use as a glycosyl donor in glycosylations to provide a range of analogues as potential glycosyltransferase inhibitors as well as related natural products.

Stereoselective syntheses of the O,N-protected subunits of the tunicamycins.

The synthesis of the title compounds is described, i.e. coupling of the ylide, generated from the iodophosphonium salt of protected N-phthaloyl-D-galactosamine with 2,3-O-isopropylidene D-ribo-aldehyde afforded an undecose in high yield. Hydroboration-oxidation reaction of the olefinic linkage in the undecose led to the desired tunicamine, as the predominant product. After conversion of the latter to a glycosyl acceptor, this was assembled with the fully protected 2-oxyimino-2-deoxy-alpha-D-arabino-hexopyranosyl bromide, leading to a trehalose-type alpha, beta-disaccharide.

Suppressive role of sialylated N-glycans in Fc receptor-mediated phagocytosis by macrophages.

By culturing with tunicamycin A1, an inhibitor of N-glycosylation, or by sialidase digestion, mouse monocytic cells P388D1 were induced to carry out Fc receptor-mediated phagocytosis of IgG-coated sheep red blood cells. There was no significant difference in the numbers of IgG-coated sheep blood cells bound to the cells at 4 degrees C before and after exposure to tunicamycin or sialidase. These results suggest that sialylated N-glycans expressed on the cell surface have a suppressive role in the induction of phagocytosis, and their decreased expression or reduced sialylation results in acquisition of the phagocytic ability of the cells by affecting some processes involved in the ingestion of the particles bound to Fc receptors.

Effects of growth factors on cell cycle arrest in dolichyl phosphate-depleted cultures.

Previously we showed that CHO cell growth is arrested in the G1 or G0 phase within 24 h after the biosynthesis of mevalonic acid is blocked. The growth-limiting factor under these conditions appeared to be dolichyl phosphate or one of its glycosylated derivatives with consequent decrease in the synthesis of N-linked glycoproteins (Doyle, J.W., and A.A. Kandutsch, 1988, J. Cell Physiol. 137:133-140; Kabakoff, B., J.W. Doyle, and A.A. Kandutsch, 1990, Arch. Biochem. Biophys. 276:382-389). We show herein that cell surface glycoproteins are depleted in the inhibited cultures and that growth arrest is delayed when supraphysiological concentrations of insulin, insulin-like growth factor-1 (IGF-1) and bFGF are added to the culture medium. Apparently an elevated level of a growth factor increases the length of time during which a threshold level of occupied receptor is maintained as the number of glycosylated receptor molecules declines. The results support the idea that cellular levels of dolichyl phosphate and its derivatives may limit cell division by controlling the numbers of functional receptors for growth factors and of other glycoproteins on the cell surface.

N-linked oligosaccharides are not required for hormone binding of the lutropin receptor in a Leydig tumor cell line and rat granulosa cells.

We have examined roles of carbohydrates of the lutropin receptor in a murine Leydig tumor cell line (MLTC) and primary cultures of rat granulosa cells. We approached this issue by deglycosylating mature receptors with glycosidases and by preventing glycosylation of nascent receptors with tunicamycin B2, an inhibitor of protein glycosylation but not protein synthesis. Deglycosylation of mature receptors with neuraminidase, N-glycanase or both did not affect ligand binding capacity. Regardless of glycosidase treatment, the number of hormone binding sites was similar. The Kas for native receptors, asialoreceptors and aglycoreceptors, are also comparable, being 2.0 x 10(9) M-1, 1.9 x 10(9) M-1 and 1.7 x 10(9) M-1 respectively. In contrast, cells treated with tunicamycin B2 failed to bind the hormone. These results demonstrate that N-oligosaccharides of mature lutropin receptors are not required for ligand binding. In addition, our data suggest, for the first time, that N-glycosylation of the receptor may be necessary for expressing functional receptors on the cell surface and that there exist striking similarities in roles of oligosaccharides of lutropin and its receptor.

Tissue factor apoprotein: intracellular transport and expression in shed membrane vesicles.

We have studied the synthesis and subcellular distribution of the glycosylated membrane protein tissue factor (TF) in human blood monocytes and the inducible monocyte like cell line, HL-60. Following induction with endotoxin or the phorbol ester, TPA, tissue factor specific activity was measured in intact cells, sonicated cells, isolated plasma membranes and shed vesicles. We have shown that TF is transported over time from the cytoplasm to the plasma membrane and finally to shed membrane vesicles. Optimal transport of TF to the plasma membrane and shedding in membrane vesicles required glycosylation as judged by partial inhibition of this process by the tunicamycin homologues B2 and C2.

Biosynthesis of the wall acidic polysaccharide in Bacillus cereus AHU 1356.

Biosynthetic studies on an acidic polysaccharide, comprising galactose, rhamnose, N-acetylglucosamine and sn-glycerol 1-phosphate, were carried out with a membrane system obtained from Bacillus cereus AHU 1356. Incubation of the membranes with UDP-[14C]Gal, TDP-[14C]Rha and UDP-[14C]GlcNAc resulted in the formation of four or more labeled-sugar-linked lipids and a labeled polysaccharide. Data on structural analysis of the sugar moieties released from the glycolipids, together with results of enzymatic conversion of [14C]galactose-linked lipid and [14C]Rha-Gal-linked lipid to higher-oligosaccharide-linked lipids and polysaccharide, led to the conclusion that the acidic polysaccharide is probably synthesized through the following pathway: (sequence in text) The glycerophosphate residues seem to be derived from phosphatidylglycerol.

Structural studies on the minor teichoic acid of Bacillus coagulans AHU 1631.

The minor teichoic acid linked to glycopeptide was isolated from lysozyme digests of Bacillus coagulans AHU 1631 cell walls, and the structure of the teichoic acid moiety and its junction with the peptidoglycan were studied. Hydrolysis of the teichoic-acid--glycopeptide complex with hydrogen fluoride gave a nonreducing oligosaccharide composed of glucose, galactose and glycerol in a molar ratio of 3:1:1 which was presumed to be dephosphorylated repeating units of the polymer chain. From the results of structural analysis involving NaIO4 oxidation, methylation and acetolysis, the above fragment was characterized as glucosyl(beta 1----3)glucosyl(beta 1----6)galactosyl(beta 1----6)glucosyl(alpha 1----1/3)glycerol. In addition, the Smith degradation of the complex yielded a phosphorus-containing fragment identified as glycerol-P-6-glucosyl(beta 1----1/3)glycerol. These results led to the most likely structure for the repeating units of the teichoic acid, -6[glucosyl(beta 1----3)]glucosyl(beta 1----6)galactosyl(beta 1----6)glucosyl(alpha 1----1/3)glycerol-P-. The minor teichoic acid, just like the major teichoic acid bound to the linkage unit, was released by heating the cell walls at pH 2.5. The mild alkaline hydrolysis of the minor teichoic acid after reduction with NaB3H4 gave labeled saccharides characterized as glucosyl(beta 1----6)galactitol and glucosyl(beta 1----3)glucosyl(beta 1----6)galactitol, together with a large amount of the unlabeled repeating units of the teichoic acid chain. Thus, the minor teichoic acid chain is believed to be directly linked to peptidoglycan at the galactose residue of the terminal repeating unit without a special linkage sugar unit.

Inhibition of the formation of lipid-linked intermediates in normal and transformed cells by a purified tunicamycin homologue.

The effects of a purified homologue of tunicamycin (B2-tunicamycin) on the biosynthesis of lipid-linked intermediates participating in protein glycosylation in normal embryonic fibroblasts, 3T3 and virally transformed (simian virus 40 and polyoma virus) mouse fibroblasts grown in culture were investigated. Long incubations (20 h) with the antibiotic caused a higher degree of inhibition of sugar incorporation into glycoproteins in transformed cells. However, the formation of lipid-linked intermediates was inhibited to a similar level in both cell types. When time dependent inhibition experiments were carried out using transformed cells, an earlier and stronger inhibition of the formation of lipid-oligosaccharides occurred (70% inhibition at 30 min). In 3T3 cells, prolonged incubation (6-8 h) was necessary in order to reach a similar degree of inhibition. Formation of lipid-sugar was also inhibited to a greater extent by B2-tunicamycin in transformed cells. This inhibition was not clearly time dependent. Analysis of the newly synthesized glycolipids in 3T3 and in transformed cells after B2-tunicamycin treatment have shown reduction in dolichyl-P-P-sugars as well as in other glycolipids. Dimethylsulfoxide (10%) and linoleic acid (0.5 mg/ml) markedly increased the level of tunicamycin activity in 3T3 cells while phosphatidylcholine (2 mg/ml) partially reversed it. The stronger and faster inhibition of the formation of lipid intermediates of the dolichyl-phosphate cycle caused by B2-tunicamycin in transformed cells, described here for the first time, may therefore be due to differences in penetration of the antibiotic into these cells.

Biosynthesis of N-acetylmannosaminuronic-acid-containing cell-wall polysaccharide of Bacillus subtilis.

The particulate enzyme from Bacillus subtilis AHU 1031 catalyzed the synthesis of a polysaccharide and glycolipids from UDP-N-acetylmannosaminuronic acid (UDP-ManNAcUA), UDP-N-acetylglucosamine (UDP-GlcNAc), and UDP-glucose (UDP-Glc). The polysaccharide synthesis required UDP-ManNAcUA and UDP-GlcNAc, proceeded optimally at pH 8.5 and in the presence of 5 mM MgCl2 and 2.5 mM dithiothreitol, and was stimulated by the addition of UDP-Glc. The molar ratio of ManNAcUA, GlcNAc, and Glc incorporated into polysaccharide was calculated to be 1:1:1.8 from chemical analysis involving reduction with water soluble carbodiimide; its relative molecular mass was estimated to be 12000. The analysis of Smith degradation products revealed that the polysaccharide backbone is composed of repeating trisaccharide units comprising ManNAcUA, GlcNAc, and Glc. Based on the data regarding the time course of the incorporation of glucose into the polysaccharide, extra glucose seems to be attached to the polysaccharide backbone as lateral branches. The saccharide moieties of the glycolipids were identified as GlcNAc, ManNAcUA-GlcNAc, and Glc-ManNAcUA-GlcNAc from several analytical criteria. The addition of antibiotic 24010, a tunicamycin-like antibiotic, at 10 micrograms/ml resulted in almost complete inhibition of the synthesis of glycolipids and polysaccharide. It is therefore concluded that the glycolipids function as intermediates in polysaccharide formation. Incubation of the ManNAcUA-GlcNAc-linked lipid. (labeled in the ManNAcUA moiety) with the particulate enzyme and UDP-Glc resulted incorporation of radioactivity into a trisaccharide-linked lipid and a polysaccharide. These results suggest that the particulate enzyme utilizes the trisaccharide moiety of the Glc-ManNAcUA-GlcNAc-linked lipid for the elongation of the main polysaccharide chain presumed to be cell wall acidic polysaccharide of this strain.

Role of glycoproteins in neuronal differentiation. Inhibition of neurite outgrowth and the major cell surface glycoprotein of murine neuroblastoma cells by a purified tunicamycin homologue.

Mouse neuroblastoma cells in culture can be induced to differentiate morphologically by serum deprivation or by dibutyryl cyclic AMP (db-cAMP), e.g. they appear flattened, adhere more firmly to the culture substratum and extend long neuritic processes, and thus represent a widely used model system for neuronal cells. This differentiation is accompanied by modulation of cell surface components, such as the induction of a high molecular weight (HMW) glycoprotein (200 kD). We have studied the role of glycoproteins in the process of neuronal differentiation, using a purified homologue of the antibiotic tunicamycin (Al-tunicamycin) and neuroblastoma N115 cells grown in culture. Al-tunicamycin markedly inhibited (up to 60-75%) the incorporation of radioactively labelled sugars into cellular proteins of differentiating neuroblastoma cells. Concomitantly, the cells altered their morphology, they became rounded and less adhesive and retracted their neurites. Changes in the appearance, glycosylation and electrophoretic mobility of several cellular and secreted glycoproteins were observed, when cells were incubated in the presence of Al-tunicamycin. The most striking effect of Al-tunicamycin on the composition of cellular glycoproteins was the marked reduction in appearance of the 200 kD glycoprotein. The findings suggest that glycoproteins and in particular the neuron-specific 200 kD glycoprotein, are related to morphological differentiation processes, mainly to cellular adhesion and neurite outgrowth.

Inhibition of protein glycosylation and selective cytotoxicity toward virally transformed fibroblasts caused by B3-tunicamycin.

The biological effect of B3-tunicamycin, the only known homologue of tunicamycin which contains a saturated fatty-acid side chain, was examined using chick embryo fibroblasts, a mouse fibroblastic line (3T3) and a virally transformed mouse fibroblastic line (SV40-3T3). This homologue inhibited the transfer of N-acetylglucosamine 1-phosphate from UDP-N-acetylglucosamine to dolichyl phosphate, catalyzed by microsomes from chick liver or from cultured mouse fibroblasts. B3-tunicamycin also inhibited the incorporation of mannose into glycoproteins synthesized by chick or mouse fibroblasts. Incorporation of the amino acids proline and tyrosine was inhibited by B3-tunicamycin to a lesser extent than the incorporation of mannose. The mannose incorporation into glycoproteins synthesized by virally transformed cells was inhibited by B3-tunicamycin to a higher degree than what was achieved in the nontransformed lines or in the chick primary fibroblasts. When the activity of B3-tunicamycin as an inhibitor of protein glycosylation was compared to other homologues of tunicamycin, it was found to be the most active. This homologue caused complete (more than 95%) inhibition of protein glycosylation at a concentration of 50 ng/ml in chick and in mouse fibroblasts and at a concentration of 10 ng/ml in transformed mouse fibroblasts. When the cytotoxic activities of tunicamycin homologues were examined on nontransformed and virally transformed 3T3 cells, it was found that B3-tunicamycin displayed the highest selective cytotoxicity toward the transformed cells. When transformed fibroblasts (10(5) cells/plate) were treated with B3-tunicamycin (100 ng/ml) for 48 h, complete cell death was observed. The viability and the proliferative activity of the nontransformed fibroblast were normal even when treated with concentrations up to 500 ng/ml of B3-tunicamycin. This suggests that B3-tunicamycin may be a suitable candidate for studies of tumor growth in animals.

Holomycin and an antibiotic (MM 19290) related to tunicamycin, metabolites of Streptomyces clavuligerus.

Streptomyces clavuligerus produces penicillin N, several cephalosporins and the beta-lactamase inhibitor clavulanic acid. The detection, isolation and properties of further metabolites of this culture, MM 21801 and MM 19290, are described. MM 21801 was identified as the antibiotic holomycin. MM 19290 was shown to be related to tunicamycin, an antibiotic complex obtained from cultures of Streptomyces lysosuperificus.

Formation and function of N-acetyloglucosamine-linked phosphoryl- and pyrophosphorylundecaprenols in membranes from Bacillus cereus.

Membranes from Bacillus cereus AHU 1356 incorporated radioactivity from UDP-N-acetyl[14C]glucosamine into three alkaline-stable and acid-labile lipids which were extracted into chloroform:methanol (2:1) and separated from each other by thin layer chromatography on silica gel plates. The major labeled lipid (Lipid 1) and a minor one (Lipid 2) were identified as N-actetylglucosaminyl phosphorylundecaprenol from several analytical criteria involving mass spectral data and from reversal of their formation by UDP. These two lipids appear to differ in geometry of their polyprenol moieties. The third labeled lipid (Lipid 3) was identified as N-acetylglucosaminyl pyrophosphorylundecaprenol. Antibiotic 24010, a tunicamycin-like antibiotic, at 1 microgram/ml was found to inhibit almost completely the formation of Lipid 3, whereas it inhibited the formation of Lipid 1 much more weakly and rather enhanced the formation of Lipid 2. Radioactivity was also incorporated into a polymer from UDP-GlcNAc and from Lipid 3. UDP-N-acetylmannosamine, UDP-N-acetylgalactosamine, and UDP-glucose supported the incorporation. Antibiotic 24010 strongly inhibited the incorporation of radioactivity from UDP-GlcNAc into polymer, whereas it did not affect the incorporation from Lipid 3. Thus, it is concluded that N-acetylglucosaminyl pyrophosphorylundecaprenol serves as a precursor in the synthesis of a polymer presumed as the cell wall polysaccharide of this bacterial strain.