Translational studies of intravenous and intracerebroventricular routes of administration for CNS cellular biodistribution for BMN 250, an enzyme replacement therapy for the treatment of Sanfilippo type B.

BMN 250 is being developed as enzyme replacement therapy for Sanfilippo type B, a primarily neurological rare disease, in which patients have deficient lysosomal alpha-N-acetylglucosaminidase (NAGLU) enzyme activity. BMN 250 is taken up in target cells by the cation-independent mannose 6-phosphate receptor (CI-MPR, insulin-like growth factor 2 receptor), which then facilitates transit to the lysosome. BMN 250 is dosed directly into the central nervous system via the intracerebroventricular (ICV) route, and the objective of this work was to compare systemic intravenous (IV) and ICV delivery of BMN 250 to confirm the value of ICV dosing. We first assess the ability of enzyme to cross a potentially compromised blood-brain barrier in the Naglu-/- mouse model and then assess the potential for CI-MPR to be employed for receptor-mediated transport across the blood-brain barrier. In wild-type and Naglu-/- mice, CI-MPR expression in brain vasculature is high during the neonatal period but virtually absent by adolescence. In contrast, CI-MPR remains expressed through adolescence in non-affected non-human primate and human brain vasculature. Combined results from IV administration of BMN 250 in Naglu-/- mice and IV and ICV administration in healthy juvenile non-human primates suggest a limitation to therapeutic benefit from IV administration because enzyme distribution is restricted to brain vascular endothelial cells: enzyme does not reach target neuronal cells following IV administration, and pharmacological response following IV administration is likely restricted to clearance of substrate in endothelial cells. In contrast, ICV administration enables central nervous system enzyme replacement with biodistribution to target cells.

Evaluation of non-reducing end pathologic glycosaminoglycan detection method for monitoring therapeutic response to enzyme replacement therapy in human mucopolysaccharidosis I.

Therapeutic development and monitoring require demonstration of effects on disease phenotype. However, due to the complexity of measuring clinically-relevant effects in rare multisystem diseases, robust biomarkers are essential. For the mucopolysaccharidoses (MPS), the measurement of glycosaminoglycan levels is relevant as glycosaminoglycan accumulation is the primary event that occurs due to reduced lysosomal enzyme activity. Traditional dye-based assays that measure total glycosaminoglycan levels have a high background, due to a normal, baseline glycosaminoglycan content in unaffected individuals. An assay that selectively detects the disease-specific non-reducing ends of heparan sulfate glycosaminoglycans that remain undegraded due to deficiency of a specific enzyme in the catabolic pathway avoids the normal background, increasing sensitivity and specificity. We evaluated glycosaminoglycan content by dye-based and non-reducing end methods using urine, serum, and cerebrospinal fluid from MPS I human samples before and after treatment with intravenous recombinant human alpha-l-iduronidase. We found that both urine total glycosaminoglycans and serum heparan sulfate derived non-reducing end levels were markedly decreased compared to baseline after 26 weeks and 52 weeks of therapy, with a significantly greater percentage reduction in serum non-reducing end (89.8% at 26 weeks and 81.3% at 52 weeks) compared to urine total glycosaminoglycans (68.3% at 26 weeks and 62.4% at 52 weeks, p < 0.001). Unexpectedly, we also observed a decrease in non-reducing end levels in cerebrospinal fluid in all five subjects for whom samples were collected (mean 41.8% reduction, p = 0.01). The non-reducing ends in cerebrospinal fluid showed a positive correlation with serum non-reducing end levels in the subjects (r2 = 0.65, p = 0.005). Results suggest utility of the non-reducing end assay in evaluating a therapeutic response in MPS I.

Nigral overexpression of α-synuclein in a rat Parkinson's disease model indicates alterations in the enteric nervous system and the gut microbiome.

BACKGROUND: A hallmark feature of Parkinson's disease (PD) is the build-up of α-synuclein protein aggregates throughout the brain; however α-synuclein is also expressed in enteric neurons. Gastrointestinal (GI) symptoms and pathology are frequently reported in PD, including constipation, increased intestinal permeability, glial pathology, and alterations to gut microbiota composition. α-synuclein can propagate through neuronal systems but the site of origin of α-synuclein pathology, whether it be the gut or the brain, is still unknown. Physical exercise is associated with alleviating symptoms of PD and with altering the composition of the gut microbiota. METHODS: This study investigated the effects of bilateral nigral injection of adeno-associated virus (AAV)-α-synuclein on enteric neurons, glia and neurochemistry, the gut microbiome, and bile acid metabolism in rats, some of whom were exposed to voluntary exercise. KEY RESULTS: Nigral overexpression of α-synuclein resulted in significant neuronal loss in the ileal submucosal plexus with no change in enteric glia. In contrast, the myenteric plexus showed a significant increase in glial expression, while neuronal numbers were maintained. Concomitant alterations were observed in the gut microbiome and related bile acid metabolism. Voluntary running protected against neuronal loss, increased enteric glial expression, and modified gut microbiome composition in the brain-injected AAV-α-synuclein PD model. CONCLUSIONS AND INFERENCES: These results show that developing nigral α-synuclein pathology in this PD model exerts significant alterations on the enteric nervous system (ENS) and gut microbiome that are receptive to modification by exercise. This highlights brain to gut communication as an important mechanism in PD pathology.

Prebiotic supplementation in frail older people affects specific gut microbiota taxa but not global diversity.

BACKGROUND: There are complex interactions between aging, frailty, diet, and the gut microbiota; modulation of the gut microbiota by diet could lead to healthier aging. The purpose of this study was to test the effect of diets differing in sugar, fat, and fiber content upon the gut microbiota of mice humanized with microbiota from healthy or frail older people. We also performed a 6-month dietary fiber supplementation in three human cohorts representing three distinct life-stages. METHODS: Mice were colonized with human microbiota and then underwent an 8-week dietary intervention with either a high-fiber/low-fat diet typical of elderly community dwellers or a low-fiber/high-fat diet typical of long-stay residential care subjects. A cross-over design was used where the diets were switched after 4 weeks to the other diet type to identify responsive taxa and innate immunity changes. In the human intervention, the subjects supplemented their normal diet with a mix of five prebiotics (wheat dextrin, resistant starch, polydextrose, soluble corn fiber, and galactooligo-saccharide) at 10 g/day combined total, for healthy subjects and 20 g/day for frail subjects, or placebo (10 g/day maltodextrin) for 26 weeks. The gut microbiota was profiled and immune responses were assayed by T cell markers in mice, and serum cytokines in humans. RESULTS: Humanized mice maintained gut microbiota types reflecting the respective healthy or frail human donor. Changes in abundance of specific taxa occurred with the diet switch. In mice with the community type microbiota, the observed differences reflected compositions previously associated with higher frailty. The dominance of Prevotella present initially in community inoculated mice was replaced by Bacteroides, Alistipes, and Oscillibacter. Frail type microbiota showed a differential effect on innate immune markers in both conventional and germ-free mice, but a moderate number of taxonomic changes occurring upon diet switch with an increase in abundance of Parabacteroides, Blautia, Clostridium cluster IV, and Phascolarctobacterium. In the human intervention, prebiotic supplementation did not drive any global changes in alpha- or beta-diversity, but the abundance of certain bacterial taxa, particularly Ruminococcaceae (Clostridium cluster IV), Parabacteroides, Phascolarctobacterium, increased, and levels of the chemokine CXCL11 were significantly lower in the frail elderly group, but increased during the wash-out period. CONCLUSIONS: Switching to a nutritionally poorer diet has a profound effect on the microbiota in mouse models, with changes in the gut microbiota from healthy donors reflecting previously observed differences between elderly frail and non-frail individuals. However, the frailty-associated gut microbiota did not reciprocally switch to a younger healthy-subject like state, and supplementation with prebiotics was associated with fewer detected effects in humans than diet adjustment in animal models.

Changes in microbiota composition, bile and fatty acid metabolism, in successful faecal microbiota transplantation for Clostridioides difficile infection.

BACKGROUND: Alteration of the gut microbiota by repeated antibiotic treatment increases susceptibility to Clostridioides difficile infection. Faecal microbiota transplantation from donors with a normal microbiota effectively treats C. difficile infection. METHODS: The study involved 10 patients with recurrent C. difficile infection, nine of whom received transplants from individual donors and one who received a donor unit from a stool bank (OpenBiome). RESULTS: All individuals demonstrated enduring post-transplant resolution of C. difficile- associated diarrhoea. Faecal microbiota diversity of recipients significantly increased, and the composition of the microbiota resembled that of the donor. Patients with C. difficile infection exhibited significantly lower faecal levels of secondary/ bile acids and higher levels of primary bile acids. Levels of secondary bile acids were restored in all transplant recipients, but to a lower degree with the OpenBiome transplant. The abundance increased of bacterial genera known from previous studies to confer resistance to growth and germination of C. difficile. These were significantly negatively associated with primary bile acid levels and positively related with secondary bile acid levels. Although reduced levels of the short chain fatty acids, butyrate, propionate and acetate, have been previously reported, here we report elevations in SCFA, pyruvic and lactic fatty acids, saturated, ω-6, monounsaturated, ω-3 and ω-6 polyunsaturated fatty acids (PUFA) in C. difficile infection. This potentially indicates one or a combination of increased dietary FA intake, microbial modification of FAs or epithelial cell damage and inflammatory cell recruitment. No reversion to donor FA profile occurred post-FMT but ω-3 to ω-6 PUFA ratios were altered in the direction of the donor. Archaeal metabolism genes were found in some samples post FMT. CONCLUSION: A consistent metabolic signature was identified in the post-transplant microbiota, with reduced primary bile acids and substantial restoration of secondary bile acid production capacity. Total FA levels were unchanged but the ratio of inflammatory to non-inflammatory FAs decreased.

Towards standards for human fecal sample processing in metagenomic studies.

Technical variation in metagenomic analysis must be minimized to confidently assess the contributions of microbiota to human health. Here we tested 21 representative DNA extraction protocols on the same fecal samples and quantified differences in observed microbial community composition. We compared them with differences due to library preparation and sample storage, which we contrasted with observed biological variation within the same specimen or within an individual over time. We found that DNA extraction had the largest effect on the outcome of metagenomic analysis. To rank DNA extraction protocols, we considered resulting DNA quantity and quality, and we ascertained biases in estimates of community diversity and the ratio between Gram-positive and Gram-negative bacteria. We recommend a standardized DNA extraction method for human fecal samples, for which transferability across labs was established and which was further benchmarked using a mock community of known composition. Its adoption will improve comparability of human gut microbiome studies and facilitate meta-analyses.

Guanidinylated Neomycin Conjugation Enhances Intranasal Enzyme Replacement in the Brain.

Iduronidase (IDUA)-deficient mice accumulate glycosaminoglycans in cells and tissues and exhibit many of the same neuropathological symptoms of patients suffering from Mucopolysaccharidosis I. Intravenous enzyme-replacement therapy for Mucopolysaccharidosis I ameliorates glycosaminoglycan storage and many of the somatic aspects of the disease but fails to treat neurological symptoms due to poor transport across the blood-brain barrier. In this study, we examined the delivery of IDUA conjugated to guanidinoneomycin (GNeo), a molecular transporter. GNeo-IDUA and IDUA injected intravenously resulted in reduced hepatic glycosaminoglycan accumulation but had no effect in the brain due to fast clearance from the circulation. In contrast, intranasally administered GNeo-IDUA entered the brain rapidly. Repetitive intranasal treatment with GNeo-IDUA reduced glycosaminoglycan storage, lysosome size and number, and neurodegenerative astrogliosis in the olfactory bulb and primary somatosensory cortex, whereas IDUA was less effective. The enhanced efficacy of GNeo-IDUA was not the result of increased nose-to-brain delivery or enzyme stability, but rather due to more efficient uptake into neurons and astrocytes. GNeo conjugation also enhanced glycosaminoglycan clearance by intranasally delivered sulfamidase to the brain of sulfamidase-deficient mice, a model of Mucopolysaccharidosis IIIA. These findings suggest the general utility of the guanidinoglycoside-based delivery system for restoring missing lysosomal enzymes in the brain.

Effect of room temperature transport vials on DNA quality and phylogenetic composition of faecal microbiota of elderly adults and infants.

BACKGROUND: Alterations in intestinal microbiota have been correlated with a growing number of diseases. Investigating the faecal microbiota is widely used as a non-invasive and ethically simple proxy for intestinal biopsies. There is an urgent need for collection and transport media that would allow faecal sampling at distance from the processing laboratory, obviating the need for same-day DNA extraction recommended by previous studies of freezing and processing methods for stool. We compared the faecal bacterial DNA quality and apparent phylogenetic composition derived using a commercial kit for stool storage and transport (DNA Genotek OMNIgene GUT) with that of freshly extracted samples, 22 from infants and 20 from older adults. RESULTS: Use of the storage vials increased the quality of extracted bacterial DNA by reduction of DNA shearing. When infant and elderly datasets were examined separately, no differences in microbiota composition were observed due to storage. When the two datasets were combined, there was a difference according to a Wilcoxon test in the relative proportions of Faecalibacterium, Sporobacter, Clostridium XVIII, and Clostridium XlVa after 1 week's storage compared to immediately extracted samples. After 2 weeks' storage, Bacteroides abundance was also significantly different, showing an apparent increase from week 1 to week 2. The microbiota composition of infant samples was more affected than that of elderly samples by storage, with significantly higher Spearman distances between paired freshly extracted and stored samples (p < 0.001). When the microbiota profiles were analysed at the operational taxonomic unit (OTU) level, three infant datasets in the study did not cluster together, while only one elderly dataset did not. The lower microbiota diversity of the infant gut microbiota compared to the elderly gut microbiota (p < 0.001) means that any alteration in the infant datasets has a proportionally larger effect. CONCLUSIONS: The commercial storage vials appear to be suitable for high diversity microbiota samples, but may be less appropriate for lower diversity samples. Differences between fresh and stored samples mean that where storage is unavoidable, a consistent storage regime should be used. We would recommend extraction ideally within the first week of storage.

Delivery of an enzyme-IGFII fusion protein to the mouse brain is therapeutic for mucopolysaccharidosis type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB, Sanfilippo syndrome type B) is a lysosomal storage disease characterized by profound intellectual disability, dementia, and a lifespan of about two decades. The cause is mutation in the gene encoding α-N-acetylglucosaminidase (NAGLU), deficiency of NAGLU, and accumulation of heparan sulfate. Impediments to enzyme replacement therapy are the absence of mannose 6-phosphate on recombinant human NAGLU and the blood-brain barrier. To overcome the first impediment, a fusion protein of recombinant NAGLU and a fragment of insulin-like growth factor II (IGFII) was prepared for endocytosis by the mannose 6-phosphate/IGFII receptor. To bypass the blood-brain barrier, the fusion protein ("enzyme") in artificial cerebrospinal fluid ("vehicle") was administered intracerebroventricularly to the brain of adult MPS IIIB mice, four times over 2 wk. The brains were analyzed 1-28 d later and compared with brains of MPS IIIB mice that received vehicle alone or control (heterozygous) mice that received vehicle. There was marked uptake of the administered enzyme in many parts of the brain, where it persisted with a half-life of approximately 10 d. Heparan sulfate, and especially disease-specific heparan sulfate, was reduced to control level. A number of secondary accumulations in neurons [ß-hexosaminidase, LAMP1(lysosome-associated membrane protein 1), SCMAS (subunit c of mitochondrial ATP synthase), glypican 5, ß-amyloid, P-tau] were reduced almost to control level. CD68, a microglial protein, was reduced halfway. A large amount of enzyme also appeared in liver cells, where it reduced heparan sulfate and ß-hexosaminidase accumulation to control levels. These results suggest the feasibility of enzyme replacement therapy for MPS IIIB.

Glycan-based biomarkers for mucopolysaccharidoses.

The mucopolysaccharidoses (MPS) result from attenuation or loss of enzyme activities required for lysosomal degradation of the glycosaminoglycans, hyaluronan, heparan sulfate, chondroitin/dermatan sulfate, and keratan sulfate. This review provides a summary of glycan biomarkers that have been used to characterize animal models of MPS, for diagnosis of patients, and for monitoring therapy based on hematopoietic stem cell transplantation and enzyme replacement therapy. Recent advances have focused on the non-reducing terminus of the glycosaminoglycans that accumulate as biomarkers, using a combination of enzymatic digestion with bacterial enzymes followed by quantitative liquid chromatography/mass spectrometry. These new methods provide a simple, rapid diagnostic strategy that can be applied to samples of urine, blood, cerebrospinal fluid, cultured cells and dried blood spots from newborn infants. Analysis of the non-reducing end glycans provides a method for monitoring enzyme replacement and substrate reduction therapies and serves as a discovery tool for uncovering novel biomarkers and new forms of mucopolysaccharidoses.

Enzyme augmentation therapy enhances the therapeutic efficacy of bone marrow transplantation in mucopolysaccharidosis type II mice.

Before the availability of an enzyme replacement therapy (ERT) for mucopolysaccharidosis type II (MPS II), patients were treated by bone marrow transplantation (BMT). However, the effectiveness of BMT for MPS II was equivocal, particularly at addressing the CNS manifestations. To study this further, we subjected a murine model of MPS II to BMT and evaluated the effect at correcting the biochemical and pathological aberrations in the viscera and CNS. Our results indicated that BMT reduced the accumulation of glycosaminoglycans (GAGs) in a variety of visceral organs, but not in the CNS. With the availability of an approved ERT for MPS II, we investigated and compared the relative merits of the two strategies either as a mono or combination therapy. We showed that the combination of BMT and ERT was additive at reducing tissue levels of GAGs in the heart, kidney and lung. Moreover, ERT conferred greater efficacy if the immunological response against the infused recombinant enzyme was low. Finally, we showed that pathologic GAGs might potentially represent a sensitive biomarker to monitor the therapeutic efficacy of therapies for MPS II.

WITHDRAWN: Glycan-based biomarkers for mucopolysaccharidoses.

Ahead of Print article withdrawn by publisher. At request of the authors, this article will be published in the journal Cancer Biomarkers (ISSN 1574-0153).

Specific antibody titer alters the effectiveness of intrathecal enzyme replacement therapy in canine mucopolysaccharidosis I.

Intrathecal enzyme replacement therapy is an experimental option to treat central nervous system disease due to lysosomal storage. Previous work shows that MPS I dogs receiving enzyme replacement with recombinant human alpha-l-iduronidase into the cisterna magna showed normal brain glycosaminoglycan (GAG) storage after three or four doses. We analyzed MPS I dogs that received intrathecal enzyme in a previous study using an assay that detects only pathologic GAG (pGAG). To quantify pGAG in MPS I, the assay measures only those GAG which display terminal iduronic acid residues on their non-reducing ends. Mean cortical brain pGAG in six untreated MPS I dogs was 60.9±5.93 pmol/mg wet weight, and was 3.83±2.64 in eight normal or unaffected carrier animals (p<0.001). Intrathecal enzyme replacement significantly reduced pGAG storage in all treated animals. Dogs with low anti-iduronidase antibody titers showed normalization or near-normalization of pGAG in the brain (mean 8.17±6.17, n=7), while in dogs with higher titers, pGAG was reduced but not normal (mean 21.9±6.02, n=4). Intrathecal enzyme therapy also led to a mean 69% reduction in cerebrospinal fluid pGAG (from 83.8±26.3 to 27.2±12.3 pmol/ml CSF). The effect was measurable one month after each dose and did not differ with antibody titer. Prevention of the immune response to enzyme may improve the efficacy of intrathecal enzyme replacement therapy for brain disease due to MPS I.

Disease-specific non-reducing end carbohydrate biomarkers for mucopolysaccharidoses.

A considerable need exists for improved biomarkers for differential diagnosis, prognosis and monitoring of therapeutic interventions for mucopolysaccharidoses (MPS), inherited metabolic disorders that involve lysosomal storage of glycosaminoglycans. Here we report a simple, reliable method based on the detection of abundant nonreducing ends of the glycosaminoglycans that accumulate in cells, blood and urine of individuals with MPS. In this method, glycosaminoglycans are enzymatically depolymerized, releasing unique mono-, di- or trisaccharides from the nonreducing ends of the chains. The composition of the released mono- and oligosaccharides depends on the nature of the lysosomal enzyme deficiency, and therefore they serve as diagnostic biomarkers. Analysis by LC/MS allowed qualitative and quantitative assessment of the biomarkers in biological samples. We provide a simple conceptual scheme for diagnosing MPS in uncharacterized samples and a method to monitor efficacy of enzyme replacement therapy or other forms of treatment.

Genistein improves neuropathology and corrects behaviour in a mouse model of neurodegenerative metabolic disease.

BACKGROUND: Neurodegenerative metabolic disorders such as mucopolysaccharidosis IIIB (MPSIIIB or Sanfilippo disease) accumulate undegraded substrates in the brain and are often unresponsive to enzyme replacement treatments due to the impermeability of the blood brain barrier to enzyme. MPSIIIB is characterised by behavioural difficulties, cognitive and later motor decline, with death in the second decade of life. Most of these neurodegenerative lysosomal storage diseases lack effective treatments. We recently described significant reductions of accumulated heparan sulphate substrate in liver of a mouse model of MPSIIIB using the tyrosine kinase inhibitor genistein. METHODOLOGY/PRINCIPAL FINDINGS: We report here that high doses of genistein aglycone, given continuously over a 9 month period to MPSIIIB mice, significantly reduce lysosomal storage, heparan sulphate substrate and neuroinflammation in the cerebral cortex and hippocampus, resulting in correction of the behavioural defects observed. Improvements in synaptic vesicle protein expression and secondary storage in the cerebral cortex were also observed. CONCLUSIONS/SIGNIFICANCE: Genistein may prove useful as a substrate reduction agent to delay clinical onset of MPSIIIB and, due to its multimodal action, may provide a treatment adjunct for several other neurodegenerative metabolic diseases.

Heparan sulfate 2-O-sulfotransferase is required for triglyceride-rich lipoprotein clearance.

Hepatic clearance of triglyceride-rich lipoproteins depends on heparan sulfate and low density lipoprotein receptors expressed on the basal membrane of hepatocytes. Binding and uptake of the lipoproteins by way of heparan sulfate depends on the degree of sulfation of the chains based on accumulation of plasma triglycerides and delayed clearance of triglyceride-rich lipoproteins in mice bearing a hepatocyte-specific alteration of N-acetylglucosamine (GlcNAc) N-deacetylase-N-sulfotransferase 1 (Ndst1) (MacArthur, J. M., Bishop, J. R., Stanford, K. I., Wang, L., Bensadoun, A., Witztum, J. L., and Esko, J. D. (2007) J. Clin. Invest. 117, 153-164). Inactivation of Ndst1 led to decreased overall sulfation of heparan sulfate due to coupling of uronyl 2-O-sulfation and glucosaminyl 6-O-sulfation to initial N-deacetylation and N-sulfation of GlcNAc residues. To determine whether lipoprotein clearance depends on 2-O-and 6-O-sulfation, we evaluated plasma triglyceride levels in mice containing loxP-flanked conditional alleles of uronyl 2-O-sulfotransferase (Hs2st(f/f)) and glucosaminyl 6-O-sulfotransferase-1 (Hs6st1(f/f)) and the bacterial Cre recombinase expressed in hepatocytes from the rat albumin (Alb) promoter. We show that Hs2st(f/f)AlbCre(+) mice accumulated plasma triglycerides and exhibited delayed clearance of intestinally derived chylomicrons and injected human very low density lipoproteins to the same extent as observed in Ndst1(f/f)AlbCre(+) mice. In contrast, Hs6st1(f/f)AlbCre(+) mice did not exhibit any changes in plasma triglycerides. Chemically modified heparins lacking N-sulfate and 2-O-sulfate groups did not block very low density lipoprotein binding and uptake in isolated hepatocytes, whereas heparin lacking 6-O-sulfate groups was as active as unaltered heparin. Our findings show that plasma lipoprotein clearance depends on specific subclasses of sulfate groups and not on overall charge of the chains.

Deoxygenated disaccharide analogs as specific inhibitors of beta1-4-galactosyltransferase 1 and selectin-mediated tumor metastasis.

The disaccharide peracetylated GlcNAcbeta1-3Galbeta-O-naphthalenemethanol (disaccharide 1) diminishes the formation of the glycan sialyl Lewis X (Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3) GlcNAc; sLe(X)) in tumor cells. Previous studies showed that the mechanism of action of disaccharide 1 involves three steps: (i) deacetylation by carboxyesterases, (ii) action as a biosynthetic intermediate for downstream enzymes involved in sLe(X) assembly, and (iii) generation of several glycans related to sLe(X). In this report, we show that GlcNAcbeta1-3Galbeta-O-naphthalenemethanol binds to the acceptor site of human beta1-4-galactosyltransferase much like the acceptor trisaccharide, GlcNAcbeta1-2Manbeta1-6Man, which is present on N-linked glycans. The 4'-deoxy analog, in which the acceptor hydroxyl group was replaced by -H, did not act as a substrate but instead acted as a competitive inhibitor of the enzyme. The acetylated form of this compound inhibited sLe(X) formation in U937 monocytic leukemia cells, suggesting that it had inhibitory activity in vivo as well. A series of synthetic acetylated analogs of 1 containing -H, -F, -N(3), -NH(2), or -OCH(3) instead of the hydroxyl groups at C-3'- and C-4'-positions of the terminal N-acetylglucosamine residue also blocked sLe(X) formation in cells. The reduction of sLe(X) by the 4'-deoxy analog also diminished experimental tumor metastasis by Lewis lung carcinoma in vivo. These data suggest that nonsubstrate disaccharides have therapeutic potential through their ability to bind to glycosyltransferases in vivo and to alter glycan-dependent pathologic processes.

Surfen, a small molecule antagonist of heparan sulfate.

In a search for small molecule antagonists of heparan sulfate, we examined the activity of bis-2-methyl-4-amino-quinolyl-6-carbamide, also known as surfen. Fluorescence-based titrations indicated that surfen bound to glycosaminoglycans, and the extent of binding increased according to charge density in the order heparin > dermatan sulfate > heparan sulfate > chondroitin sulfate. All charged groups in heparin (N-sulfates, O-sulfates, and carboxyl groups) contributed to binding, consistent with the idea that surfen interacted electrostatically. Surfen neutralized the anticoagulant activity of both unfractionated and low molecular weight heparins and inhibited enzymatic sulfation and degradation reactions in vitro. Addition of surfen to cultured cells blocked FGF2-binding and signaling that depended on cell surface heparan sulfate and prevented both FGF2- and VEGF(165)-mediated sprouting of endothelial cells in Matrigel. Surfen also blocked heparan sulfate-mediated cell adhesion to the Hep-II domain of fibronectin and prevented infection by HSV-1 that depended on glycoprotein D interaction with heparan sulfate. These findings demonstrate the feasibility of identifying small molecule antagonists of heparan sulfate and raise the possibility of developing pharmacological agents to treat disorders that involve glycosaminoglycan-protein interactions.

Glycan antagonists and inhibitors: a fount for drug discovery.

Glycans, the carbohydrate chains of glycoproteins, proteoglycans, and glycolipids, represent a relatively unexploited area for drug development compared with other macromolecules. This review describes the major classes of glycans synthesized by animal cells, their mode of assembly, and available inhibitors for blocking their biosynthesis and function. Many of these agents have proven useful for studying the biological activities of glycans in isolated cells, during embryological development, and in physiology. Some are being used to develop drugs for treating metabolic disorders, cancer, and infection, suggesting that glycans are excellent targets for future drug development.

Environmental source of Candida dubliniensis.

We isolated Candida dubliniensis from a nonhuman source, namely, tick samples from an Irish seabird colony. The species was unambiguously identifi ed by phenotypic and genotypic means. Analysis of the 5.8S rRNA gene showed that the environmental isolates belong to C. dubliniensis genotype 1.