TARGETING GLYCOSYLATION AS A THERAPEUTIC APPROACH

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1 TARGETING GLYCOSYLATION AS A THERAPEUTIC APPROACH Raymond A. Dwek, Terry D. Butters, Frances M. Platt and Nicole Zitzmann Increased understanding of the role of protein- and lipid-linked carbohydrates in a wide range of biological processes has led to interest in drugs that target the enzymes involved in glycosylation. But given the importance of carbohydrates in fundamental cellular processes such as protein folding, therapeutic strategies that modulate, rather than ablate, the activity of enzymes involved in glycosylation are likely to be a necessity. Two such approaches that use imino sugars to affect glycosylation enzymes now show considerable promise in the treatment of viral infections, such as hepatitis B, and glucosphingolipid storage disorders, such as Gaucher disease. GLYCAN A polymer consisting of monosaccharides linked together by glycosidic bonds. ENDOPLASMIC RETICULUM Membrane-bounded compartment in the cytoplasm of eukaryotic cells, in which lipids, membrane-bound and secreted proteins are synthesized. GOLGI APPARATUS Membrane-bounded organelle in eukaryotic cells, in which lipids and proteins made in the endoplasmic reticulum are modified and sorted. Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Correspondence to R.A.D. rad@bioch.ox.ac.uk DOI: /nrd708 Current estimates indicate that genes that regulate N- and O-glycosylation of glycoproteins make up 1 2% of the human genome. In the case of N-linked GLYCANS, more than 30 enzymes, located in the cytosol, the ENDOPLASMIC RETICULUM (ER) and the GOLGI APPARATUS, are required to generate, attach and process the oligosaccharides (FIG. 1). Many functions have been described for protein glycosylation, including promoting protein folding in the ER 1, stabilizing cell-surface glycoproteins 2, and providing recognition epitopes that activate the innate immune system 3. It is therefore not surprising that genetic mutations that decrease or eliminate the activity of GLYCOSYLTRANSFERASES and GLYCOSIDASES can lead to serious physiological disorders and can be lethal in animals as well as in humans 4. Glycoproteins generally exist as populations of glycosylated variants of a single polypeptide, known as glycoforms 5. The attachment and processing of sugars is not random, but exquisitely controlled by both the cell and the three-dimensional structure of the protein itself. Changes in protein glycosylation are early indicators of cellular changes in many diseases, most notably cancer 6 and rheumatoid arthritis 7, providing useful diagnostic markers and insights into disease progression and pathogenesis. Important physiological functions have also been established for glycosylated sphingolipids, which form two distinct families: the glucosphingolipids and the galactosphingolipids. Here, we will focus exclusively on the glucosphingolipids (GSLs), which are synthesized from the lipid ceramide by several carbohydrate-processing enzymes. GSLs are present on the plasma membranes of all mammalian cells, and are essential during embryonic development and differentiation 8. They are also exploited as receptors by some bacteria and viruses. Gangliosides a subclass of GSLs that contain sialic acid are particularly enriched in neuronal tissues and are important for proper nervous-system function. Maintenance of a balanced population of GSLs on cell membranes requires stringent control of GSL biosynthesis and degradation. However, in GSL storage diseases such as Tay Sachs disease, defects in the degradation pathway result in the accumulation of GSLs in cells, particularly in neurons, which causes neurodegeneration and a shortened lifespan 9. When assessing the potential of drugs that target protein and lipid glycosylation, the overriding consideration must be the importance of host glycosylation. It is only realistic to use drugs that completely ablate the activity of carbohydrate-processing enzymes in cases in which the target enzyme has no mammalian counterpart; for example, pathogen-specific enzymes involved in the synthesis of the carbohydrate components of bacterial and fungal cell walls. Otherwise, specific strategies that do not involve totally eliminating enzyme activity are necessary. Here, we describe two NATURE REVIEWS DRUG DISCOVERY VOLUME 1 JANUARY

2 Glucose Mannose 1-P DPP-GlcNAc 2 Man DPP-GlcNAc 2 Man 9 Glc 3 Oligosaccharide transfer ER α-mannosidase ER α-glucosidase II ER α-glucosidase II ER α-glucosidase I Golgi α-mannosidase I Golgi endomannosidase Golgi α-mannosidase I N-acetylglucosaminyltransferase I GlcNAc Man Golgi α-mannosidase II N-acetylglucosaminyltransferase II Further glycosyltransferasecatalysed reactions Mature glycoproteins Glc Figure 1 Biosynthesis of glycoproteins that possess N-linked glycans. As the nascent glycoprotein enters the endoplasmic reticulum (ER), a preformed oligosaccharide known as the dolicol-phosphate precursor (DPP) is attached co-translationally to some Asn residues that are part of the consensus sequence Asn-Xaa-Ser/Thr. The biosynthesis of this precursor, its attachment to Asn residues and the subsequent steps of its processing in the ER and the Golgi, are performed by a series of glycosidases and glycosyltransferases. Asn, asparagine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose; P, phosphate; Ser, serine; Thr, threonine; Xaa, any amino acid. GLYCOSYLTRANSFERASE Glycosyltransferases produce glycosidic bonds by transferring a glycosyl group any group formed by detaching the glycosidic hydroxyl group from the cyclic form of a monosaccharide, oligosaccharide or derivatives. GLYCOSIDASE An enzyme that hydrolyses glycosidic bonds. LYSOSOME Membrane-bounded organelle in eukaryotic cells responsible for controlled intracellular digestion of macromolecules. Lysosomes contain a wide range of hydrolytic enzymes, including glycosidases. CHARGE-TRANSITION-STATE ANALOGUES A structural mimic of the transition state between reactant(s) and product(s) for a given reaction. Transition-state analogues make good inhibitors because they are bound to the enzyme more tightly than the substrates. such approaches that have been used successfully: the first interferes with the life cycle of several viruses, such as hepatitis B virus (HBV), and the second compensates for inherited genetic defects in enzymes of the LYSOSOMAL degradation pathway for GSLs. The drugs that have been evaluated for these diseases are a family of sugar mimetics termed imino sugars and are the focus of this review. Imino-sugar inhibitors The imino-sugar family of compounds, many of which occur naturally in certain plants and microorganisms, have had several important uses for glycobiologists 10, and more recently their activities have been exploited in the potential treatment of human diseases. Imino sugars are monosaccharide mimics, with a nitrogen atom in place of the ring oxygen (TABLE 1). They act as CHARGE-TRANSITION-STATE ANALOGUES and are submicromolarrange inhibitors of many hydrolytic enzymes, including ER α-glucosidases I and II, which are involved in processing glycoproteins 11,12 (FIG. 1). The nitrogen atom provides a further point for modification, and simple alkylation of imino-sugar glucose or galactose analogues confers unexpected activities for other enzyme targets, such as the pathway that is mediated by ceramide-specific glucosyltransferase (CerGlcT; UDP-glucose: N-acylsphingosine D-glucosyltransferase), which is crucial for GSL biosynthesis 11 (FIG. 2). Imino sugars in metabolic control The potent enzyme-inhibitory activity of imino sugars has been applied to diseases in which the control of oligosaccharide metabolism is linked to cellular dysfunction 13. The successful treatment of non-insulindependent diabetes with the imino sugar N-hydroxyethyldeoxynojirimycin (Miglitol) depends on partial inhibition of intestinal disaccharidases to reduce the level of postprandial glucose 14. Another use of imino sugars in drug development for metabolic control is in the modification of N-linked oligosaccharides on cell-surface proteins to reduce tumour-cell metastasis 15. Imino sugars with antiviral activity The proper folding and controlled assembly of many nascent glycoproteins depends on interactions with chaperones, such as calnexin, in the ER (FIG. 3). Stepwise removal by ER α-glucosidases of terminal glucose residues from N-glycan chains attached to nascent glycoproteins enables the glycoproteins to interact with the chaperones calnexin and calreticulin, which bind exclusively to monoglucosylated glycoproteins 16,17 (FIGS 1 and 3). Interaction with calnexin is crucial for the correct folding of some, but not all, glycoproteins, and inhibitors of the ER α-glucosidases can be used specifically to target proteins that depend on this interaction 18. Mammalian viruses are not known to encode their own carbohydrate-modifying enzymes; they use the 66 JANUARY 2002 VOLUME 1

3 ENVELOPE A lipoprotein-bilayer outer membrane of many viruses. Envelope proteins are often heavily glycosylated. host-cell glycosylation machinery to modify their ENVELOPE proteins. As with some cellular host proteins, the folding of certain viral glycoproteins has been shown to be calnexin-dependent So, targeting the ER α-glucosidases at a low level could disrupt the folding of these proteins, and potentially be of therapeutic use in treating viral infections, without affecting host-cell viability. Indeed, inhibitors of the N-linked glycosylation pathway (FIG. 1) have been widely tested for antiviral activity (TABLE 2). In general, inhibitors of ER α-glucosidases I and II might inhibit the replication of some viruses, whereas inhibitors of Golgi α-mannosidases I and II rarely have any effect. However, it is difficult to generalize on the effectiveness of ER α-glucosidase inhibitors. For example, the replication of certain viruses, such as human immunodeficiency virus (HIV), Moloney murine leukaemia virus, mouse hepatitis virus, HBV, cytomegalovirus and Sindbis virus, is greatly inhibited in vitro, whereas the replication of viruses such as Rous Sarcoma virus, influenza A virus and Semliki forest virus is not affected. As suggested above, inhibition of viral replication by ER α-glucosidase inhibitors is likely to be explained by the retention of glucosylated precursor oligosaccharides on the viral glycoproteins, which if these proteins depend on the folding pathway mediated by calnexin/calreticulin leads to their misfolding. Perhaps the most important long-term advantage of this approach is that, as the enzymes are host-cell- and not virus-encoded, emergence of drug-resistant viruses is less likely to occur. No general rules can be made about the effects the change in N-glycan composition of envelope glycoproteins or their (partial) misfolding can have on the viral life cycle. Misfolding, especially if restricted to only small areas of the protein, might not necessarily lead to degradation. For many viruses, it results in impairment of the maturation of envelope precursor proteins 24 28, which, in turn, can lead to a decrease in viral assembly and release 26,29 and/or viral infectivity 24.However,for other viruses, effects on viral assembly and infectivity are independent of envelope-precursor cleavage 22,30. Three examples that highlight the processing of N-linked glycans as a target for antiviral intervention are the HIV model, the HBV model, and bovine viral diarrhoea virus (BVDV), a model organism for the human hepatitis C virus (HCV). HIV. HIV, the causative agent of AIDS, encodes two envelope glycoproteins (gp120 and gp41), which are derived from a precursor protein (gp160) by endoproteolytic Table 1 The imino-sugar family Imino sugar Compound name Site of action Therapeutic use/ Clinical status potential N (CH 2 ) 2 N CH 2 (CH 2 ) 3 CH 3 CH 2 N-hydroxyethyl-DNJ Intestinal Non-insulin-dependent Approved since 1996 (Glyset, Miglitol) disaccharidases diabetes (Bayer Group) N-butyl-DNJ CerGlcT GSL lysosomal Submitted for NDA (OGT918) disorders 68 for Gaucher disease (OGS) ER α-glucosidase I Hepatitis B 39 N CH 2 N-butyl-DGJ CerGlcT GSL lysosomal Preclinical/ (OGT923) disorders 46 development (OGS) Gangliosidoses (CH 2 ) 3 CH 3 N CH 2 (CH 2 ) 8 CH 3 N-nonyl-DNJ CerGlcT GSL lysosomal disorders Preclinical/prototype ER α-glucosidase I Hepatitis B 47 Hepatitis C surrogate 30 N CH 2 N-nonyl-DGJ CerGlcT GSL lysosomal disorders Preclinical/prototype Not determined Hepatitis B 47 Hepatitis C surrogate 30 (CH 2 ) 8 CH 3 N CH 3 (CH 2 ) 7 OCH 2 CH 3 N-7-oxanonyl-6- Not an enzyme Hepatitis C surrogate 30 Preclinical Me-DGJ inhibitor development/ind (Synergy Pharmaceuticals) CerGIcT, ceramide-specific glucosyltransferase; DGJ, deoxygalactonojirimycin; DNJ, deoxynojirimycin; GSL, glucosphingolipid; IND, investigational new drug application; NDA, new drug application; OGS, Oxford GlycoSciences. NATURE REVIEWS DRUG DISCOVERY VOLUME 1 JANUARY

4 NB-DNJ NB-DGJ NN-DNJ NN-DGJ UDP-glucose O HN Ganglio-series UDP O O HN L-serine, palmitoyl-coenzyme A O Ceramide Glucosylceramide Lactosylceramide Lacto (neo)-series UDP-glucose: N-acylsphingosine D-glucosyltransferase (CerGlcT) Globo-series Figure 2 Biosynthesis of glucosphingolipids. The first committed step in glucosphingolipid biosynthesis is the transfer of glucose to ceramide by the ceramide-specific glucosyltransferase (CerGlcT; UDP-glucose: N-acylsphingosine D-glucosyltransferase). This step is inhibited by the imino sugars N-butyldeoxynojirimycin (NB-DNJ) and N-nonyldeoxynojirimycin (NN-DNJ), and their galactose analogues N-butyldeoxygalactonojirimycin (NB-DGJ) and N-nonyldeoxygalactonojirimycin (NN-DGJ). SYNCYTIUM A mass of cytoplasm containing several separate nuclei enclosed in a continuous membrane resulting from the fusion of individual cells. V1/V2 LOOP The gp120 protein has eleven defined loop segments, five of which are termed variable (designated V1 V5). The tip of one of the six non-variable loops forms a β-hairpin that hydrogen bonds with two parallel strands from the V1/V2 loop to form a β-sheet that effectively connects the inner and outer domains. VIRAEMIA The presence of viruses in the blood. VIRION A mature infectious virus particle. cleavage in the cis-golgi 31. Although proteolytically cleaved, gp120 remains non-covalently attached to the transmembrane protein gp41, which serves as an anchor for the complex. During infection, gp120, which is fully exposed on the outer face of the viral envelope, binds to its cellular receptor (CD4), and undergoes a conformational change that exposes gp41. This, in turn, allows fusion with the cellular membrane and entry of the virus into the cell. With 30 potential N-glycan sites between them, gp41 and gp120 are among the most heavily N-glycosylated viral proteins known. They normally carry a mixture of oligomannose and complex glycans. Treatment of HIV-1 with N-butyldeoxynojirimycin (NB-DNJ), an ER α-glucosidase inhibitor (TABLE 1), suppresses viral infectivity and SYNCYTIUM formation in vitro 32. By contrast, treatment with deoxymannojirimycin (DMJ), a Golgi mannosidase inhibitor (FIG. 1), has no effect on the secretion of infectious virus 33, emphasizing the need of the virus for ER α-glucosidase processing and interaction with calnexin and/or calreticulin. The reduction in viral infectivity caused by NB-DNJ is the result of impairments in post-cd4 binding steps 32,34. Although binding to CD4 occurs, the conformational shift of gp120 that results in exposure of gp41 does not occur, and the process of viral fusion is therefore prevented. Consistent with this is the finding that, in the presence of NB-DNJ, there is a regional misfolding in the V1/V2 LOOP of gp120 (REF. 35). This structural change in gp120 does not prevent the transport of the protein to the plasma membrane or viral budding, but it is sufficient to inhibit viral fusion, a crucial step in the HIV life cycle. NB-DNJ was evaluated in Phase II clinical trials as an anti-hiv agent, but it was not possible to achieve sufficiently high serum concentrations of the drug 36,37, and no major impact was observed on VIRAEMIA 37.However, important information was gained in the trials. NB-DNJ was well tolerated, with the main side effect being gastrointestinal-tract distress due to intestinal disaccharidase inhibition, resulting in osmotic diarrhoea. HBV. HBV infects over 350 million people worldwide and can cause liver disease and hepatocellular carcinoma 38. The HBV genome encodes three envelope proteins: large (L), middle (M) and small (S), which are derived from a single open reading frame by use of alternative translational start sites. In addition to forming the main component of the viral envelope, these proteins are also secreted in the form of DNA-free non-infectious subviral particles, which can outnumber VIRIONS a million to one. In contrast to the HIV envelope glycoproteins, HBV envelope M proteins contain only two glycosylation sites. However, just as with HIV, HBV is sensitive to inhibitors of the ER α-glucosidases. The role of the N-glycans of HBV has been probed using inhibitors of the N-glycosylation pathway (tunicamycin, NB-DNJ and DMJ) 29,39 and site-directed mutagenesis. In an HBV-secreting cell line (HepG2.2.15), neither ER α-glucosidase processing nor interaction with calnexin/calreticulin is required for the correct folding of the S and L proteins 40. Even in the presence of NB-DNJ, subviral particles containing S and L proteins are secreted with a full array of complex glycan structures that have been processed through the Golgi endomannosidase pathway (in the presence of the glucosidase blockade, the Golgi endomannosidase removes all three glucose residues in one step 41 ; FIG. 1). However, the M protein crucially depends on calnexin interaction for proper folding. If this interaction is prevented, the M protein does not fold correctly and formation of the viral envelope is prevented. There is no efficient infectivity assay available for HBV at present. However, the woodchuck chronically infected at birth with woodchuck hepatitis virus (WHV), a close relative of HBV, is recognized as a good animal model to test potential antiviral treatments for the human disease. When chronically infected woodchucks were treated with N-nonyl-DNJ (NN-DNJ), a nine-carbon alkyl derivative of DNJ (TABLE 1) that has been shown in cell-based assays to be times more potent at inhibiting HBV secretion than NB-DNJ, virus levels were reduced in a dose-dependent manner 42. Interestingly, at NN-DNJ concentrations sufficient to prevent WHV secretion, the glycosylation of most serum glycoproteins seemed to be unaffected, indicating 68 JANUARY 2002 VOLUME 1

5 PLAQUE-REDUCTION ASSAY Virus-induced cell death causes a plaque in a cell monolayer. Plaques can be counted to indicate how much virus is present. MULTIPLICITY OF INFECTION (MOI). The (average) number of virus particles that infect each cell in an experiment. Glc Man P α-glucosidase I & II NB-DNJ NN-DNJ Glucosyltransferase that this class of therapeutics might be selective against HBV. Alternatively, a mechanism other than, and in addition to, ER α-glucosidase inhibition might be responsible in part for the antiviral effect observed. This additional mechanism, which seems to be associated with the length of the alkyl side chain attached to the iminosugar headgroup of the inhibitor, was described in detail for another viral system, BVDV. BVDV. Because of its similarity to HCV and the fact that there is no efficient cell-culture system available to support HCV replication, BVDV 43 has been used as a model organism in infectivity assays for the screening of potential anti-hcv drugs. Most BVDV and HCV proteins are functionally homologous. The envelope glycoproteins E1 and E2 interact either non-covalently (HCV) 44 or through disulphide bonds (BVDV) 22 to form a dimer, which has been proposed to be the functional complex present on the surface of mature virions. The envelope glycoproteins of both HCV and BVDV interact with calnexin during productive folding 22,23. NB-DNJ and NN-DNJ have an antiviral effect against BVDV in vitro, with the long alkyl-chain derivative NN-DNJ being at least tenfold more potent than NB-DNJ 30,45. NB-DNJ and NN-DNJ have at least three features that might be involved in their antiviral activity: they are inhibitors not only of the ER α-glucosidases, but also of CerGlcT, which catalyses the first committed step in Endoplasmic reticulum P P Other chaperones G II α-glucosidase II P NB-DNJ NN-DNJ Cx Protein folding on/off calnexin P Golgi Figure 3 The role of glycosylation in protein folding. The folding and assembly of many newly synthesized glycoproteins depends on interactions with chaperones. Processing of the attached common oligosaccharide precursor GlcNAc 2 Man 9 Glc 3 (which is N-linked; see FIG. 1) in the endoplasmic reticulum (ER) by α-glucosidases I and II gives GlcNAc 2 Man 9 Glc, which can bind to chaperones such as calnexin (Cx). Chaperones act as quality-control factors by retaining glycoproteins in the ER until they are correctly folded. Inhibition of the α-glucosidases by the imino sugars N-butyldeoxynojirimycin (NB-DNJ) and N-nonyldeoxynojirimycin (NN-DNJ) can interfere with this process, leading to misfolded proteins. G II, ER α-glucosidase II; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose; P, protein. glucosphingolipid biosynthesis (FIG. 2). In addition, the alkyl side chains might influence their antiviral behaviour (TABLE 1). For BVDV, these different activities have been investigated in more detail than for the other viral systems outlined above. When N-butyldeoxygalactonojirimycin (NB-DGJ; the galactose analogue of NB-DNJ), an inhibitor that targets only CerGlcT (not ER α-glucosidases I and II), was used in PLAQUE-REDUCTION ASSAYS, no effect was observed on BVDV plaque formation, even at concentrations high enough to completely inhibit the enzyme 45.As NB-DNJ and NB-DGJ carry the same alkyl side chain, this implies that the antiviral effect observed using NB-DNJ can be attributed to the inhibition of the ER α-glucosidases involved in N-glycan processing, not the inhibition of GSL synthesis. This result was confirmed by experiments showing that, in the presence of NB-DNJ, the interaction of BVDV E1 and E2 with calnexin is prevented, which leads to the misfolding of the envelope glycoproteins and inefficient formation of the E1 E2 heterodimers. For NB-DNJ, the degree of this effect correlates with the dose-dependent antiviral effect observed 22. However, for the long alkyl-chain compound NN-DNJ, the situation is not as straightforward. There is a lack of correlation between the ability of long alkylchain DNJ derivatives to inhibit ER α-glucosidases and their antiviral effect against BVDV, ruling out ER α-glucosidase inhibition as the sole antiviral mechanism responsible. For example, the long alkyl-chain compound NN-DGJ, which is not an ER α-glucosidase inhibitor, is just as effective against BVDV as NN-DNJ when a low MULTIPLICITY OF INFECTION (MOI) is used in the plaque-reduction assays, with NN-DNJ showing superiority only at higher MOIs 30. These results hint at an entirely new mechanism by which imino-sugar derivatives carrying longer alkyl side chains might exert their antiviral effect. Using short and long alkyl-chain DNJ- and DGJ-derivatives, the possibility that this mechanism could be acting at the level of replication, protein synthesis or protein processing was ruled out 30. Long alkyl-chain derivatives induce an increase in the accumulation of E2 E2 dimers in the ER, and these homodimers are subsequently also enriched in secreted virus particles; further investigations will show whether this causes or reflects the antiviral mechanism. NN-DNJ caused a reduction in viral secretion (probably due to misfolding of viral envelope glycoproteins caused by ER α-glucosidase inhibition), as well as a reduction in the infectivity of newly released virions. NN-DGJ exerted its antiviral effect solely through the production of particles with reduced infectivity 30.The fact that NN-DNJ combines both activities could explain its superiority at higher MOIs. However, these inhibitors are being investigated as potential antiviral drugs against hepatitis C, a chronic disease that is not usually characterized by high viral titres in the blood (although it is not possible to estimate local virus concentrations; for example, in the liver). In addition, patients might require long-term treatment, in which case a DGJ-based compound, which avoids some of the known side effects NATURE REVIEWS DRUG DISCOVERY VOLUME 1 JANUARY

6 Table 2 N-glycosylation inhibitors as potential antivirals Virus family Examples of viruses tested References Retroviruses Human immunodeficiency virus 71 Moloney murine leukaemia virus 71,72 Hepadnaviruses Hepatitis B virus 28,38 Coronaviruses Murine hepatitis virus 23 Herpesviruses Herpes simplex virus type 1 73 Herpes simplex virus type 2 74 Cytomegalovirus 75,76 Alphaviruses Sindbis virus 24,77,78 Semliki forest virus 79,80 Rhabdoviruses Vesicular stomatitis virus 81 Orthomyxoviruses Influenza A virus 77 Paramyxoviruses Measles virus 82 Flaviviruses Dengue virus 83 Japanese encephalitis 84 Pestiviruses Bovine viral diarrhoea virus 22,45 Arenaviruses Lymphocytic choriomeningitis virus 85 Junin virus 86 Baculoviruses Autographa californica multicapsid 87 nuclear polyhedrosis virus associated with DNJ-based compounds, might be preferable 46. Treatment of an MDBK cell line chronically infected with non-cytopathic BVDV using long alkylchain imino-sugar derivatives that were chemically modified to reduce in vitro toxicity, showed that both DNJ and DGJ derivatives can cure the infection (D. Durantel and N. Z., unpublished observations). Promising results from in vivo preclinical toxicology and pharmacokinetic studies with one of these compounds could lead to it being evaluated in clinical studies for treatment against HCV in the near future, subject to a successful investigational new drug filing (IND). The results obtained using ER α-glucosidase inhibitors and non-inhibitors in the BVDV system might prompt investigators to revisit the HIV, HBV 47 and WHV systems described above, using long alkylchain derivatives of DGJ as well as DNJ. The mechanism of action in each case is not a trivial task to evaluate, and a mixture of different mechanisms could well be responsible for the antiviral effects achieved. The fact that imino-sugar derivatives can disrupt the general cellular function of glycoprotein processing gave rise to both the hope that they could be used as therapeutics for various diseases and the fear that the significant mammalian toxicity of the compounds would be a hindrance to their usefulness. However, this is frequently true of many drug candidates and it is not unreasonable to expect that an appropriate therapeutic window can be found. Improvement of their pharmacokinetic properties should result in lower dose rates being necessary so that undesirable side effects are limited. Imino-sugar inhibitors and GSL storage diseases The GSL storage diseases are a family of progressive disorders in which GSL species are stored in the lysosome as a result of defects in the GSL degradation pathway (FIG. 4). In their most severe forms, they cause progressive neurodegeneration and are fatal in early infancy. GSL storage diseases occur at a collective frequency of 1 in 18,000 live births and are the most common cause of neurodegenerative disease in infants and children 48. They result from the inheritance of mutations in genes that encode acid glycosidases or their protein cofactors, which participate in the sequential removal of monosaccharide units from GSLs in the lysosome. Most are autosomal recessive diseases, with the exception of Fabry disease, which is X-linked. The specific diseases are: Gaucher types 1, 2 and 3, Fabry,Tay Sachs, Sandhoff and GM2 gangliosidosis. With the exception of type 1 Gaucher disease, all are associated with GSL storage in the nervous system 9, reflecting the particular abundance of GSLs in neural tissue. Individual mutations have different consequences on the residual activity of the specific enzyme in question, which provides a guide as to the severity of clinical manifestations. The infantile-onset disease variants have low or undetectable residual enzyme activity, the juvenile-onset patients have detectable but low enzymatic activities, whereas the adult-onset group have moderate residual enzyme activity. The more residual enzyme activity an individual has, the longer it takes for storage of GSLs to build up to pathological levels. The molecular cell pathology of the GSL storage diseases is not clearly understood and, until recently, no appropriate small animal models of these diseases were available for experimental study. The options for treating the GSL storage disorders are limited at present and, for most affected patients, no specific therapy is available. This difficulty is further compounded by the inaccessibility of the central nervous system (CNS). To date, most research has focused on methods to augment the level of enzymatic activity within the lysosome by direct enzyme replacement 49, bone-marrow transplantation (BMT) or gene therapy. Enzyme-replacement therapy is an established treatment for non-neuropathic Gaucher disease, but delivery of the enzyme is intravenous and therefore invasive, and lack of uptake of enzymes across the blood brain barrier limits the potential of this approach in neuropathic diseases. BMT has had mixed success in this group of disorders; it requires matched donors and is a procedure associated with high mortality rates. Gene-therapy strategies, although promising, pose many unanswered questions about efficacy and safety, and major technical difficulties mean that they are many years from clinical implementation. So, is there any place for more conventional drugbased strategies for the management of these diseases? One attractive approach proposed by Radin and colleagues was the concept of partially inhibiting GSL synthesis using a pharmacological agent 50,51. Slowing the rate of synthesis of GSLs will lead to fewer GSLs entering the lysosome for catabolism, reducing the rate of storage. In principle, complete balance would be achieved if sufficient residual enzyme activity were present. However, even if enzyme levels were low or undetectable, it is anticipated that severe disease could be converted into a milder, slower-progressing form. Many terms have been given to this approach, including substrate deprivation, 70 JANUARY 2002 VOLUME 1

7 a substrate inhibition and substrate balance. For the purpose of this discussion, it will be termed substrate reduction therapy (SRT). There are three main goals for this approach: first, to use an oral drug; second, the drug should penetrate the CNS; and third, an early step in the GSL biosynthetic pathway should be targeted, such b GM2 ganglioside GalNAcβ4 Galβ4GlcCer GalNAcβ3Galα4Galβ4GlcCer NeuAcα3 β-hexosaminidase A β-hexosaminidase A β-hexosaminidase B Tay Sachs disease Efflux Sphingomyelinase Niemann Pick disease Diseased cell (lysosomal storage of GSLs) Nucleus NeuAcα3Galβ4GlcCer Sphingomyelin Golgi Lysosome Impaired enzyme degradation Sandhoff disease Galβ4GlcCer GlcCer Ceramide Synthesis Influx Galα4Galβ4GlcCer β-glucocerebrosidase Gaucher disease Membrane GSLs α-galactosidase Fabry disease Figure 4 The glucosphingolipid cycle and glucosphingolipid storage diseases. a Glucosphingolipids (GSLs) are synthesized from ceramide (FIG. 2) by the sequential addition of monosaccharides in the Golgi, and are then transported to the cell plasma membrane. As part of the normal turnover of components of the plasma membrane, GSLs are transported to the lysosome for degradation. GSL storage diseases, such as Tay Sachs disease, are caused by defects in enzymes in the degradation pathway, which result in accumulation of GSL species in the lysosome. b Selected steps in the GSL degradation pathway in humans, showing the defective enzyme in several GSL storage diseases. Gal, galactose; GalNAc, N-acetylgalactosamine; GlcCer, glucosylceramide; NeuAc, N-acetylneuraminic acid. that a single drug could treat a family of GSL storage diseases, without the need for disease-specific intervention. Imino-sugar inhibitors of GSL biosynthesis N-alkylated imino sugars with glucose and galactose stereochemistries inhibit GSL biosynthesis by inhibiting CerGlcT, which catalyses the transfer of glucose to ceramide 11 (FIG. 2). This is the first committed step of the GSL biosynthetic pathway, and glucosylceramide is the precursor for all glucosphingolipids, including neutral GSLs and gangliosides. These analogues do not inhibit the ceramide galactosyltransferase that synthesizes galactosyl ceramide, which along with its sulphated derivative, sulphatide is an important lipid in myelin 36. The prototypic compound, NB-DNJ (TABLE 1) 52,53, was developed as an antiviral agent (see above). Imino sugars were not identified as inhibitors of CerGlcT until 1994 (REF. 54), despite the fact they were in common use as inhibitors of enzymes that process N-glycans 10,55. Inhibition of GSL biosynthesis was observed only in cells treated with compounds that had a minimal alkylchain length of at least three carbons 54,56. Molecular modelling studies indicated that ceramide mimicry might contribute, at least in part, to the mechanism of inhibition of CerGlcT by NB-DNJ 11,36 ; the nature of the ring moiety is also important. So, in addition to the glucose analogue NB-DNJ, the galactose analogue NB-DGJ (TABLE 1) inhibits CerGlcT, but the mannose, fucose and N-acetylglucosamine derivatives do not 56. Whereas α-glucosidases I and II are resident in the ER, CerGlcT is located on an early Golgi compartment with its catalytic domain exposed to the cytosol. Although NB-DNJ is a more potent inhibitor of ER α-glucosidases I and II than it is of CerGlcT, both in vitro and in vivo, the dominant activity of the compound is against CerGlcT as, owing to its cytosolic orientation, this enzyme is much more accessible to the drug 36. Evaluation of SRT in mouse models Several knockout mouse models of GSL storage diseases have been generated 57, allowing in vivo studies of SRT to be performed. Before evaluating SRT in disease models, it was shown that healthy adult mice treated orally with NB-DNJ could tolerate partial GSL depletion 58,even though mice null for GSLs do not develop 8. Tay Sachs disease and Sandhoff disease are caused by the accumulation of the GM2 ganglioside due to a deficiency in the degradation enzyme, β-hexosaminidase (FIG. 4). There are two main isoenzymes of β-hexosaminidase hexosaminidase A, a heterodimer formed from an α-chain and a β-chain, and hexosaminidase B, a β β homodimer. Tay Sachs disease and Sandhoff disease are caused by mutations in the genes coding for the α-chain (HEXA) and β-chain (HEXB), respectively. In the mouse model of Tay Sachs disease (Hexa knockout), the mice store GM2 ganglioside in a progressive fashion, but the levels never exceed the threshold required to elicit neurodegeneration 59,60. This is because in mice (but not in humans), a lysosomal sialidase is sufficiently abundant or active to convert GM2 into GA2, which can then be NATURE REVIEWS DRUG DISCOVERY VOLUME 1 JANUARY

8 catabolised by the unaffected hexosaminidase-b isoenzyme 61. Disruption of Hexb to produce a mouse model of Sandhoff disease knocks out both hexosaminidase A and B, resulting in the storage of GM2 and GA2 gangliosides in the CNS and periphery 61.The Sandhoff-disease mouse has very low levels of residual enzyme activity, conferred by the minor hexosaminidase S (α α) isoenzyme. The mice cannot bypass the block in catabolism, however, and so undergo rapid, progressive neurodegeneration and die at 4 5 months of age 61. The Tay Sachs mouse model therefore showed whether or not SRT could prevent storage in the brain and the Sandhoff mouse provided a model in which to study whether SRT would affect clinical pathology and extend survival by slowing the rate of storage. SRT in a mouse model of Tay Sachs disease. Tay Sachs mice were reared on food containing NB-DNJ and monitored for 12 weeks. A reduction in stored GM2 ganglioside was observed in all animals from the NB-DNJ-treated group (50% reduction in GM2 ganglioside in the brains of treated mice relative to the untreated controls) 62. NB-DNJ was therefore able to cross the blood brain barrier to an extent that prevented storage 62. SRT in a mouse model of Sandhoff disease. When Sandhoff mice were treated with NB-DNJ, their life expectancy was increased by 40% and GSL storage was reduced in peripheral tissues and in the CNS 63. Following the onset of symptoms, the rate of decline was significantly slower in NB-DNJ-treated mice, and the age at which deterioration could first be detected was delayed (approximately 100 days for untreated mice and approximately 135 days for NB-DNJ-treated mice). However, the terminal stage of the disease (when the mice are moribund) was prolonged in NB-DNJ-treated mice. When GSL storage levels were measured in the untreated and NB-DNJ-treated Sandhoff mice at their end points (at 125 days and 170 days, respectively), the levels of GM2 and GA2 were comparable, indicating that death correlated with the same levels of GSL storage in the brains of the two groups of mice. Histological examination of the mice at 120 days showed reduced storage in the brain of NB-DNJ-treated mice, consistent with the clinical improvement. Combination therapy in the Sandhoff mouse. Sandhoff-disease mice treated with both BMT and NB-DNJ survived significantly longer than those treated with BMT or NB-DNJ alone. When the mice were subdivided into two groups on the basis of the enzyme levels in their CNS, which were derived from donor bone marrow, the high enzyme group had a greater degree of synergy (25%) than the group as a whole (13%). Combination therapy might therefore be the strategy of choice for treating the infantile-onset disease variants in which lack of enzyme limits the potential of SRT 64. Proof of concept in a genetic model of SRT. The efficacy of SRT was further shown in the Sandhoff mouse model by Proia and colleagues, who crossed the Sandhoff mouse with a mouse engineered to block synthesis of the storage GSL 65. The resulting mice, which had defects in both GM2 ganglioside synthesis and catabolism, no longer stored GSL, lived much longer and had greatly improved neurological function. It is interesting to note that these mice, which were deficient in both the hexosaminidase A and B isoenzymes, eventually developed storage of oligosaccharides derived from the catabolism of N-linked glycans. These are additional substrates for β-hexosaminidases and are known to accumulate in Sandhoff-disease patients. This was the first clear indication that stored N-glycans might also contribute to neuropathology in this disease. This study elegantly highlighted the limitation of SRT in diseases in which the defective enzyme has other non-gsl substrates 65. SRT in a mouse model of NPC disease. GSL accumulation is not restricted to the classical GSL storage diseases in which the inherited defect is in a lysosomal hydrolase. Other lysosomal disorders, including Niemann Pick type C (NPC) disease, result in the accumulation of GSLs in the brain, secondary to the primary defect. In NPC disease, the primary defect is in the NPC1 gene (and, less commonly, the NPC2 gene). NPC1 is localized to vesicles that are thought to recycle unesterified cholesterol from late endosomes or lysosomes to the ER and Golgi. For this reason, NPC has historically been considered to be a disorder of cholesterol transport. However, the fact that certain GSLs are stored in the brain in NPC disease indicates that NPC1 might also be involved in GSL homeostasis. A crucial question in NPC disease is the cause of the neurodegenerative phenotype. As GSL accumulation in the classical GSL storage diseases is known to lead to neuronal dysfunction and death (although precisely how remains to be determined), Walkley and colleagues proposed that the stored GSLs might be major contributors to the neuropathology associated with this devastating disease 66. Two classes of GSL accumulate in the brain in NPC disease: neutral GSL species and gangliosides. The dominant species is GM2, which is known to cause progressive neurodegeneration when it is stored in Tay Sachs and Sandhoff disease. Proia and colleagues therefore investigated the contribution of the gangliosides to the pathology of NPC disease by crossing the NPC mouse (spontaneous model with a lesion in the Npc1 gene) with a mouse genetically engineered to prevent it synthesizing GM2 and some other higher GSL species 67. Neuronal GM2 storage was prevented, but it did not alter the disease course. So, if GSLs are involved in pathogenesis, it must be those that remain in the double-mutant mice, namely GM3, lactosylceramide, glucosylceramide and free sphingosine. Walkley and colleagues used NB-DNJ treatment in the NPC mouse model to determine whether or not any glucosylceramide-based GSLs contributed to the pathology 66, and found that life expectancy of the NPC 72 JANUARY 2002 VOLUME 1

9 Box 1 Clinical trial of substrate reduction therapy in type I Gaucher disease The trial was coordinated by Oxford GlycoSciences and N-butyldeoxynojirimycin (NB-DNJ) was referred to as OGT918. The trial enrolled 28 adult patients (14 females and 14 males); 7 of whom had had previous splenectomies. All patients were unable or unwilling to take enzyme-replacement therapy. Side effects. The main known side effect of OGT918 is diarrhoea, which was noted in the previous trial in which this compound was used as an agent against human immunodeficiency virus (HIV) 37. In the Gaucher study, a tenfold lower dose of drug was used relative to the HIV trial. Although most patients reported gastrointestinal (GI)-tract symptoms as soon as they started taking OGT918, the diarrhoea spontaneously resolved in most patients within several weeks and did not, in general, pose a significant problem 68. Of the 28 patients enrolled in the trial, 6 withdrew (2 owing to the GI-tract side effects, 2 owing to pre-existing medical conditions and 2 for personal reasons). The remaining 22 patients were monitored at 6 and 12 months for signs of clinical improvement. Two further patients withdrew because of symptoms of peripheral neuropathy. All other patients on OGT918 have been investigated by electromyography, and to date no other cases of peripheral neuropathy have been identified. Beyond the 12-month study, 18 patients have continued to receive OGT918 in an extended-treatment protocol, with some patients having so far taken therapy for 2.5 years. Clinical efficacy. Both spleen and liver volumes showed a statistically significant reduction (15% and 7%, respectively) after 6 months of therapy. At 12 months, the decreases from baseline were 19% and 12%, respectively 66. This was comparable to the response observed in patients of similar baseline disease severity receiving enzyme-replacement therapy 70. Chitotriosidase activity, a marker of disease activity, was reduced in a time-dependent manner, indicating a reduction in the total pool of Gaucher cells within the patients treated with OGT918 (REF. 68). Haemoglobin and platelet counts showed trends towards improvement, with a greater improvement in haemoglobin noted in patients who were anaemic at baseline. A statistically significant improvement in platelet counts was achieved following 12 months of treatment 68. Assessment of 18 patients in the extended-use protocol has shown continued reduction of organ volume, further improvements in platelet and haemoglobin counts (all values now statistically significant) and continued decline in chitotriosidase activity (A. Zimran, presented at the Fourth European Working Group of Gaucher Disease (EWGED) Workshop, 2000). MACROPHAGE A type of white blood cell that is specialized for the uptake of material by phagocytosis. mouse was significantly extended and the neuropathology significantly delayed. They obtained similar data in the NPC cat model 66. These observations indicate that GSLs are involved in the neuropathology of NPC, although precisely which stored GSL species contribute is unknown. The other possibility is that NB-DNJ mediates the clinical improvement owing to an as yet unidentified activity of this drug, independent of GSL depletion. However, the imino sugar NB-DGJ, which also inhibits GSL biosynthesis, but does not cause any of the side effects attributable to NB-DNJ, has the same effect in the NPC mouse. Life expectancy was extended to the same extent, making a mechanism based on GSL depletion highly likely 66. The study of NB-DNJ in the NPC mouse has led to the planning of clinical trials of NB-DNJ in NPC patients, due to start in Clinical trial of SRT in type 1 Gaucher disease Gaucher disease results from mutations in the gene encoding glucocerebrosidase, the enzyme that removes glucose from ceramide (FIG. 4). The symptoms of Gaucher disease are primarily due to Gaucher cells (MACROPHAGES engorged with glucosylceramide), which commonly collect in the spleen and liver, resulting in enlargement of these organs, as well as blood abnormalities, such as anaemia. Type I Gaucher disease was chosen for the first clinical trial of SRT because it has no CNS involvement, has well-defined clinical endpoints and there is an existing effective therapy to make comparisons with (intravenous enzyme-replacement therapy). Therefore, from 1998 to 1999, patients with type I Gaucher (non-neuropathic) disease were recruited at four centres Cambridge, Amsterdam, Prague and Jerusalem into a 1-year open-label clinical trial of NB-DNJ. Data from this trial 68 (BOX 1), and subsequent studies in an extended-use protocol, strongly indicate that GSL depletion improves all key clinical features of Gaucher disease. The data from the preclinical, clinical and toxicology studies have recently been submitted to the regulatory authorities in Europe and the United States for consideration for approval. Future prospects In principle, it would be predicted that, in the same way that BMT and substrate deprivation are synergistic in their action in the Sandhoff mouse model 64, combining intravenous enzyme replacement and SRT in Gaucher patients would be a rational treatment option 69.Several permutations could be envisaged, including monotherapy, sequential therapy that is, enzyme followed by NB-DNJ maintenance or co-administration; that is, continuous NB-DNJ administration with periodic enzyme administration. The preclinical studies in mouse models of Tay Sachs and Sandhoff disease 62,63 offer the prospect that SRT might be of benefit to patients with CNS involvement, at least those with the juvenile- and adult-onset variants of these disorders. With the advent of more effective means of delivering enzymes to the CNS (BMT, gene therapy and neuronal stem-cell therapy), several strategies might become available for improving the lives of patients suffering from these devastating neurological diseases. Combining substrate-reducing drugs with enzymeaugmenting therapies could even make therapy in the very severe infantile-onset patients a reality. NATURE REVIEWS DRUG DISCOVERY VOLUME 1 JANUARY

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