Efficient synthesis of a 3,6-branched mannose hepta- and octasaccharide

Alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)]-alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)]-D-Manp and alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)]-alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)]-D-Manp, were synthesized as their methyl glycosides in a regio- and stereoselective way.     

IL-4 modulates transcriptional control of the mannose receptor in mouse FSDC dendritic cells

The mannose receptor is a 175 kDa protein found on the surface of macrophages and dendritic cells whose functions include clearance of extracellular hydrolases, internalization of pathogens, and antigen capture. Receptor expression is closely linked to the functional state of these cells and is regulated by cytokines. Previous work has shown that treatment of macrophages and dendritic cells with interleukin-4 leads to increased mannose receptor expression. We have examined the mechanism of this IL-4-mediated up-regulation in the murine dendritic cell line FSDC. IL-4 increased mannose receptor activity, protein, and mRNA. The mannose receptor promoter was functional in FSDCs using transient transfection assays, and IL-4 treatment increased promoter activity 2.6-fold. The responsive region was localized to the proximal 228 bp. Electrophoretic mobility shift assays detected an IL-4-inducible protein that bound to the mannose receptor promoter at a site spanning the region between -147 and -108 bp. The sequence TTAC(N)4CACC (-135 and -124 bp) is similar to the IL-4 response region in the Fc receptor II. Mutation of the flanking TT and CC in this motif blocked IL-4 responsiveness and binding of the IL-4-induced mannose receptor binding protein. This protein does not appear to be STAT6 since neither an anti-STAT6 antibody nor a STAT6 consensus oligonucleotide altered factor binding.     

Drug-induced phospholipidosis is caused by blockade of mannose 6-phosphate receptor-mediated targeting of lysosomal enzymes

Cationic amphiphilic drugs (CADs) cause massive intracellular accumulation of phospholipids, thereby resulting in phospholipidosis (PLD); however, the molecular mechanism underlying CAD-induced PLD remains to be resolved. Here, we found that treatment of normal rat kidney cells with CADs known to induce PLD caused redistribution of a mannose 6-phosphate/IGF-II receptor (MPR300) from the TGN to endosomes and concomitantly increased the secretion of lysosomal enzymes, resulting in a decline of intracellular lysosomal enzyme levels. These results enable the interpretation of why CADs cause excessive accumulation of undegraded substrates, including phospholipids in lysosomes, and led to the conclusion that the impaired MPR300-mediated sorting system of lysosomal enzymes reflects the general mechanism of CAD-induced PLD. In addition, our findings suggest that the measurement of lysosomal enzyme activity secreted into culture medium is useful as a rapid and convenient in vitro early screening system to predict drugs that can induce PLD.     

Biochemical and functional characterization of cation dependent (Mr 46,000) goat mannose 6-phosphate receptor

The Mannose 6-phosphate receptor (MPR’s) proteins are important for transporting lysosomal enzymes from trans-golgi to the pre-lysosomal compartment. These are conserved in the vertebrates from fish to mammals. We have cloned the full length cDNA for the goat MPR 46 protein and compared its sequences to the other known vertebrate MPR 46 proteins. In the present study the full-length cDNA for the goat MPR 46 protein was expressed in MPR deficient cells. The expressed protein was purified on the multivalent phosphomannan gel in the presence of divalent metal ions. The apparent molecular mass of the expressed protein was found to be ∼46 kDa and also exhibits oligomeric nature as observed in the other species, by using an MSC1 antibody (that recognizes the MPR 46 from molluscs to mammals) as well as with a peptide specific antibody corresponding to amino acid residues (218–237) of the cytoplasmic tail of human MPR 46 protein. Furthermore the distribution of the expressed protein was visualized by immunofluorescence using MSC1 and LAMP1 antibody. Additionally in the goat MPR 46 expressing cells, the sorting function of the expressed protein to sort cathepsin D to lysosomes was studied by confocal microscopy using cathepsin D antiserum and LAMP1 antibody. The binding of goat MPR 46 to cathepsin D was shown in far Western blotting and the mannose 6-phosphate dependent binding was shown by co-immunoprecipitation.     

A facile regio- and stereoselective synthesis of mannose octasaccharide of the N-glycan in human CD2 and mannose hexasaccharide antigenic factor 13b

A highly concise and effective synthesis of the mannose octasaccharide of the N-linked glycan in the adhesion domain of human CD2 was achieved via TMSOTf-promoted selective 6-glycosylation of a trisaccharide 4,6-diol acceptor with a pentasaccharide donor, followed by deprotection. The pentasaccharide was constructed by selective 3,6-diglycosylation of 1,2-O-ethylidene-beta-D-mannopyranose with 2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate, while the trisaccharide was obtained by selective 3-O-glycosylation of allyl 4,6-O-benzylidene-alpha-D-mannopyranoside with the same disaccharide trichloroacetimidate, followed by debenzylidenation. The mannose hexasaccharide antigenic factor 13b was synthesized by condensation of a trisaccharide donor, 2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-4,6-di-O-acetyl-2-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate, with a trisaccharide acceptor, methyl 3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranoside, followed by deprotection.     

78 kDa receptor for Man6P-independent lysosomal enzyme targeting: biosynthetic transport from endoplasmic reticulum to "high-density vesicles"

Recent work has shown that the cation-independent mannose 6-phosphate and the 78 kDa receptors for lysosomal enzyme targeting are located in different cell compartments. While the mannose 6-phosphate receptor is enriched in the Percoll fractions that contain Golgi apparatus, most of the 78 kDa receptor is localized in a heavy fraction at the bottom of the Percoll gradient. This report presents the biosynthetic transport of the 78 kDa receptor. Newly synthesized 78 kDa receptor was transported to Golgi from endoplasmic reticulum with a half life of 5 min. From the Golgi apparatus, the receptor takes two routes; about 15-25% is transported to the plasma membrane, and the rest migrates to late endosomes, subsequently to prelysosomes and finally to the dense vesicles. The 78 kDa receptor starts appearing at the dense vesicles 120 min after biosynthesis and reaches a maximum of 40-50% of the total receptor. Treatment of cells with NH4Cl causes depletion of the receptor from the dense vesicles and prelysosomes and corresponding augmentation in endosomes and plasma membrane. These results suggest that the 78 kDa receptor cycles between compartments and that the dense vesicles seem to represent the most distal compartment in the biosynthetic pathway of this receptor.     

Optimization of lipase-catalyzed synthesis of novel galactosyl ligands for selective targeting of liposomes to the asialoglycoprotein receptor

The asialoglycoprotein receptor (ASGPR) is a potential target in the search for hepatic cancer drugs. However, application of ASGPR targeting in the clinic is limited by inefficient synthetic methods for the ligand. In this study, we designed and synthesized a novel galactosylated lipid with a mono-galactoside moiety using a lipase. Then we investigated the optimal reaction conditions and analyzed the targeting ability of liposomes modified with the galactosylated lipid. In an organic phase system, different lipases were used as catalysts to synthesize (5-cholesten-3b-yl) [(4-O-β-D-galactopyranosyl)D-glucitol-6] sebacate (CHS-SE-LA). Variables in enzymatic esterification, such as the type of enzyme and solvent, were explored by single-factor experiments. Optimal reaction conditions were determined through response surface methodology. The (CHS-SE-LA)-incorporated galactosylated liposome containing fluorescent dye was then prepared by thin-film hydration and a HepG2 cell transfection test used to confirm the targeting efficiency of galactosylated liposomes to hepatic cancer cells. The structure of CHS-SE-LA was identified by electrospray ionization or ESI and nuclear magnetic resonance or NMR. Under optimal conditions, the predicted yield of CHS-SE-LA was 94.3%, and the actual experimental value was 95.6 ± 1.35%, n = 3. For HepG2 cells, the cellular fluorescence intensities of liposomes modified with CHS-SE-LA (galactosylated liposomes [GAL-FL]) were as much as 2.6-fold (P < 0.01) the control liposomes (FL). Moreover, the presence of excess galactose significantly inhibited the uptake of GAL-FL suggesting ASGPR mediated uptake. The novel galactosylated ligand was synthesized enzymatically with high efficiency under mild conditions. Liposomes containing CHS-SE-LA have great potential as drug delivery carriers for hepatocyte-selective targeting.     

The Thomsen-Friedenreich disaccharide as antigen for in vivo tumor targeting with multivalent scFvs

The Thomsen-Friedenreich disaccharide (TF_α) is a promising antigen for tumor immunotargeting, since it is almost exclusively expressed on carcinoma tissues. So far, an obstacle preventing the exploitation of TF for immunotargeting has been the lack of suitable (non-IgM) antibodies with high affinity and specificity. Recently we reported on a novel strategy for generating antibodies toward small uncharged carbohydrates and the generation of recombinant antibodies toward TF. Among them, two multivalent scFv antibodies showed sub-micromolar functional affinities and appeared well suited for immunotargeting. In the present study, the trimeric scFv(1aa) and the tetrameric scFv(0aa) have been further developed for radioimmunotargeting. The scFvs were radiolabeled with ¹¹¹In using DTPA as chelator without losing binding activity or molecular stoichiometry. Binding affinities as high as 1 × 10⁻⁷ M toward TF displayed on living cells were determined. Antibody biodistribution and tumor targeting efficacy were studied in TF-positive human breast cancer (ZR-75-1) bearing mice. TF was successfully targeted in vivo with tumor uptakes of ∼11 and 8% ID/g after 24 h for the trimeric and tetrameric scFv, respectively. These results validate TF as a potent antigen for tumor targeting. The biodistribution of the scFvs was comparable to that reported for IgGs. In contrast to the IgGs, the serum clearance of the scFvs was very fast, which could be an advantage in a therapeutic setting. Furthermore, kidney uptake, which is often critical for small recombinant antibodies labeled with radio-metals, was low with the tetramer (11% ID/g). We conclude that the multimeric anti-TF scFvs are promising candidates to be further developed toward therapeutic application. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Design and synthesis of a multivalent homing device for targeting to murine CD22

CD22 is a cell-surface glycoprotein uniquely located on mature B-cells and B-cell derived tumour cells. Current evidence suggests that binding of endogenous ligands to CD22 leads to modulation of B-cell activation by antigen. Incidentally, however, B-cell activation may derail. and lead to an undesired immune response, for example in cases of allergy, rheumatoid arthritis and Crohn's disease. In this situation, synthetic high-affinity ligands for CD22 may be of therapeutic value as inhibitors of B-cell activation. Recent studies have revealed that natural ligands for CD22 contain the trisaccharide NeuAc alpha-2,6-Lac as the basic binding motif. In addition, it has been demonstrated that binding to CD22 is strongly enhanced by multivalent presentation of the basic binding motif (cluster effect). In this paper. the stepwise development of a novel multivalent high-affinity ligand for CD22 is described. In the first stage, a series of monovalent NeuAc alpha-2,6-Glc(Y)X type binding motifs was prepared, and their affinity for murine CD22 was monitored, to obtain more insight into the effect of separate structure elements on ligand recognition. In the second stage, we prepared a trivalent cluster, based on the monovalent motif that displayed the highest affinity for CD22, NeuAc alpha-2,6-GlcNBzNO2OMe (7). This cluster, TRIS(NeuAc alpha-2,6-GlcNBzNO2)3 (52), displayed a more than 58-fold higher affinity for CD22 than the reference structure NeuAc alpha-2,6-LacOMe (10). To our knowledge, the cluster 52 is one of the most potent antagonists for CD22 yet synthesised.     

Design,synthesis, and effects of novel phenylpyrimidines as glucagon receptor antagonists

The hormone glucagon increases blood glucose levels through increasing hepatic glucose output. In diabetic patients, dysregulation of glucagon secretion contributes to hyperglycemia. Thus, the inhibition of glucagon receptor is one target for the treatment of hyperglycemia in type 2 diabetes. Here we designed and synthesized a series of small molecules based on phenylpyrimidine. Of these, the compound (R)-7a most significantly decreased the glucagon-induced cAMP production and glucagon-induced glucose production during in vitro and in vivo assays. In addition, (R)-7a showed good efficacy in glucagon challenge tests and lowered blood glucose levels in diabetic db/db mice. Our results suggest that the compound (R)-7a could be a potential glucose-lowering agent for treating type 2 diabetes.     

Design,synthesis and biological evaluation of novel analgesic agents targeting both cyclooxygenase and TRPV1

Multitarget-directed ligands might offer certain advantages over traditional single-target drugs and/or drug combinations. In the present study, a series of novel analgesic agents targeting both cyclooxygenase and TRPV1 were prepared and evaluated in an effort to optimize properties of previously described lead compounds from piperazine, ethanediamine cores. These compounds were evaluated for antagonism of hTRPV1 activation by capsaicin and the ability to inhibit Ovine COX-1 and human recombinant COX-2 in vitro. The favorable potentials of these test compounds were further characterized in preliminary analgesic and side-effects tests in vivo. On the basis of comprehensive evaluations, compound 8d which showed strong TRPV1 antagonistic activity, middle COX-2 inhibition, weak ulcerogenic action and had no hyperthermia side-effect was considered as a safe candidate for the further development of analgesic drugs.     

Synthesis of a mannose nonasaccharide existing in the exopolysaccharide of Cryphonectria parasitica

alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)[alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)]-alpha-D-Manp-(1-->6)-[alpha-D-Manp-(1-->2)]-alpha-D-Manp, existing in the exopolysaccharide of Cryphonectria parasitica was synthesized as its allyl glycoside in a regio- and stereoselective manner.     

Selective targeting of avidin/mannose 6-phosphate receptor chimeras to early or late endosomes

In this study we have used the Semliki forest virus expression system to transiently express chimeric proteins that contain transmembrane and cytoplasmic domains of the cation-independent mannose 6-phosphate receptor (CI-MPR) fused to chicken avidin. Immunofluorescence and electron microscopy studies showed that the chimeric protein with the entire cytoplasmic domain of CI-MPR was transported to late endosomes, where it accumulated. We made use of the biotin-binding capacity of lumenal avidin, and found that, in agreement with this distribution, the chimeric protein could be labelled with biotinylated HRP endocytosed for a long, but not a brief, period of time. However, truncation of the C-terminal tail distal to the rapid endocytosis motif (YKYSKV), caused the truncated chimera to be transported to, and accumulated within, early endosomes. This truncated chimera did not reach recycling early endosomes labelled with internalised transferrin, to any significant extent, but was accessible to biotinylated HRP internalised for 5 min (or for longer periods at 19 degrees C). Coinfection of these chimeras showed that they follow the same route from the TGN to the early endosomes. We conclude that the sequence distal to the endocytosis motif contains the signals which are required for efficient transport to late endosomes. Our results also suggest that the YKYSKV sequence close to the CI-MPR transmembrane segment is sufficient for targeting to sorting early endosomes.     

A novel gene delivery system targeting urokinase receptor

Viral vectors are widely used in gene therapy due totheir high efficiency of gene transfer. However, majordisadvantages of viral vectors for gene transfer include thelimitation of cell type specificity and the size of incor-porated DNA, the potential risk…     

2-Deoxyglucose sensitizes melanoma cells to TRAIL-induced apoptosis which is reduced by mannose

While melanoma cell lines use aerobic glycolysis, addition of a competitive inhibitor such as 2-deoxyglucose (2DG) by itself achieved only modest killing. To overcome high levels of pro-survival proteins in melanoma cells, 2DG or glucose deprivation (GD) was combined with tumor necrosis factor-related apoptosis inducing-ligand (TRAIL). TRAIL treatment by itself also only induced modest killing, but combining TRAIL with 2DG or GD triggered a synergistic pro-apoptotic response in melanoma lines but not melanocytes. In melanoma cells, there was cleavage of caspases 3, 8 and Bid. Killing by combination treatments was completely blocked by a pan-caspase inhibitor, z-VAD. Mechanistically, 2DG and GD enhanced surface levels for both death receptors (DR4 and DR5); which was accompanied by reductions in levels of Mcl-1, Bcl-2 and survivin. Mannose pre-treatment reduced enhanced killing by combination treatments, accompanied by reduced DR5 levels. These results indicate melanoma cells in which there is altered glucose-related metabolomics can be exploited by interfering with glucose metabolism in combination with TRAIL; thereby overcoming the notorious death resistance of melanoma. Thus, a new therapeutic window is open for future clinical trials using agents targeting the glucose-related metabolome, in combination with agents triggering death receptors in patients with melanoma.     

Antigenic targeting of the human mannose receptor induces tumor immunity

Pattern recognition receptors are preferentially expressed on APCs allowing selective uptake of pathogens for the initiation of antimicrobial immunity. In particular, C-type lectin receptors, including the mannose receptor (MR), facilitate APC-mediated adsorptive endocytosis of microbial glyconjugates. We have investigated the potential of antigenic targeting to the MR as a means to induce Ag-specific humoral and cellular immunity. hMR transgenic (hMR Tg) mice were generated to allow specific targeting with the anti-hMR Ab, B11. We show that hMR targeting induced both humoral and cellular antigenic specific immunity. Immunization of hMR Tg mice with B11 mAbs induced potent humoral responses independent of adjuvant. Injection of hMR Tg mice with mouse anti-hMR Ab clone 19.2 elicited anti-Id-specific humoral immunity while non-Tg mice were unresponsive. B11-OVA fusion proteins (B11-OVA) were efficiently presented to OVA-specific CD4 and CD8 T cells in MR Tg, but not in non-Tg, mice. Effector differentiation of responding T cells in MR Tg mice was significantly enhanced with concomitant immunization with the TLR agonist, CpG. Administration of both CpG and B11-OVA to hMR Tg mice induced OVA-specific tumor immunity while WT mice remained unprotected. These studies support the clinical development of immunotherapeutic approaches in cancer using pattern recognition receptor targeting systems for the selective delivery of tumor Ags to APCs.     

Identification and synthesis of an efficient multivalent E. coli heat labile toxin inhibitor __ A dynamic combinatorial chemistry approach

A polymer based dynamic combinatorial library (DCL) was generated through condensation between aldehyde functionalized linear poly(glycidol) (APG) and galactose containing acylhydrazide derivatives. Pentameric E. coli heat labile enterotoxin B subunit (LTB) was subsequently applied to the DCL as external stimulus, resulting in amplification of a specific acylhydrazone side chain that was further used for the synthesis of a multivalent LTB inhibitor. In the in vitro biological evaluation, this inhibitor exhibited strong inhibition properties as well as low cytotoxicity.     

Design and synthesis of a novel photoaffinity probe for labelling EGF receptor tyrosine kinases

The epidermal growth factor receptor (EGFR) and HER2 are two important tyrosine kinases that play crucial roles in signal transduction pathways that regulate numerous cellular functions including proliferation, differentiation, migration, and angiogenesis. In the past 20?years, many proteomic methods have emerged as powerful methods to evaluate proteins in biological processes and human disease states. Among them, activity-based protein profiling (ABPP) is one useful approach for the functional analysis of proteins. In this study, a novel photoaffinity probe 11 was designed and synthesised to assess the target profiling of the reactive group in the photoaffinity probe 11. Biological evaluation was performed, and the results showed that the novel photoaffinity probe binds to EGFR and HER2 proteins and it hits targets by the reactive group.