Glycobiology Advance Access originally published online on May 18, 2005
Glycobiology 2005 15(10):43R-52R; doi:10.1093/glycob/cwi076
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REVIEW |
Imino sugar inhibitors for treating the lysosomal glycosphingolipidoses
Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
1 To whom correspondence should be addressed; e-mail: terry.butters{at}bioch.ox.ac.uk
Accepted on May 10, 2005
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Abstract |
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The inherited metabolic disorders of glycosphingolipid (GSL) metabolism are a relatively rare group of diseases that have diverse and often neurodegenerative phenotypes. Typically, a deficiency in catabolic enzyme activity leads to lysosomal storage of GSL substrates and in many diseases, several other glycoconjugates. A novel generic approach to treating these diseases has been termed substrate reduction therapy (SRT), and the discovery and development of N-alkylated imino sugars as effective and approved drugs is discussed. An understanding of the molecular mechanism for the inhibition of the key enzyme in GSL biosynthesis, ceramide glucosyltransferase (CGT) by N-alkylated imino sugars, has also lead to compound design for improvements to inhibitory potency, bioavailability, enzyme selectivity, and biological safety. Following a successful clinical evaluation of one compound, N-butyl-deoxynojirimycin [(NB-DNJ), miglustat, Zavesca], for treating type I Gaucher disease, issues regarding the significance of side effects and CNS access have been addressed as exposure of drug to patients has increased. An alternative experimental approach to treat specific glycosphingolipid (GSL) lysosomal storage diseases is to use imino sugars as molecular chaperons that assist protein folding and stability of mutant enzymes. The principles of chaperon-mediated therapy (CMT) are described, and the potential efficacy and preclinical status of imino sugars is compared with substrate reduction therapy (SRT). The increasing use of imino sugars for clinical evaluation of a group of storage diseases that are complex and often intractable disorders to treat has considerable benefit. This is particularly so given the ability of small molecules to be orally available, penetrate the central nervous system (CNS), and have well-characterized biological and pharmacological properties.
Key words: enzyme inhibitor / Gaucher disease / glycosphingolipid / lysosomal storage diseases / novel therapeutics
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Glycolipid lysosomal storage diseases |
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In all eukaryotic cells, the cycle of biosynthesis and catabolism operates to ensure that mature macromolecules are removed and degraded efficiently, thus preserving the integrity of biological function. When this cycle is broken, by an insufficiency in either a biosynthetic or a catabolic enzyme, a number of pathological conditions arise that result in disease phenotypes in man. The glycosphingolipidoses are a group of inherited diseases that are caused by mutations in catabolic enzymes or other cofactors that reduce the efficiency of substrate GSL hydrolysis resulting in lysosomal storage (Kolter and Sandhoff, 1999
; Vellodi, 2005). To aid hydrolysis of the membrane-embedded GSL, a number of activator proteins, Saposins A, B, C, and D, and GM2 activator protein (Kolter and Sandhoff, 1998) are required to partially extract the GSL from the membrane and present the oligosaccharide to the enzyme (Furst and Sandhoff, 1992).
The sequential breakdown of GSL oligosaccharide by glycosidases follows the reverse pathway to that of biosynthesis, mediated by glycosyltransferases (Figure 1). If catabolic activity in the lysosome is reduced, a feedback mechanism that signals the endoplasmic reticulum (ER) and Golgi to reduce biosynthesis accordingly may not be sufficient to prevent storage (Sano et al., 2005). As a consequence, GSL substrate concentration increases, far in excess of the rate at which it can be hydrolysed by the catalytically impaired enzyme. When the catalytic activity of the enzyme falls below a critical threshold value (Kolter and Sandhoff, 1998), substrate will accumulate to pathological levels, creating an intralysosomal lipoprotein complex that restricts access of enzyme to substrate, accelerating GSL storage.
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Lysosomal storage of GSL produces disorders in man that differ in the storage product, cell and tissue types affected, and disease severity. The degree of pathology depends on the mutational impairment of catalytic activity. Low levels of enzyme activity usually predict that symptom onset occurs more rapidly leading to infantile or juvenile disease. Where the major site for accumulation is neural tissue, as in the gangliosidoses, Sandhoff and Tay-Sachs disease for example, neurodegeneration is acute and often leads to premature death (Jeyakumar et al., 2002; Platt and Walkley, 2004). In Fabry disease, cardiac and renal storage of GSL leads to premature death due to cardiovascular disease and/or kidney failure (Desnick et al., 2001). The hepatosplenomegally, haematological disturbance, and bone destruction seen in Gaucher disease are caused by the accumulation of glucosylceramide in macrophages where the phagocytic activity of these cells induces an additional lysosomal burden of glucosylceramide (Zimran, 1997). Lysosomal or extra-lysosomal GSL either acts as a primary stimulus for a disruption in cell signalling events inducing cell death or the storage lipids (or their metabolites) may be directly cytotoxic. A proinflammatory cascade, also demonstrated in neural tissue mediated by microglial cells (Jeyakumar et al., 2003), could be a common mechanism for pathogenesis in all glycosphingolipidoses (Mizukami et al., 2002). In support of this hypothesis, the administration of nonsteroidal antiinflammatory drugs (NSAID) to mice with Sandhoff disease reduced the rate of disease progression (Jeyakumar et al., 2004). The accumulation of ganglioside may also result in neuronal damage driven by an apoptotic mechanism following the activation of an unfolded protein response (UPR) in cells. This pathway appears to be induced directly by the accumulated GM1 depleting ER calcium stores (del Tessitore et al., 2004).
Storage GSLs or their metabolites may not be the exclusive candidates for disease induction. In Sandhoff disease, the hexosaminidase deficiency also causes the lysosomal accumulation of free N-linked oligosaccharides with terminal hexosamine groups. These oligosaccharides are observed in the brain of the presymptomatic Sandhoff disease mouse and appear to correlate with microglial cell activation and inflammatory chemokine production (Tsuji et al., 2005). Mouse models for disease have been a powerful tool for evaluating disease progression in response to therapy (Norflus et al., 1998; Jeyakumar et al., 2002; Platt et al., 2003). Although engineered to be null for a specific enzyme activity, transgenic mice show typical pathological traits observed in man where human disorders of lysosomal storage disorders have some residual enzyme activity. However, in models where storage is observed without apparent disease phenotype, such as Fabry and Tay-Sachs, these offer good experimental paradigms for addressing proof of principle therapeutic studies at the biochemical level (Platt et al., 1997; Suzuki et al., 1998; Liu et al., 1999; Abe et al., 2000; Platt et al., 2003).
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Therapy |
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Treatment of rare orphan diseases has received increasing interest despite the incidence of disease being very low. The most prevalent of the glycosphingolipidoses is type I Gaucher disease, where in the Ashkenazi Jewish population, mutation frequency in glucocerebrosidase is 3%. A point mutation in the glucocerebrosidase gene at amino acid position 370 (substitution of serine for asparagine) leads to a decrease in catalytic rate constants for the glucocerebroside (glucosylceramide, Figure 1), substrate, and the interaction of the enzyme with Saposin C activator protein and the lysosomal membrane anionic phospholipid (Salvioli et al., 2005
). Although N370S homozygosity occurs with high frequency, the heterogeneous nature of this disorder leads to an asymptomatic course of disease in a quarter of this population. Predictions for the number of live births of individuals with a glycosphingolipidosis based on disease incidence may therefore not accurately estimate numbers but could be as high as 1:18,000 worldwide (Meikle et al., 1999). Enzyme replacement therapy (ERT) is a disease-specific intervention that uses direct infusion of purified enzyme, bone marrow transplantation, or gene delivery. Of these, type I Gaucher disease, where glucocerebrosidase has been a registered drug since 1992, and ERT for Fabry disease by using recombinant 
-galactosidase, licensed in Europe in 2003, are the most successful to date (Desnick and Schuchman, 2002; Brady, 2003).
This review brings together two novel approaches for using imino sugars therapeutically for a group of glycolipid disorders that present several obstacles for conventional treatment. The first termed SRT is a generic strategy and aimed to partially inhibit the biosynthetic cycle to reduce GSL substrate influx into the catabolically compromised lysosome. This research has progressed to regulatory-approved drug (Platt and Butters, 1998; Butters et al., 2000a; Butters et al., 2003a,b). The second, more experimental approach uses imino sugars to target the protein folding and trafficking pathways of glycosidase to assist correction of lysosomal enzyme activity, CMT. Both methods can be considered as an intervention in normal cellular processes to produce a partial effect on enzyme activity. SRT reduces the synthesis of GSL by using imino sugar concentrations that never completely depletes these critical components. A partial increase in enzyme activity by CMT may be sufficient to adjust the critical threshold activity of enzyme (Kolter and Sandhoff, 1998) to levels where GSL storage in the lysosome is reduced to nonpathological concentrations, under noninhibitory conditions.
These novel approaches to imino sugar therapy for the glycosphingolipidoses could also be used in combination to provide a simple oral solution to the physiological deficit observed. The increasing clinical data that supports the safe administration of imino sugars for lifelong therapy should generate interest in understanding mechanism and promote further the evaluation of small molecule inhibitors for early intervention in treating lysosomal storage disorders.
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Imino sugars for SRT |
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The hypothesis that a reduction in the concentration of GSL substrate in the lysosome could be achieved by using an inhibitor of biosynthesis was originally proposed by Radin several years ago. The compounds that Radin and colleagues synthesized to inhibit the pivotal enzyme in GSL biosynthesis, ceramide glucosyltransferase (CGT), were structural mimics of ceramide with good binding affinities (Vunnam and Radin, 1980
; Radin, 1996; Lee et al., 1999). Whilst these compounds were excellent tools for the biochemist to study the biological function and significance of GSL, limited in vivo, data has been obtained to support a clinical trial in man.
The effects of GSL depletion have been experimentally determined in mouse knockout studies, and a null phenotype for CGT is embryonically lethal (Yamashita et al., 1999). The deletion of genes coding glycosyltransferases downstream from glucosylceramide (Figure 1) also leads to neurological disturbance (Allende and Proia, 2002; Proia, 2003). Important lessons have been learnt from these studies and serve to highlight the difference between species when using mouse models for understanding disease pathology. In the GM3 synthase knockout mouse, the animals are viable with some neurological/cell signally differences observed, whereas in man a GM3 deficiency leads to infantile epilepsy and dramatic neurological dysfunction (Simpson et al., 2004). In recognition of the important roles played by many GSL, the aim of SRT is to reduce biosynthesis to balance the decreased lysosomal hydrolysis of GSL. A significant reduction in GSL composition, although tolerated in single cells, is not desirable in vivo and is not necessary to achieve efficacy.
The discovery that N-alkylated imino sugars with the correct chirality inhibited CGT (Platt et al., 1994a,b; Butters et al., 2000b) was rapidly exploited for development as a novel therapeutic for small molecule treatment of the glycosphingolipidoses. Deoxynojirimycin (DNJ) and N-alkylated derivatives were identified as potent -glucosidase inhibitors, where fundamental studies had shown the role of these enzymes in early stages of glycoprotein N-linked oligosaccharide processing in the ER (Elbein, 1991). Because most enveloped viruses use the same pathway for glycoprotein synthesis in infected cells, modulation of the oligosaccharide to induce a reduction in infectivity became a pharmacological target, particularly when initiatives for HIV therapy were required. From the rapid expansion in imino sugar synthesis to discover therapeutically beneficial inhibitors, N-butyl-DNJ (NB-DNJ) (Figure 2), was identified by Searle/Monsanto as a leading candidate for HIV treatment, culminating in clinical trials in man as a monotherapy (Tierney et al., 1995) and in combination with AZT (Fischl et al., 1994).
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These clinical trials were unsuccessful, because the antiviral concentrations required might not be achieved in man. The precise mechanism by which -glucosidase inhibitors were antiviral was not known at this time. However, the trials provided valuable medicinal safety information, which was vital for subsequent studies for SRT. Very few imino sugars have reached the clinic, and until NB-DNJ was approved for use, Bayer’s N-methoxy-DNJ (Miglitol) for noninsulin dependent (NID) diabetes was the sole member of this class of compound to be in conventional use as a drug.
The later identification of the quality control mechanism for protein folding that involves monoglucosylated glycans interacting with ER-chaperons such as calnexin (Helenius and Aebi, 2001) has revealed the mechanism for viral glycoprotein misfolding and reduction in infectivity observed following glucosidase inhibitor treatment (Fischer et al., 1995, 1996). Despite the lack of a significant effect as a therapeutic for HIV, the use of imino sugars to misfold viral glycoprotein as a therapeutic approach has now been applied to hepatitis B (Block et al., 1998) and hepatitis C (Zitzmann et al., 1999) and gained pharmaceutical industry interest (Dwek et al., 2002). Clearly the result of misfolding of glycoprotein monomers will influence the outcome for viral glycoprotein function, and the antiviral effect will be dependent on oligomerization and assembly of these misfolded proteins in the virion.
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Imino sugars as inhibitors of glycolipid biosynthesis |
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It was an unexpected finding that imino sugars with a certain chirality and N-substituted with groups containing a minimum of three carbon atoms had an additional property, inhibition of GSL biosynthesis, to their known inhibition of glycosidase enzyme activity. Fortunately, NB-DNJ, which was being used as an
-glucosidase inhibitor, had all the requirements for blocking the metabolic pathway for GSL biosynthesis in cells. Subsequent studies showed that the molecular basis of this activity was the inhibition of CGT and that NB-DNJ was a reversible, competitive inhibitor for ceramide and noncompetitive for the sugar donor, UDP-glucose, in the reaction scheme. A partial explanation was revealed following molecular modelling studies by using the crystal structure of ceramide and the nuclear magnetic resonance (NMR) solution structure of NB-DNJ (Butters et al., 2000b). Although not sufficient to explain all the structure/function data (Butters et al., 2000b), it has acted as a useful springboard for synthesizing analogues with more potent CGT inhibitory activity. Many DNJ compounds have recently been described that were synthesised to engender greater ceramide molecular mimicry. The model predicts that the alignment of the N-acyl chain of ceramide and the N-alkyl chain of the imino sugar contributes significantly to potency. The addition of a further alkyl chain could improve mimicry, hence inhibitory potency, but this was not proved by experiment (Boucheron et al., 2005). The likely explanation is that the conformational space of the alkyl and acyl chains is restricted by the DNJ cyclic ring and does not allow close alignment with ceramide (Boucheron et al., 2005) (Figure 3). Increasing the hydrophobicity of the N-substituent does increase potency, however, and provides further improvement by virtue of membrane adsorption, persistence in tissues and greater brain penetrance (Mellor et al., 2002). By using recombinant CGT, kinetic measurements of the interaction between N-alkylated imino sugars and the native ceramide substrate revealed subtle difference between the modes of inhibition. Compounds with longer alkyl chains, for example, N-nonyl-DNJ, have a noncompetitive mode of inhibition for ceramide suggesting that there may be more than one binding site on the recombinant enzyme.
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No crystal structure of the CGT enzyme has been obtained, but an in silico model has provided further information regarding the interaction of N-alkylated imino sugars with the enzyme (Butters et al., 2003c). Short chain N-alkylated inhibitors bind to a site with a more hydrophilic environment than a membrane-associated hydrophobic site that accepts longer chain (C > 5) inhibitors (Butters et al., 2003c).
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Preclinical studies for imino sugar SRT |
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 TopAbstractGlycolipid lysosomal storage...TherapyImino sugars for SRTImino sugars as inhibitors... Preclinical studies for imino... Clinical studies for imino...Imino sugars for CMTSummaryReferences |
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At least three chiral centres in the six-membered piperidines are required for the inhibition of CGT, and those with glucose and galactose (D and L configuration) stereochemistry are active. This has allowed further compounds to be evaluated for SRT, the most notable being the galactose analogue, N-butyl-deoxygalactonojirimycin (N-butyl-DGJ) (Platt et al., 1994b
) (Figure 2). Development of this compound has followed a route similar to NB-DNJ with efficacy shown in tissue culture models of Gaucher disease (Platt et al., 1994b), in vivo tolerability (Andersson et al., 2000) and in an animal model of Sandhoff disease (Andersson et al., 2004). The galactose analogue is equally effective as NB-DNJ at reducing substrate burden in the brain has greater benefit in increasing life expectancy due to the lack of drug side effects (Jeyakumar et al., 1999; Andersson et al., 2004). The potential side effects of NB-DNJ, such as gastrointestinal (GI) malabsorption and weight loss in mouse, and inhibition of ER glucosidases are lacking in N-butyl-DGJ indicating this compound to be more selective and tolerated better. These factors would make a powerful inducement for a clinical evaluation of this compound, particularly in paediatric cases where early intervention is required before significant neuropathology has occurred (Table I).
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A genetic model of SRT in Sandhoff disease mice has validated most of the arguments supporting this approach whilst revealing potential limitations (Liu et al., 1999; Tifft and Proia, 2000). As described earlier (Tsuji et al., 2005), oligosaccharides have been implicated in the inflammatory disease process in Sandhoff disease, and the depletion of GSL substrate by either genetic manipulation to block GSL glycosyltransferase activity (Liu et al., 1999) or NB-DNJ (Jeyakumar et al., 1999) results in reduced GSL storage only. Oligosaccharides resulting from impaired degradation of glycoproteins and proteoglycans continue to accumulate, possibly to pathology-inducing amounts. The contribution of these glycans to late stage cellular pathology remains to be determined, as does the potential for early GSL accumulation as causing irreversible neurological damage (Jeyakumar et al., 2002).
The application of SRT to rare disorders where a deficiency in activator protein (Figure 1) also leads to GSL storage and pathology (Fujita et al., 1996; Liu et al., 1997) may provide a therapeutic benefit, particularly where other approaches may be less advanced. Similarly, in diseases where GSL accumulation is secondary to the primary lesion, such as Niemann-Pick type C (NP-C) disease (Table I), SRT would be predicted to reduce the burden of GSL and downstream pathology.
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Clinical studies for imino sugar SRT |
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The preclinical and clinical data (from the HIV trial) obtained for NB-DNJ allowed a 12-month assessment for efficacy in type I Gaucher disease sponsored by Oxford GlycoSciences (Abingdon, Oxon, UK). Following publication of the results showing improvements of organ volumes and haematological parameters (Cox et al., 2000
), NB-DNJ gained approval in Europe, USA, and Israel for use of type I patients who were unable or unwilling to take ERT. Clinical benefit was demonstrated, and although many issues regarding safety were raised (Pastores and Barnett, 2003), this proof of principle study allowed further trials to evaluate low dose administration (Heitner et al., 2002) and a 3-year continuation study (Elstein et al., 2004). Statistically significant improvements in all the major efficacy endpoints were achieved in the latter extension study indicating that consistent with the mechanism of action of miglustat as a modulator of GSL biosynthesis, treatment was increasingly effective with time. No serious adverse events have been reported; diarrhoea and weight loss decreased, and no new case of peripheral neuropathy, noted in the first trial, was found.
In a small group of type I Gaucher patients, these data are remarkable in showing clinical improvement with a relatively low dose of inhibitor. A treatment regime of 100–300 mg three times per day produced a stable plasma concentration of 6–8 µM, a concentration that was predicted to partially inhibit CGT leading to substrate reduction in cellulo (Platt et al., 1994a) (Table II). Concerns regarding the safety and tolerability of long-term imino sugar treatment now appear to be reduced and may be explained by underlying and diverse manifestations of Gaucher disease that complicate small clinical trials (Sidransky, 2005). The next challenge for imino sugar SRT will be treatment of neuronopathic diseases, and one recent study has demonstrated the biochemical, but not clinical efficacy of Zavesca for these "untreatable" disorders and provides further support for the translocation of imino sugars across the blood/brain barrier in species other than rodents (Lachmann et al., 2004). In this study, a dose of 100 mg/day was sufficient to reduce the pathological burden of GSL that is apparent in an NP-C patient. The lipid disorder in NP-C is thought to be abnormal trafficking of cholesterol but many studies have identified the accumulation of GSL and point to these as being the signal for downstream pathological events in neural tissue (Zervas et al., 2001; Jeyakumar et al., 2002). After 7 months of therapy, 
20% (0.6 µM) of the circulating plasma concentration (3 µM) was found in the CSF similar to figures obtained when mice are orally administered NB-DNJ (Lachmann et al., 2004). These data provide some optimism that other neurodegenerative disorders such as types II/III Gaucher disease and the gangliosidoses (Table I) may respond to SRT providing that sufficient residual enzyme activity is present.
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Imino sugars for CMT |
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CMT using imino sugars relies on reversible, tight-binding constants for an imino sugar inhibitor, and it’s enzyme substrate. Because many imino sugars are active site directed with good affinities (10-6 to 10-9 M), co- or posttranslational binding to the enzyme may provide sufficient protection against misfolding or inactivation. This could occur either in the ER or during transfer to the lysosome. Most lysosomal glycosidases use N-glycans (mannose 6-phosphate) for targeting to the lysosome, requiring ER-luminal biosynthesis and vesicular transport. For imino sugars to have efficacy in the ER to stabilise misfolded proteins, significant concentrations need to be maintained in the lumen working against the observed concentration gradient.
By using enzymes as cellular markers for imino sugar entry to cells, it has been possible to determine the effective cell organelle concentration for inhibition and in a time-dependent manner. In many cells, the concentration of N-alkylated DNJ (N-alkyl-DNJ) analogues to inhibit ER resident -glucosidases is 1000–100,000 fold greater than the measured affinity constant in vitro (Butters et al., 2000b; Mellor et al., 2002; Mellor et al., 2004a). By contrast, the effective concentration of the same analogues required to inhibit CGT in cells, where the active site of the enzyme is cytoplasmically exposed, is similar to in vitro inhibition constants (Mellor et al., 2004b).
Entry of N-alkyl-DNJ compounds to cells across the plasma membrane is rapid (<1 min), independent of alkyl chain length and is similar to other amphipathic molecules that translocate by a flip-flop mechanism rather than by facilitated transport (Mellor et al., 2004b). The failure of these molecules to sustain cytoplasmic concentrations in the ER could be due to a restricted entry or protein facilitated transport out of the lumen by molecules analogous to the multidrug resistant efflux pump, P-glycoprotein (P-gp). P-gp expression and GSL metabolism appear to be linked following observations that multidrug resistant cell lines have elevated levels of GSL (Morjani et al., 2001; Norris-Cervetto et al., 2004). No direct evidence for the translocation of GSL by P-gp has been obtained in cellulo, although the link appears to be tangible (Raggers et al., 2000), and when reconstituted in a proteoliposome, P-gp acts as a flippase for simple neutral GSL, including glucosylceramide (Eckford and Sharom, 2005). If N-alkylated imino sugars that inhibit CGT structurally mimic ceramide, is it possible that these also are recognised by P-gp and translocated? Recent data does not support such an interaction because N-alkylated imino sugars are unable to compete with other hydrophobic substrates for binding in vitro (Norris-Cervetto et al., 2004) and are not translocated in P-gp containing proteoliposomes (E. Norris-Cervetto et al., unpublished). Long alkyl chain imino sugars that deplete GSL do not, however, render multidrug resistant cells sensitive to drug cytotoxicity (Norris-Cervetto et al., 2004), indicating that the role of P-gp in modulating GSL metabolism and organelle trafficking in cancer cells may be more complex than previously thought (Radin, 2003).
For chaperon-mediated therapeutic applications, entry to the ER is necessary and concentrations need to be determined to regulate dose and efficacy (Fan, 2003). However, there appear to be few data that either address this issue or consider cell type or tissue ER access variability. At the experimental level, partial correction of activity to misfolded enzyme has been achieved for Gaucher ß-glucosidase (Sawkar et al., 2002), Fabry -galactosidase (Fan et al., 1999; Asano et al., 2000; Yam et al., 2005), GM1 gangliosidosis (Tominaga et al., 2001), and Tay-Sachs/Sandhoff ß-hexosaminidase (Tropak et al., 2004) (see Tables I and II for details).
The effective concentration of imino sugar that needs to be delivered to cells in these studies is variable (5–500 µM), and high concentrations would not translate to a potential therapeutic dose in man. The type I Gaucher trial of NB-DNJ was successful because the effective concentration to partially reduce the activity of CGT in cells (5 µM) (Platt et al., 1994a) was achievable in plasma following oral dosing 300 mg NB-DNJ/day in patients (steady state concentration 6 µM) (Cox et al., 2000). Where enzyme correction requires >20 µM of compound, the ER barrier may preclude an effective outcome, particularly where at high doses, compounds have additional activities that produce unwanted side effects. N-alkylated DNJ compounds (Figure 2 and Table II) inhibit CGT in addition to both - and ß-glucosidases and are effective for SRT and CMT. One complication (gastrointestinal distress) during oral delivery of NB-DNJ was because of the inhibition of the -glucosidase activity of the intestinal sucrase/isomaltase complex. At low dose (3 x 100 mg/day) in the first Gaucher trial, diarrhoea was transient and responded well to therapy but higher doses or use of compounds that are more potent against this enzyme or to those that are not cleared from the gut could cause significant problems. More hydrophobic DNJ analogues, such as N-nonyl-DNJ reported as being potentially beneficial for CMT (Sawkar et al., 2002), were retained for longer time in the gastrointestinal system when these were given orally to mice (Mellor et al., 2002) and produce potential toxic membrane perturbations that are unrelated to target enzyme inhibition (Mellor et al., 2003).
One study has shown that the addition of DGJ in Fabry mutant cells can redirect -galactosidase to the lysosome, instead of ER-associated degradation (ERAD) of misfolded protein. The increased lysosomal activity of this enzyme results in reduced storage of substrate, globotriaosylceramide, Gb3 (Yam et al., 2005). In many other studies, including those where mutations have been engineered in animals, the mutant cells or tissues do not store GSL substrate, so the efficacy of small molecule chaperons to clear lysosomal GSL is difficult to assess. Factors that influence lysosomal GSL storage are composition, and the turnover rate of these molecules as they are cycled from the plasma membrane to the lysosome for degradation. These factors will be tissue and cell dependent and by using cells with partially active mutant enzyme and low GSL content or reduced lysosomal, influx rates do not adequately reflect disease phenotype. Despite this, all studies where imino sugars have been used to chaperone mutant enzymes, an increase in catalytic activity has been demonstrated, albeit using synthetic substrates and concentrations of imino sugar far in excess of their inhibition constants (Table II).
This apparent paradox requires some explanation as one might expect that long-term administration of imino sugars at inhibitory concentrations could generate rather than correct a disease phenotype. Imino sugars are weak amines and are rapidly taken up by the lysosome where concentration equilibrium between the cytosol and lysosomal lumen is established. This is evident from experiments where hexosaminidase inhibitors have been administered to cells at concentrations similar to the inhibition constant. Under these conditions, hexosamine-containing GSL are increased 5-fold following 15 days of treatment (T. D. Butters and A. Armitage, unpublished). The low pH environment of the lysosome is clearly no barrier to inhibition, as suggested by Yam et al. (2005), and quite how a protonated, tight binding, potent inhibitor, for example DGJ (Table II) where the pKa is 7.1 (Legler and Pohl, 1986), dissociates from a partially active enzyme in the lysosome is difficult to understand. The Km of the enzyme for the natural GSL substrate in the lysosome is probably extremely low and can rarely be analyzed accurately in vitro where synthetic substrates and detergents poorly emulate the lysosomal environment. Substrate concentrations are always in excess of Km, especially when storage becomes pronounced, and the competition for binding will favour substrate rather than imino sugar leading to inhibitor displacement. Lysosomal cofactors potentiate substrate hydrolysis moving the kinetic balance of imino sugar treatment towards GSL catalysis by the mutant enzyme, not inhibition. In support of this proposal, DGJ when administered for very short periods to transgenic mice that express the R301Q mutation on an -galactosidase (Fabry disease) knockout background increased the activity in enzyme in the heart without inducing lysosomal storage of globoside, Gb3 (Ishii et al., 2004) (Table II). It remains to be seen that after longer treatment with imino sugar chaperon, where substrate becomes depleted in the lysosome to below Km, enzyme activity is able to balance hydrolysis at the expense of inhibition.
A further complication with a chaperon-mediated approach is that many of the imino sugars used for the correction of a misfolded enzyme also inhibit other lysosomal enzymes. For example, DGJ inhibits both - and ß-galactosidases (Table II), and the correction of Fabry -galactosidase enzyme may induce storage of ß-galactose terminating glycoconjugates, as observed in GM1 gangliosidosis, due to the inhibition of ß-galactosidase (Table II). Target enzyme discrimination can be made on a concentration basis, however, as with NB-DNJ which inhibits CGT and lysosomal ß-glucocerebrosidase. In humans, a plasma circulating concentration of 5–6 µM is sufficient to inhibit CGT (IC50 = 20 µM) (Butters et al., 2000b), whereas the inhibition of ß-glucocerebrosidase requires 25-fold higher concentrations (IC50 = 520 µM). In agreement with the chaperon-mediated effects of active site directed imino sugars, NB-DNJ also has a positive effect on peak activity and half-life of ß-glucocerebrosidase in the mouse (Priestman et al., 2000), although this compound did not increase enzyme activity in N370S mutant Gaucher cells at 100 µM (Sawkar et al., 2002). The recently obtained crystal structure of glucocerebrosidase bound to the irreversible inhibitor, conduritol ß-epoxide, may permit the design of more specific active site inhibitors, but data on mutant enzymes is lacking to allow predictions for improvement to protein folding (Premkumar et al., 2005).
Further structure/activity relationships need to be established for imino sugar compounds where anomeric specificity can be maintained. However, the possibility that inhibitors can be designed to target specific glycosphingolipidoses extends the exciting repertoire of these novel therapeutics.
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Summary |
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The results of clinical trials with NB-DNJ, and published data where this drug has been used to treat specific cases of disease, provide optimism for continued and long-term use in glycosphingolipidoses patients potentially where neuropathology remains a major obstacle for conventional treatment. Refinements to this drug may be necessary to further decrease the side effects shown by miglustat, particularly where paediatric use is indicated, but more potent drugs may not be required to obtain corrective treatment. Access of these drugs to the central nervous system has been demonstrated to be restricted to a fraction of the circulating concentration in plasma but effective in reducing GSL burden. A further understanding for the basis of cell and organelle targeting is necessary before small molecules can be designed for optimal use.
Many imino sugars are in preclinical or phase I/II clinical trials for the evaluation of molecular chaperone therapy for the glycosphingolipidoses (Amicus Therapeutics, North Brunswick, NJ) increasing the options to treat specific diseases. Imino sugar exposure to patients with different disease phenotypes is expected to increase with both SRT and CMT, and these approaches may provide tolerable therapeutic alternatives for treating the glycosphingolipidoses.
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Acknowledgements |
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The authors thank Dr. Mark Wormald for providing Figure 3 and the Oxford Glycobiology Institute for support.
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Abbreviations |
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CGT, ceramide glucosyltransferase; CMT, chaperon-mediated therapy; CNS, central nervous system; DGJ-deoxygalactonojirimycin; ER, endoplasmic reticulum; ERT, enzyme replacement therapy; GSL, glycosphingolipid; NB-DNJ, N-butyl-deoxynojirimycin; NMR, nuclear magnetic resonance; P-gp, P-glycoprotein; SRT, substrate reduction therapy
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References |
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