Linear PADRE T helper epitope and carbohydrate B cell epitope conjugates induce specific high titer IgG antibody responses.

Linear carbohydrate-peptide constructs based on the 13 amino acid nonnatural pan DR epitope (PADRE) and carbohydrate B cell epitopes are demonstrated to be potent immunogens. These data support our belief that PADRE should be considered as an alternative to more complex carriers for use in prophylaxis and therapeutic vaccines. Two model carbohydrate-PADRE glycoconjugates were used to demonstrate that PADRE could effectively provide T cell help for carbohydrate-specific Ab responses. Conjugates of PADRE covalently linked to the human milk oligosaccharide, lacto-N-fucopentose II or a dodecasaccharide derived from Salmonella typhimurium O-Ag induced high titer IgG Ab responses in mice, which were comparable to glycoconjugates employing human serum albumin (HSA) as the carrier protein. Different adjuvants, in combination with PADRE conjugates, allowed for the modulation of the isotype profile with alum supporting an IgG1 profile; QS-21 an IgG2a, 2b profile, while an alum/QS-21 mixture generated a balanced IgG1/IgG2b isotype profile. As defined by binding to synthetic glycoconjugates, dodecasaccharide-specific Abs exhibited fine specificity similar to protective polyclonal Ab responses previously reported for dodecasaccharide-protein conjugates. The same Abs bound to intact S. typhimurium cells, suggesting that biologically relevant specificities were produced. The affinity of the dodecasaccharide-specific Abs was further shown to be comparable to that of a well-characterized, high affinity monoclonal anti-carbohydrate Ab recognizing the same epitope.

The synthesis of sialylated oligosaccharides using a CMP-Neu5Ac synthetase/sialyltransferase fusion.

Large-scale enzymatic synthesis of oligosaccharides, which contain terminal N-acetyl-neuraminic acid residues requires large amounts of the sialyltransferase and the corresponding sugar-nucleotide synthetase, which is required for the synthesis of the sugar-nucleotide donor, CMP-Neu5Ac. Using genes cloned from Neisseria meningitidis, we constructed a fusion protein that has both CMP-Neu5Ac synthetase and alpha-2,3-sialyltransferase activities. The fusion protein was produced in high yields (over 1200 U/L, measured using an alpha-2,3-sialyltransferase assay) in Escherichia coli and functionally pure enzyme could be obtained using a simple protocol. In small-scale enzymatic syntheses, the fusion protein could sialylate various oligosaccharide acceptors (branched and linear) with N-acetyl-neuraminic acid as well as N-glycolyl- and N-propionyl-neuraminic acid in high conversion yield. The fusion protein was also used to produce alpha-2,3-sialyllactose at the 100 g scale using a sugar nucleotide cycle reaction, starting from lactose, sialic acid, phosphoenolpyruvate, and catalytic amounts of ATP and CMP.

Poly(ethylene glycol)-grafted liposomes with oligopeptide or oligosaccharide ligands appended to the termini of the polymer chains.

Novel conjugates tailor-made for inclusion in liposomal formulations, containing distearoylphosphatidylethanolamine (DSPE) as a lipid anchor, heterobifunctional polyethylene glycol (PEG) with a molecular weight of 2000 as a linking moiety, and a biological cell adhesive ligand [YIGSR peptide or Sialyl Lewis(X) oligosaccharide (SLX)], were synthesized. They were characterized by NMR, chromatography, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS). Inclusion of either of the ligand-PEG-lipid conjugates (2 mol %) in a lecithin/cholesterol/ methoxy-PEG2000-DSPE (55:40:3 mole ratio) lipid mixture followed by preparation of unilamellar vesicles (100 nm) resulted in positioning of 55% of the YIGSR and 63% of the SLX ligands on the periphery of the outer surface-grafted polymeric "brush", as determined by a combination of specific enzymatic alterations of each ligand and HPLC. Similar densities of ligand-bearing PEG chains were incorporated into liposomes by simply incubating (37 degrees C, 5 h) either one of the ligand-PEG-lipid conjugates with preformed lipid vesicles. This conjugate insertion process was aggregation free. Using enzymatic derivatization-HPLC, it was demonstrated that all the ligands incorporated into lipid membranes by this new approach were positioned exclusively on the outer leaflet of the liposomal bilayers. Since liposomes of this type are intended for in vivo use as long-circulating, ligand-presenting platforms, the insertion approach is preferable because of the more efficient utilization of ligand-PEG-lipid conjugates.

Novel synthetic analogs of sialyl Lewis X can inhibit angiogenesis in vitro and in vivo.

Selectins have been shown to play an important role in angiogenesis. We have set out to synthesize multiple analogs of the selectin ligand sialyl Lewis X. We show here that analogs of sialyl Lewis X at concentrations of 50 micrograms/ml or greater significantly inhibited angiogenesis in in vitro assays of endothelial cell migration and proliferation (p < 0.05). These analogs also exerted significant inhibitory effects in in vivo angiogenesis assays using the chick chorioallantoic membrane at doses of 2 mg/chick or greater (p < 0.05). In contrast, the related carbohydrate Lewis X did not inhibit angiogenesis in both in vitro and in vivo assays. These data indicate that angiogenesis can be inhibited specifically by synthetic analogs of sialyl Lewis X. These analogs may potentially be useful in the treatment of angiogenesis-related diseases.

Ligand interactions with E-selectin. Identification of a new binding site for recognition of N-acyl aromatic glucosamine substituents of sialyl Lewis X.

Several N-acylglucosamine derivatives of sialyl Lewis X (1-3) were prepared using a combined chemical enzymatic approach and evaluated as an inhibitor of E-selectin-mediated cellular adhesion. Compounds with aromatic functionality, 1 and 2, were found to be 3-10 times more potent than the N-acetyl derivative (14) in an ELISA E-selectin cell adhesion assay. Conformational analysis with NMR indicated that the sialyl Lewis x domain of 1 retained the conformation of the N-acetyl derivative (14) despite the presence of the N-naphthamido group. The dramatic order of magnitude increase in potency of these monovalent structures can be utilized to design more potent selectin-based cell adhesion inhibitors.

Cardioprotection by liposome-conjugated sialyl Lewisx-oligosaccharide in myocardial ischaemia and reperfusion injury.

OBJECTIVES: Selectins are important adhesion molecules which utilize a carbohydrate ligand such as sialyl Lewisx (SLex). Our objective was to study the effects of a liposome-conjugated SLex (Lipo-SLex) in myocardial ischaemia (MI) and reperfusion (R) injury in order to further clarify the actions of this carbohydrate. METHODS: We studied the efficacy of Lipo-SLex in a feline model of MI (90 min) and R (270 min) injury in vivo. Lipo-SLex (400 micrograms SLex/kg, iv) was administered intravenously 10 min prior to R. We also utilized an in vitro system of neutrophil adherence to thrombin-stimulated coronary endothelium to validate the efficacy of Lipo-SLex. RESULTS: Lipo-SLex significantly attenuated myocardial necrosis (8.6 +/- 1.2 vs. 29.5 +/- 3.1% of area-at-risk, P < 0.01) and plasma creatine kinase activities (P < 0.01) compared to vehicle (liposome alone). Moreover, endothelium-dependent relaxation to acetylcholine and A23187 in ischaemic-reperfused coronary rings obtained from cats treated with Lipo-SLex was significantly preserved compared to cats given liposomes without SLex (P < 0.01). After reperfusion, ex vivo PMN adherence to ischaemic-reperfused coronary endothelium was significantly increased in vehicle-treated cats, however, this was significantly attenuated in Lipo-SLex-treated cats (82 +/- 7 vs. 28 +/- 3 PMNs/mm2, P < 0.01). Myeloperoxidase activity in the ischaemic myocardium, a marker of PMN accumulation, was also significantly attenuated in Lipo-SLex-treated cats compared to liposomes without SLex (P < 0.01). CONCLUSIONS: Liposome-conjugated SLex-oligosaccharide attenuates myocardial necrosis and preserves coronary endothelial function following MI/R in vivo. The mechanism appears to be mediated by inhibition of the initial PMN-endothelial interaction and eventual accumulation into the ischaemic cardiac tissue. The liposome-SLex complex may be an efficient drug formulation for acute inflammatory diseases.

Structure--activity relationships of sialyl Lewis x-containing oligosaccharides. 1. Effect of modifications of the fucose moiety.

Leukocyte adhesion to the vasculature is mediated by E-, P-, and L-selectins. The natural ligands for E- and P-selectins have not been fully characterized but have been shown to contain the tetrasaccharide sialyl Lewis x structure (SLe(x)). To determine the importance of the fucose moiety of SLe(x), various analogs of SLe(x) containing modifications thereof were prepared and tested as inhibitors of E-selectin-mediated cell adhesion. Cellular experiments indicate that replacement of the hydroxyl groups of fucose by hydrogen abrogated E-selectin binding. However, the arabinose analog of fucose (CH3 delta H) inhibited cell adhesion but was 5-fold less potent than native SLe(x). This data suggests that modifications of fucose on SLe(x) are generally deleterious toward E-selectin binding.

Role of H1 receptors and P-selectin in histamine-induced leukocyte rolling and adhesion in postcapillary venules.

The objective of this study was to define the nature, magnitude, and mechanisms of histamine-induced leukocyte-endothelial cell interactions in postcapillary venules of the rat mesentery using intravital microscopic techniques. Superfusion of the mesentery with histamine (10(-7)-10(-5) M) resulted in a dose-related increase in the number of rolling leukocytes, a reduction in rolling velocity, and an increased clearance of FITC-labeled rat albumin from blood to superfusate. The histamine-induced recruitment of rolling leukocytes and increased albumin clearance were prevented by histamine H1 (hydroxyzine, diphenhydramine) but not H2 (cimetidine) receptor antagonists. Because histamine induces expression of the adhesion molecule P-selectin in cultured endothelial cells, a monoclonal antibody directed against rat P-selectin and soluble sialyl-LewisX oligosaccharide (the carbohydrate ligand to P-selectin) were also tested as inhibitors. Both were effective in preventing the histamine-induced recruitment of rolling leukocytes, but neither agent attenuated the increased albumin clearance. These observations suggest that (a) histamine recruits rolling leukocytes and increases albumin leakage in postcapillary venules via H1 receptor activation, (b) histamine-induced recruitment of rolling leukocytes is mediated in part by P-selectin expressed on the endothelial cell surface, and (c) the histamine-induced vascular albumin leakage is unrelated to leukocyte-endothelial cell adhesion. Our results are consistent with the view that histamine may act as a mediator of acute inflammatory reactions.

Protective effects of sialylated oligosaccharides in immune complex-induced acute lung injury.

Using sialyl Lewisx (SLX) oligosaccharides derived from fucosyl transferase-expressing cells or generated synthetically, the ability of these compounds to protect against acute lung damage after deposition of immunoglobulin (Ig)G or IgA immune complexes has been determined. The synthetic compounds were tetra- and pentasaccharide derivates of SLX as well as the nonfucosylated forms of SLX as controls. In the IgG immune complex model of lung injury, which is E-selectin dependent, SLX preparations provided dose-dependent protective effects, as assessed by changes in lung vascular permeability and hemorrhage. Protective effects were associated with diminished tissue accumulation of neutrophils in lungs (as assessed by myeloperoxidase). Morphological assessment revealed reduced physical contact of neutrophils with the pulmonary vascular endothelium and reduced tissue accumulation of neutrophils. In the model of IgA immune complex-induced lung injury, which does not involve participation of neutrophils and is independent of the requirement for E-selectin, SLX preparations were not protective. These data suggest that, in neutrophil-mediated and E-selectin-dependent lung injury, SLX preparations provide significant, protective effects against inflammatory vascular injury. The ability to achieve antiinflammatory outcomes in vivo with appropriate oligosaccharides suggests a new approach to the blocking of acute inflammatory responses.

Metabolic activation of 2-substituted derivatives of myristic acid to form potent inhibitors of myristoyl CoA:protein N-myristoyltransferase.

The importance of myristoylation for the proper biological functioning of many acylated proteins has generated interest in the enzymes of the myristoylation pathway and their interactions with substrates and inhibitors. Previous observations that S-(2-oxopentadecyl)-CoA, a nonhydrolyzable methylene-bridged analogue of myristoyl-CoA, was a potent inhibitor of myristoyl-CoA:protein N-myristoyltransferase (NMT) [Paige, L. A., Zheng, G.-q., DeFrees, S. A., Cassady, J. M., & Geahlen, R. L. (1989) J. Med. Chem. 32, 1665] prompted a closer examination of the effect of substituents at the 2-position on the interactions of myristic acid and myristoyl-CoA analogues with NMT. As an initial approach, three myristic acid derivatives bearing different substituents at the 2-position, 2-fluoromyristic acid, 2-bromomyristic acid, and 2-hydroxymyristic acid, were selected for study. Both 2-bromomyristic acid and 2-hydroxymyristic acid were available commercially; 2-fluoromyristic acid was prepared synthetically. All three compounds were found to be only weak inhibitors of NMT in vitro. Of the three, 2-bromomyristic acid was the most potent (Ki = 100 microM). In cultured cells, however, 2-hydroxymyristic acid was by far the more effective inhibitor of protein myristoylation. Neither 2-hydroxymyristic acid nor 2-bromomyristic acid significantly inhibited protein palmitoylation in cultured cells, indicating that inhibition was not occurring at the level of acyl-CoA synthetase. Activation of the 2-substituted myristic acid derivatives to their corresponding acyl-CoA thioesters by acyl-CoA synthetase resulted in inhibitors of greatly increased potency. The 2-substituted acyl-CoA analogues, 2-hydroxymyristoyl-CoA, 2-bromomyristoyl-CoA, and 2-fluoromyristoyl-CoA, were synthesized and shown to be competitive inhibitors of NMT in vitro (Ki's = 45, 450, and 200 nM, respectively). These data suggested that the enhanced inhibitory potency of 2-hydroxymyristic acid seen in cells was most probably a result of its metabolic activation to the CoA thioester. The presence of substituents at the 2-position also affected the ability of the acyl group to be transferred by NMT to a peptide substrate. Of the three acyl-CoA analogues, only 2-fluoromyristoyl-CoA served as a substrate for NMT.

Structure-activity relationships of pyrimidines as dihydroorotate dehydrogenase inhibitors.

The activity of dihydroorotate dehydrogenase (DHO-dehase) has been reported to decrease both in vitro and in vivo in hepatocellular carcinomas. DHO-dehase, the fourth enzyme of the de novo pyrimidine biosynthetic pathway, is a mitochondrial enzyme which is both a potential rate-limiting reaction in the de novo pyrimidine biosynthetic pathway and a potential therapeutic target for tumor inhibitors. This paper reports results on a series of pyrimidine analogs of dihydroorotate (DHO) and orotic acid (OA) as inhibitors of DHO-dehase. The enzyme test results established that the intact amide and imide groups of the pyrimidine ring and the 6-carboxylic acid are required for significant enzyme inhibition. The testing of several functional groups similar in characteristics to that of the carboxylic acid, such as sulfonamide, tetrazole and phosphate, indicated that the carboxylic acid group is preferred by the enzyme. Using various 5-substituted OA and DHO derivatives, it was shown that there is a steric limitation of a methyl group at this position. The compound D,L-5-trans-methyl DHO (7) (Ki of 45 microM) was both an inhibitor and a weak substrate for the enzyme, demonstrating that mechanism-based enzyme inhibitors should be effective. The testing results further suggest that a negatively charged enzyme substituent may be present near the 5-position of the pyrimidine ring and that there may be an enzyme-substrate metal coordination site near the N-1 and carboxylic acid positions of the pyrimidine ring. The combined testing results were then used to define both conformational and steric substrate enzyme binding requirements from which a model was proposed for the binding of DHO and OA to the DHO-dehase active site.

Cyclic modulation of enzymes of pyrimidine nucleotide biosynthesis precedes sialoglycoconjugate changes during 2-acetylaminofluorene-induced hepatocarcinogenesis in the rat.

Three enzymatic activities associated with pyrimidine nucleotide biosynthesis were monitored at weekly or bi-weekly intervals during 2-acetylaminofluorene- (0.025% in a Farber Basal Carcinogenic diet) induced hepatocarcinogenesis in the rat. Dihydroorotate dehydrogenase, the fourth of six enzymes in de novo pyrimidine biosynthesis, declined in activity while UDP kinase and CTP synthetase showed sequential increases in activity. The alterations in activity appeared to be cyclic, followed by a full or partial return to control values. Three full cycles were monitored. The first cycle preceded nodule formation. The second cycle accompanied nodule formation and preceded sialoglycoconjugate changes reported previously. The third cycle accompanied the early glycoconjugate changes. The cyclic pattern was reproducible in three separate experiments. In each cycle, the order of events was as follows: decrease in dihydroorotate dehydrogenase, sequential increases in UDP kinase, CTP synthetase and CMPsialic acid synthase, and finally increases in the enzyme lactosylceramide: CMPsialic acid sialyltransferase, lipid-soluble sialic acid and total sialic acid. In livers of animals fed 1.87% of the hepatotoxin, 4-acetamidophenol, no biochemical alterations resembling those induced by 2-acetylaminofluorene were obtained, despite acute centrilobular necrosis of the livers. The findings point to a biochemical cascade beginning with administration of carcinogen and continuing through the development of hyperplastic nodules and of frank carcinomas resulting not from hepatotoxicity but as events associated with the hepatocarcinogenic progression.