Glycobiology vol. 2 no. 3 pp. 199-210, 1992

MINI REVIEW

Amino-sugar glycosidase inhibitors: versatile tools for glycobiologists

Bryan Winchester and George W.J.Fleet1 Division of Biochemistry and Metabolism, Institute of Child Health (University of London), 30 Guilford Street, London WC1N 1EH, UK and 'Dyson Perrins Laboratory and Oxford Centre for Molecular Sciences, South Parks Road. Oxford 0X1 3QY, UK

Introduction Amino-sugar glycosidase inhibitors are analogues of monosaccharides in which the ring oxygen has been replaced by nitrogen. This substitution renders the compounds metabolically inert, but does not prevent their recognition by glycosidases and other carbohydrate-recognizing proteins. They inhibit glycosidases by mimicking the pyranosyl and furanosyl moiety of the corresponding substrates. Nature seems to have learnt this piece of chemistry because many inhibitory aminosugars occur naturally in microorganisms and plants (Fellows etal, 1989, 1992; Fellows and Nash, 1990). They include polyhydroxylated derivatives of piperidine, pyrrolidine, indolizidine and pyrrolizidine. Glycosidases are involved in several important biological processes, such as intestinal digestion and the catabolism and post-translational modification of glycoproteins. The realization that amino-sugar glycosidase inhibitors might have enormous therapeutic potential in many disease or protective mechanisms by altering the glycosylation or catabolism of glycoproteins, or by blocking the recognition of specific sugars, has led to a tremendous interest and demand for these compounds. Although ~25 amino-sugar glycosidase inhibitors have been isolated from natural sources, their extraction and purification are time consuming and costly. Furthermore, some of the natural sources are scarce or difficult to obtain. Consequently, many natural products and synthetic analogues have been synthesized (Legler and Julich, 1984; Fleet, 1988, 1989; Fleet etal., 1988a, 1990), including some by routes using enzymes (Kajimoto etal., 1991). A miniindustry has been created to meet the demand. Several hundred amino-sugars have been synthesized but only a small proportion is available for research in the public domain. In this review, the structural basis of the specificity of inhibition of the aminosugars and their current and potential application to biomedical and biotechnological problems will be reviewed.

Piperidines The archetypal amino-sugar inhibitor and the first to be described was nojirimycin (Ishida etal., 1967), which was isolated from bacteria (Streptomyces). It is the polyhydroxylated piperidine corresponding to glucose in the pyranose configuration, and is a potent competitive inhibitor of a- and /3-glucosidases © Oxford University Press

The structural basis of the inhibition of glycosidases by these compounds is obvious, but subtle differences in the specificity of inhibition of isoenzymes in the same cell or in different species occur. Multiple forms of the enzyme a-mannosidase occur in mammalian tissues (Winchester, 1984), but only the Golgi a-mannosidase I form in rat liver is inhibited by deoxymannojirimycin at micromolar concentrations (Bischoff and Kornfeld, 1984). The Golgi a-mannosidase II, endoplasmic reticulum, lysosomal and neutral forms of the enzyme in rat liver are not affected. The selective inhibition of Golgi a-mannosidase I is consistent with the blocking by DMJ of the conversion of high-mannose asparagine-linked glycans to complex glycans in cells in culture (Fuhrmann et al., 1984). DMJ is only a relatively weak competitive inhibitor of jack bean a-mannosidase (K\ 0.4 mM at pH 4.5), whereas the less stable mannojirimycin has a K, value of 6.5 /tM under the same conditions (Legler and Julich, 1984). In fact, DMJ is a more potent inhibitor of a-L-fucosidase (AT, 5 /*M) than of a-D-mannosidase (750 jiM) (Evans etal., 1985; Winchester etal., 1990). Figure 2 shows the structural basis of this specificity and the minimal requirements for the inhibition of a-L-fucosidase by piperidine derivatives (Winchester et al., 1990). DNJ also displays a wide range of potency towards a-glucosidases from a variety of organisms and tissues. The isolation or synthesis of analogues of deoxynojirimycin with one or more hydroxyl groups modified has permitted the definition of the structural requirements in a polyhydroxylated piperidine necessary for the inhibition of the main mammalian hexosidases. For example,' fagomine (Figure 1), which is equivalent to DNJ or DMJ minus the C-2 hydroxyl group, does not inhibit most a- or /3-glucosidases or -mannosidases, but it does inhibit mouse gut a-glucosidases (Scofield et al, 1986). This shows the relative importance of this chiral centre to the substrate specificities of these enzymes. Such information has been invaluable in the design of novel and more specific inhibitors. 199

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Key words: amino-sugars/biomedical applications/biotechnological applications/glycosidase inhibition

from various sources. The naturally occurring polyhydroxylated piperidine analogues of mannose and galactose, nojirimycin B and /3-galactostatin (Figure 1), are inhibitors of a-mannosidase (Niwa et al., 1984) and /3-galactosidase (Miyake and Ebata, 1988), respectively. The removal of the anomeric hydroxyl group to form the 1-deoxy derivatives does not destroy the inhibitory properties. 1-Deoxynojirimycin was first made by reduction of nojirimycin (Inouye et al., 1968), but it has subsequently been isolated from bacterial cultures (Murao and Miyata, 1980) and the mulberry (Yagi et al., 1976) and synthesized chemically (Kinast and Schnedel, 1981). It is more stable than nojirimycin and has become the model compound in this area of research, giving rise to a trivial but very informative nomenclature for the 1-deoxy analogues of other amino-sugars, e.g. deoxymannojirimycin (DMJ) or deoxyfuconojirimycin (DFJ) (see Figure 1). Amino-sugars can also be described systematically as derivatives of the parent heterocyclic compound or sugar, e.g. DNJ is 25-hydroxymethyl-3./?,4^?,5S-trihydroxy-piperidine or 1,5 dideoxy-1,5imino-D-glucitol.

B.Winchester and G.W.J.FIeet CH 2 OH

CH 2 OH

CH 2 OH

CH 2 OH

OH Noprimycin 5-amino.5-deoxy-Dglucopyranose CH 2 OH

Deoxynojirimycin, DNJ 1,5-dideoxy-i ,5-imino-D-glucitol or 2S-hydroxymethyl-3R,4R,5Strihydroxypiperidine

CH 2 OH

CH 2 OH

Fagorrune

CHjOH

•NCH3

\

c=o

OH

OH

a-Homonojinmycin

Nojirimycin B

CH,OH The corresponding 1,5-dideoxy-1,5-imino-hexitols ol fucose(DFJ), D-galactose, D-glucuronic acid, Liduronic acid, N-acetyl-D-gfucosamine and -galaclosamine and D-and L-rhamnose have been synthesised

Deo»ymanno|irimyein (OMJ )

Fig. 1. Structures of polyhydroxylated pipendines: analogues of pyranose sugars

OH OH

1

Pyrrolidines J3H

a-L-Fucose Deoxytuconojnmycm (DFJ) Ki. 0 01 M M

OH

CH2OH

OH !

..OH

^

Deoxymannoionmycin (OMJ)

D-Rhamno|inmyan (RMJ) Ki, 7

OH OH

N H L-Fuconic-5-lactam Ki. 0.4mM

2,3,4,-Trihydroxy p p e r d n e Ki, 8yM

,»OH

1-Deoxy-6-8a-diepicastanospermine

Fig. 2. Amino-sugar inhibitors of a-L-fucosidase.

200

Ki, 1.3

Polyhydroxylated pyrrolidines resemble sugars in the furanose configuration (Figure 3) and are usually potent inhibitors of the corresponding glycosidase. 2,5-Dihydroxymethyl-3,4 dihydroxypyrrolidine (DMDP), a naturally occurring analogue of /3-D-fructofuranose (Welter et ai, 1976), is a potent inhibitor of invertase (Evans et ai, 1985), but it does not inhibit the corresponding mammalian enzyme (sucrase), illustrating the differences in substrate binding or mechanism of action of isoenzymes. DMDP lacks the equivalent of the anomeric hydroxyl group, as do all the other natural furanose analogues except the pyrroline analogue of D-arabinose. Interestingly, DMDP can also inhibit glycosidases usually considered to catalyse the hydrolysis of glycosidic linkages involving sugars in the pyranose configuration, e.g. mammalian a-glucosidase I (Elbein et at, 1984) and human a- and /3-D-glucosidases (Cenci di Bello et ai, 1985). Pyrrolidines corresponding to several pentoses have also been isolated or synthesized (Fleet et ai, 1988b). They generally inhibit the appropriate glycosidase, but can also inhibit structurally related hexosidases. l,4-Dideoxy-l,4-imino-L-arabinitol is an inhibitor of yeast a-glucosidase (Fleet et ai, 1985) as well as of a-L-arabinosidase (Axamawaty et al., 1990). Loss of either of its two ring hydroxy groups abolishes the inhibition. In view of this inhibition of hexosidases by furanose analogues, several 1,4-dideoxy-l,4-imino-hexitols, i.e. analogues of hexoses in the furanose configuration, have been synthesized (Figure 3). l,4-Dideoxy-l,4-imino-D-mannitol, the analogue of mannofuranose, is a potent competitive inhibitor of jack bean a-Dmannosidase (K, 0.8 /*M) (Fleet etai, 1984) and human lysosomal a-D-mannosidase (K, 13 /tM), but is a poor inhibitor of a-D-mannosidase I and other processing a-mannosidases. It has the opposite specificity to DMJ, suggesting that the amannosidases fall into two groups on the basis of their inhibition by furanose and pyranose analogues. The mechanism by which furanose analogues inhibit pyranosidases is unknown,

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N-Methyldeoxynojinmycin

Amino-sugar glycosidase inhibitors

CH2OH HOH 2C HOCH

HOCH

CH2OH

HOH2C OH a-O-Glucofuranose

OH a-D-Mannofuranose

a-L-Arabinoluranose

HOH2C

2R,5R-Dihydroxymethyl3R,4R-dihydroxypyrrolidine (DMDP)

1,4-Dideoxy-1,4-iminoD-glucitol (DIG)

1,4-Dideoxy-1,4-imino L-arabinitol (LAB )

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HOHjC

CH2OH

1,4-0ideoxy-1,4-imino D-mannitol (DIM)

Fig. 3. Polyhydroxylated pyrrolidines: analogues of furanose sugars.

but it is clear that at least three correct chiral centres are essential for inhibition, whether they are in the furanose or pyranose analogue.

INDOUZIDINES

FUSED PIPERIDINE AND PYRROLIDINE

OH

Indolizidines A great impetus to research on amino-sugars and their application was the discovery that a polyhydroxylated indolizidine was the causative agent in swainsona toxicosis, a neurological disorder in grazing animals in Australia caused by ingestion of plants of the genus Swainsona (Dorling etai, 1978). They showed that this alkaloid, which they called swainsonine (Figure 4), was a potent inhibitor of lysosomal a-mannosidase. Its ingestion induced a phenocopy of the genetic lysosomal storage disease, a-mannosidosis, in which the deficiency of lysosomal a-mannosidase leads to the progressive accumulation in lysosomes of mannose-rich oligosaccharides. The inhibition of lysosomal a-mannosidase by swainsonine has been attributed to the similarity between the stereochemistry of the hydroxyl groups in the alkaloid and in mannose in the furanose configuration (Cenci di Bello et ai, 1983). Swainsonine was also found to inhibit the Golgi processing a-mannosidase II, preventing the formation of complex asparagine-linked glycans, but it did not inhibit Golgi a-mannosidase I (Tulsiani et al., 1982). The specificity of inhibition of mammalian a-D-mannosidases by swainsonine at low concentrations (micromolar) resembles that of the furanose analogue, l,4-dideoxy-l,4imino-D-mannitol (DIM), rather than that of the pyranose analogue, DMJ. This reinforces the hypothesis that mammalian a-D-mannosidases may fall into two classes on the basis of their mechanism. However swainsonine is a much more potent inhibitor than either DIM or DMJ and most human glycosidases are inhibited by 1 mM swainsonine. The bicyclic ring may impose rigidity on the structure that resembles a common intermediate in the hydrolytic pathway. Swainsonine is also present in plants of the species Astragalus L and Oxytropis DC, which cause locoweed poisoning in the USA (Molyneux and James, 1982). Several isomers or deoxy-derivatives of swain-

-

2

\

OH

Swainsonine IS^R.SR.SaR-trihydroxyindolizidine

Castanospermine 1S,6S,7R,8H,8aR-1,6,7,8tetrahydroxyindolizidine OH

CH 2 OH CH2OH NH

H6\OH

1,4-Dideoxy-1,4-imino D-mannitol (DIM) PYRROLIDINE ANALOGUE

Deoxynojirimycin (DNJ) PIPERIDINE ANALOGUE

Fig. 4. Polyhydroxylated indolizidines (fused piperidine and pyrrolidine).

sonine have been synthesized. 2-Episwainsonine (or Glcswainsonine), which resembles gluco-furanose, is a weak competitive inhibitor of fungal a-glucosidase (K, 50 /iM) (Elbein et al., 1987), but its naturally occurring derivative, 8-deoxyGlc-swainsonine (lentiginosine), has greater inhibitory activity 201

B.Winchester and G.W.J.Fleet

PYRROLIZIDINES

-

inhibition shown by isoenzymes towards monocyclic furanose and pyranose analogues are retained in their susceptibility to inhibition by indolizidines.

Pyrrolizidines An inhibitory, polyhydroxylated pyrrolidine ring can also be part of a pyrrolizidine, which consists of two fused pyrrolidines (Figure 5). Several inhibitory naturally occurring polyhydroxylated pyrrolizidines with a 1C substitution on an unusual position (C3) have been isolated from Castanospermum australe (Molyneux etal., 1988; Nash etal, 1990) and the South American sp. Alexa (Nash et al., 1988). Australine, its name reflecting the importance of the Antipodes as a source of these compounds, can be regarded as a ring-contracted form of castanospermine (Figure 5). Its strong structural resemblance to DMDP probably explains its ability to inhibit fungal a-glucosidase and processing a-glucosidase I (Tropea et al., 1989). In contrast, the pyrrolizidines equivalent to ring-contracted swainsonine (Carpenter et al., 1989) and cyclized DIM (Fairbanks etal., 1991) are not good inhibitors of a-D-mannosidases (Figure 5). Several natural isomers of alexine (Figure 5), the first pyrrolizidine with a carbon branch at C3 to be isolated, are weak inhibitors of mouse disaccharidases, but are strong inhibitors of fungal a-glucosidase (Nash et al., 1990).

Derivatives with substituents at the anomeric position Although 5-amino-5-deoxy-hexopyranoses such as nojirimycin and mannojirimycin tend to be more potent inhibitors than the corresponding l,5-dideoxy-l,5-iminohexitols (Legler, 1990), they are generally less stable. Oxidation produces the 5-lactam (Figures 1 and 2). The aldonolactams of L-fucose, D-galactose,

TWO FUSED PYRROLIDINES Both inhibit amyloglucosidase

2)

OH

CH 2 OH

CASTANOSPERMINE

2>

OH 2)

Cycllsed DIM

OH

CH2OH ALEXINE , 7a-epiAUSTRALINE

Fig. 5. Polyhydroxylated pyrrolizidines—two fused pyrrolidines.

202

CH 2 OH

AUSTRALINE (1 R,2R,3R,7S,7aR)-3-hydroxymethyl1,2,7-trihydroxypyrrolizidine

DMDP 2R,5R-dihydroxymethyl3R,4R-dihydroxypyrrolidine

»OH

Ring-contracted SWAINSONINE

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towards the enzyme {K, 10 jtM) (Pastzuszak et al., 1990). In contrast 8-deoxy-swainsonine (Colegate et al., 1984) and 8-episwainsonine (Cenci di Bello et al., 1989) are inactive towards a-mannosidase. Analysis of the inhibition of a-mannosidase by a range of epimers of swainsonine and DIM has shown that the configuration of the substituents on the C-atoms corresponding to atoms 2, 3, 4 and 5 on mannose determine the potency of inhibition of furanose analogues (Cenci di Bello et al., 1989). Indolizidine can be considered as a fused pyrrolidine and piperidine, and the versatility of plants in synthesizing useful aminosugars is illustrated by the subsequent isolation from another antipodean legume of the polyhydroxylated indolizidine, castanospermine (Hohenschutz etal., 1981). The hydroxylation of the piperidine moiety in castanospermine resembles the pyranose form of glucose (Figure 4). Castanospermine is a potent inhibitor of lysosomal a- and j3-glucosidases (K, 0.1 and 7 nM, respectively) (Saul et al., 1983; Cenci di Bello etal., 1988) and processing a-glucosidases I and II (Pan et al., 1983). Several epimers and derivatives of castanospermine have also been isolated (Molyneux, 1990) or synthesized. The mannoanalogue, 6-epicastanospermine, is a good inhibitor of human neutral a-mannosidase and the l-deoxy-6-epiderivative inhibits lysosomal a-mannosidase (Winchester et al., 1990), exemplifying the subtlety of inhibition of isoenzymes. 6-Epicastanospermine inhibits yeast a-amyloglucosidase (Molyneux et al., 1986), but not human lysosomal a-glucosidase (Winchester et al., 1990). 1-Deoxy, 6,8a diepicastanospermine (Figure 2) is a very potent and specific inhibitor of a-L-fucosidase {K{ 1.3 /tM) (Winchester et al., 1990) and 6-acetamido-6-deoxy castanospermine specifically inhibits jS-TV-acetylglucosaminidase from various sources (/50 = /tM) (Liu etal., 1991; B.Winchester, unpublished results). Thus, either the polyhydroxylated piperidine resembling a pyranose or pyrrolidine ring resembling a furanose of an indolizidine can constitute an aminosugar glycosidase inhibitor. The differences in specificity of

Amino-sugar glycosidase inhibitors

Applications Enzymology of glycosidases Affinity chromatography ligands. Amino-sugars bind to specific glycosidases in a reversible manner. This makes them ideal affinity chromatography ligands for the purification of glycosidases, as long as they can be linked to the spacer arm of the inert chromatographic support without disrupting the binding. As the hydroxyl groups are needed for specific recognition, linkage has to be through the ring nitrogen or by the addition of another functional group. Alkylation of the ring nitrogen of polyhydroxylated piperidines does not abolish their inhibition, although it may decrease the affinity and alter the specificity. Mono- and dimethylation of DNJ increases the Kx for aglucosidase I ~ 10-fold and markedly decreases the affinity for a-glucosidase II (Hettkamp et al., 1984). The N-carboxypentyl derivatives of deoxynojirimycin and its epimers retain their inhibitory properties and have been coupled to Sepharose to make affinity chromatography supports for the purification of a-glucosidase I (Hettkamp etai, 1984), an early processing a-mannosidase (Schweden et al., 1986; Schweden and Bause, 1989; Kaushal et al., 1990) and a-L-fucosidase (Paulsen and Matzke, 1988; Scudder et al., 1990). An alternative method for the assembly of the ligands has been published (Bernotas and Ganem, 1990). Alkylation of the ring nitrogen of the furanose analogues (l,4-dideoxy-l,4-iminohexitols and pentitols, see Figure 3), generally abolishes inhibition (Fairbanks et al., 1991), but can also alter the specificity. N-Benzylation of l,4-dideoxy-l,4-imino-L-allitol (DIA), a moderately good inhibitor of human liver a-mannosidases and a-fucosidase, destroys the inhibition of a-mannosidase, but creates a specific and more potent inhibition of a-fucosidase (Al Daher et al.,

1989). Substitution of the ring nitrogen of indolizidine generates a quaternary ammonium ion, but Af-alkylated castanospermine retains activity towards a-glucosidase, but not j3-glucosidase (B.Winchester, unpublished results). The Akixides of castanospermine (Saul et al., 1983) and swainsonine (Molyneux and James, 1982) are also active. Attempts to make an affinity support of swainsonine by N-alkylation have been unsuccessful (B. Winchester, unpublished results). Differential assay ofisoenzymes. The ability of an amino-sugar to inhibit isoenzymes selectively can be exploited to assay one isoenzyme in the presence of another. Multiple forms of /3-D-galactosidase occur in mammalian tissues. The lysosomal form acts exclusively on the /3-D-galactosidic linkage, whereas the abundant cytosolic form has /3-D-galactosidase, /3-D-glucosidase, j3-D-xylosidase and a-L-arabinosidase activities (Chester et al., 1976). By assaying crude tissue extracts with the jS-galactoside substrate in the presence of a specific j3-Dglucosidase inhibitor (DNJ or DMDP) which inhibits the broad specificity form, it is possible to assay the specific lysosomal /3-galactosidase selectively (Chinchetru etai., 1986). Such differential assays may pre-empt the need to separate isoenzymes for the detection of genetic deficiencies of the various forms of a glycosidase. Seminal a-glucosidase can be used as a marker of epididymal function and castanospermine is used to improve the specificity of the assay (Cooper et al., 1990). The various inhibitors of a-D-mannosidase have been used to block competing activities in the investigation of the specificity of a-mannosidase isoenzymes towards natural substrates (DeGasperi etai, 1992). Mechanism of action of glycosidases. Enzyme inhibitors can often provide information about the mechanism of action and chemical topography of the active sites of enzymes. The most reliable information comes from studies with purified enzymes, preferably those for which the crystal structure is known. This situation does not pertain for glycosidases, except lysozyme, particularly for the more biomedically important mammalian glycosidases. However, many human glycosidases have been cloned recently and structure/function relationships are emerging from mutation analysis in genetic disorders. Despite this limitation, several detailed studies of the inhibition of plant glycosidases by amino-sugars have been carried out (Kang and Elbein, 1983; Legler and Julich, 1984; Saul et al., 1984; Dale etai., 1985; Fleet, 1985; Axamawaty etai., 1990: Legler, 1990). The common structural feature of amino-sugars is the ring nitrogen, and its effect on the conformation and electrostatic properties of the inhibitors has been considered. The inhibition of glycosidases by amino-sugars increases with pH, reflecting ionization of groups on the inhibitor or enzyme. Taking into account the pKa values of the ring nitrogens and the acidic activity-pH range of many glycosidases, the most likely explanation is the formation of an ion pair between a protonated inhibitor and an anionic group (probably a carboxylate anion) in the active site. The protonated amino-sugar will resemble the glycosyl cation postulated to be formed during the action of glycosidases. However, the precise basis of inhibition will depend on the pKa of the inhibitor and the pH optimum of the enzyme. For example, the mechanisms of inhibition of human acidic and neutral a-mannosidase by swainsonine are probably different (Cenci di Bello et al., 1989). Elucidation of a plausible mechanism will require measurement of the pH dependence of kinetic parameters using pure enzyme. The conformation, size and chemical modification of the amino-sugar are also 203

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D-glucose, D-glucuronic acid, D- and L-mannose, N-acetylD-galactosamine, N-acetyl-D-glucosamine and D- and Lrhamnose have been synthesized, and are all inhibitors of the relevant glycosidase (Fleet etai, 1988a; Legler, 1990; Fairbanks et al., 1992). In general, both the a- and /3-D-glycosidases corresponding to the lactam are inhibited, but the /3-glycosidase is inhibited more strongly than a-glycosidases. Thus, the D-mannonolactam inhibits both human liver lysosomal a- and /?-mannosidase (/50 50 and 10 /tM, respectively) (Fairbanks et al., 1992) and the K, values for inhibition of Escherichia coli a- and /3-galactosidase by Dgalactonolactam are 4 and 0.07 mM, respectively (Legler, 1990). This may suggest that the stereochemistry of the lactams more closely resembles that of the /3-anomers. The addition of a substituent to the anomeric position may enhance the potency or specificity of inhibition by an amino-sugar. a-Homodeoxynojirimycin (Figure 1), a naturally occurring derivative of DNJ, has similar inhibitory properties to the parent compound (Kite et al., 1988). The synthetic a-homomannojirimycin is a weak inhibitor of mammalian a-mannosidases, as is DMJ, but unlike DMJ it is not inhibitory towards a-L-fucosidase (Bruce et al., 1989), even though the chirality of the secondary hydroxyl groups is correct (Figure 2). Addition of a hydroxymethyl group to the corresponding furanose analogue, DIM, also has very little effect on the inhibition of a-mannosidases (B. Winchester and G.W.J. Fleet, unpublished results). The synthetic /3-glucopyranosyl derivative of a-homonojirimycin (Liu, 1987), is a powerful disaccharidase inhibitor. Other disaccharide analogues have been prepared (see below).

B.Winchester and G.W.J.Fleet

Biological effects Glycosidases are involved in a wide range of important biological processes. The possibility of modifying or blocking these processes with amino-sugars for a therapeutic or biotechnological application has attracted a lot of attention. This strategy has largely been applied, so far, to glycosidases involved in intestinal digestion, post-translational processing of glycoproteins and the lysosomal catabolism of glycoconjugates.

Digestive glycosidases The mammalian gut produces several disaccharidases and oligosaccharidases to digest dietary carbohydrate to monosaccharides which are absorbed through the gut wall. They include sucrase, maltase, lactase, trehalase and isomaltase. Inhibition of all or some of these activities by amino-sugars could regulate the absorption of carbohydrate. The original isolation of deoxynojirimycin was prompted by the knowledge that extracts of mulberry were able to suppress the rise in blood glucose that follows eating and that this component might be beneficial to diabetics. Castanospermine, DNJ and derivatives of DNJ, such as Af-methyl, -hydroxyethyl and /3-glucosyl a-homodeoxynojirimycin have been shown to delay the hyperglycaemic response to oral sucrose in normal and diabetic (streptozotocin-induced) rats (Lembcke et al., 1985; Taylor etai, 1986; Rhinehart etai, 1987; Samulitis et al., 1987; Robinson et al., 1990). Several glucosyl derivatives of castanospermine have been synthesized to obtain selectivity of inhibition of the disaccharidases in vitro (Rhinehart et al., 1990), but prediction of specificity is not reliable. As well as differences in specificity between inhibitors towards digestive disaccharidases, there are marked differences between species (Campbell et al. ,1987; Fellows et al., 1989). Castanospermine 204

does not inhibit caterpillar sucrase and DMDP, which is ineffective against mammalian gut a-glucosidases, is a potent inhibitor of beetle a-glucosidases. It is hoped to use these differences, together with chemical modification of aminosugars, to develop selective pesticides. An exciting corollary to this research is the observation that some amino-sugars appear to deter insect feeding, DMDP and castanospermine having this effect on locust nymphs and aphids, respectively (Blaney et al., 1984; Dreyer etai, 1985). This feeding deterrence does not result from inhibition of glucosidases, but from recognition of these sugar analogues by taste receptors on sensillae on the insect mouthparts. Recognition of normal sugars, such as fructose or sucrose, evokes a response to eat, but DMDP (the fructose analogue) causes a non-feed response which can persist for a couple of hours (Fellows et al., 1989). The molecular basis of this signalling is under active investigation. Again, there is enormous potential for crop protection. Perhaps this is one of the reasons for their biosynthesis in plants. This phenomenon also illustrates another, as yet hardly exploited, potential application of amino-sugars as inhibitors or stimulators of processes mediated by carbohydrate-recognizing proteins, e.g. lectins, transport proteins.

Inhibition of lysosomal glycosidases The discovery that swainsonine can induce a reversible phenocopy of the genetic lysosomal storage disease, a-mannosidosis, in animals has led to the use of chemically induced deficiencies of lysosomal hydrolases as models for studying the pathogenesis of lysosomal storage diseases. The rate of intralysosomal accumulation of storage products, and the consequent changes in cellular and tissue structure and function, can be studied. The induced deficiency of the enzyme can be reversed and the endogenous enzyme activity restored by removing the amino-sugar from the diet of animals or culture medium of cells. The effect of the restored activity on the accumulated storage products and cellular and tissue changes can then be followed, i.e. a model of enzyme replacement therapy. Swainsonine-induced a-mannosidosis has been studied extensively in animals and in human cells in culture (Cenci di Bello etai., 1983). Reversible and irreversible pathological changes have been defined in the rat (Dorling et al., 1978), and the intracellular substrate specificity of lysosomal a-mannosidase has been elucidated (Daniel et al., 1992). Swainsonine is particularly effective in inducing a lysosomal storage disease because of its potency of inhibition of lysosomal a-mannosidase (Kx 70 nM) and its lysosomotropic behaviour (Chotai et al., 1983). It is a weak base with a pKa of 7.4, which ensures that it is taken up rapidly into cells by permeation and accumulates intralysosomally. However, it does also alter the glycosylation of glycoproteins by its potent inhibition of Golgi a-mannosidase II, causing the production of hybrid rather than complex asparagine-linked glycans. Thus swainsonine-induced mannosidosis is not an exact model of genetic a-mannosidosis. Castanospermine also disturbs the lysosomal catabolism of glycogen (Saul et al., 1985) and glycolipids (Cenci di Bello et al., 1988) by inhibiting lysosomal a-glucosidase in animals or /3-D-glucosidase in cells, respectively. The N-hydroxyethyl derivative of DNJ induces the accumulation of glycogen in normal human fibroblasts and the polarized HepG2 cells (Wisselaar et al., 1989). It also delays the processing of the endoplasmic reticulum a-glucosidase I and II. Thus, all the induced lysosomal storage diseases are complicated by the

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important. Many lactams, which are uncharged, are good inhibitors of glycosidases and it is possible that their conformation more closely resembles the transition state. However, not all piperidine lactams, e.g. L-fuconic 6-lactam (K{ 0.4 mM compared with a K, of 10 nM for deoxyfuconojirimycin, Fleet et al., 1988a), are potent inhibitors, suggesting that the relative contribution of electrostatic and steric factors varies from one glycosidase to another. Studies of the inhibition due to a series of derivatives of one inhibitor has permitted evaluation of the contribution of hydroxy groups to binding and the definition of essential structures for the inhibition of glycosidases (Cenci di Bello et al., 1989; Winchester et al., 1990). Some interesting observations have resulted from such studies. The addition of a bulky hydrophobic group near the 'anomeric' carbon or to the ring nitrogen of piperidine derivatives generates inhibition of A'-acetyl-^-D-hexosaminidase, suggesting a complementary hydrophobic area in the active site of the enzyme. The requirements for inhibition of mammalian /3-D-pyranosidases are more stringent than those for the corresponding a-D-pyranosidase, as evidenced by the lack of inhibition of /3-D-mannosidase by the many potent a-D-mannosidase inhibitors. This information about each glycosidase can be used to design more potent and selective inhibitors. It will be interesting to compare the chemical picture of the active sites of glycosidases built up by inhibitor studies with the X-ray crystallographic studies of the purified enzymes.

Amino-sugar glycosidase inhibitors

RER

Fig. 6. Effects of inhibitors on glycoprotein processing

effects of the amino-sugar inhibitors on processing glycosidases. Conversely, attempts to use amino-sugar glycosidase inhibitors to alter the glycosylation of proteins or inhibit intestinal digestion for therapeutic purposes may have induced lysosomal storage as a chronic complication. There is some evidence, though, that the doses of inhibitors used in experimental therapy may be too low to induce lysosomal storage (Rhinehart et al., 1991). Processing glycosidases The a-D-glucosidases and a-D-mannosidases involved in the post-translational processing of asparagine-linked glycans of glycoproteins can be selectively inhibited by different aminosugars [for reviews, see Fuhrmann et al. (1985), Elbein (1987), McDowell and Schwarz (1988) and Schwarz (1991)] (Figure 6). Using these inhibitors, the consequences of altering the glycosylation of a particular glycoprotein or the glycotype of cells in a defined way can be studied. Deoxynojirimycin (a-glucosidase I and II), castanospermine (a-glucosidase I), deoxymannojirimycin (a-mannosidase I) and swainsonine (a-mannosidase II) are available commercially and have become standard reagents. These inhibitors are not absolutely specific nor do they completely block processing unless relatively high concentrations are used, which in itself increases the lack of specificity. Therefore, there is great interest in finding more specific inhibitors. A'-Methyl-deoxynojirimycin has a lower ^ for a-glucosidase I than deoxynojirimycin and vice versa for a-glucosidase II (Hettkamp et al., 1984). Castano-

spermine has a similar K, for a-glucosidase I to N-methyldeoxynojirimycin (Pan et al., 1983), suggesting that substitution of the piperidine ring nitrogen favours the inhibition of a-glucosidase I. In contrast, the j3-D-glucopyranosyl derivative of a-homodeoxynojirimycin [2,6-dideoxy-2,6-imino-7-0 (|S-Dglucopyranosyl)-D-glycero-L-guloheptitol] (or MDL for short) exclusively inhibits a-glucosidase II at a concentration of 250 ^g/ml (Kaushal et al., 1988). Similarly kifunensine, a fungal alkaloid, which can be regarded as the cyclic oxamide of 1-amino-deoxymannonojirimycin, is a better (/50 50 nM) and more specific inhibitor of Golgi a-mannosidase I than deoxymannojirimycin (/50 10 /*M) (Elbein et al., 1990). a-Mannosidase I is particularly susceptible to amino-sugar pyranose analogues and perhaps the cyclic oxamide structure 'freezes' the kifunensine into a favourable conformation for inhibition. Although swainsonine is a potent inhibitor of a-mannosidase II, it does inhibit other processing a-mannosidases in human cells in culture (Cenci di Bello etal, 1983). 6-Deoxy-DIM (l,4,6-trideoxy-l,4-imino-D-mannitol) is a less potent inhibitor of a-mannosidase II, but may be more selective than swainsonine (G.W.J. Fleet and B. Winchester, unpublished work). The specificity of inhibition of the processing of many of the synthetic amino-sugars has not yet been evaluated, but many are more specific in vitro than their parent compounds. It would be impossible to describe all the applications of glycoprotein processing inhibitors. Suffice it to say that the effect of the common ones, e.g. swainsonine and castanospermine, on the biosynthesis, intracellular transport and function of most well characterized glycoproteins has been 205

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CS=Castanospermine SW=Swainsonine

B.Winchester and G.W.J.Fleet

evaluated. Their effects on many cellular processes dependent on glycosylation have also been studied, but perhaps the two areas of most excitement and interest are cancer and virus replication. Cancer

These effects can also be reproduced in vivo in animal models (Kino et al., 1985). The addition of swainsonine to the drinking water of mice prevents the colonization of injected B-16 BL6 melanoma to liver and lung (Newton et al., 1989). The effect of swainsonine on the highly malignant murine lymphoma MDAY-D2, in vitro and in vivo, indicates how swainsonine and other amino-sugars might act to prevent metastasis dissemination (Dennis, 1986). Swainsonine enhanced the antiproliferative effects of alfi interferon in vitro and in vivo, and vice versa, especially for cell lines expressing (3(1-6) branched asparagine-linked oligosaccharides, whose formation is prevented by swainsonine. Swainsonine and interferon a-2 also have a synergistic effect on the growth of the human H29 colon carcinoma in vitro and in vivo (Dennis et al., 1989). There is much evidence that swainsonine augments the natural tumoricidal activity of the immune system in vitro (Hino et al., 206

It has been suggested that secreted lysosomal hydrolases may be involved in tumour cell invasion by degrading glycoconjugates in the extracellular matrix (Bernacki et al., 1985). The amino-sugar, 2-acetamido-l,5-imino-l,2,5-trideoxy-Dglucitol (or the deoxynojirimycin analogue of ./V-acetyl-D-glucosamine) is a competitive inhibitor of the /3-Af-acetylglucosaminidase isoenzymes (2-8 /*M) in and secreted by human ovarian carcinoma cells (Woynarowska etal., 1992). It also inhibits the degradation of radiolabelled extracellular matrix by /3-A^-acetylhexosaminidase and the cells. The relevance of these observations to metastasis remains to be investigated. Interestingly, swainsonine decreases a normal cellular invasion process, the invasion of the basement membrane by firsttrimester trophoblasts, by preventing the formation of /S(l-6) branched AMinked glycans (Yagel etal, 1990). The specificity, potency and inertness of the amino-sugar glycosidase inhibitors make them attractive anti-cancer agents if they can be targeted appropriately. Antiviral activity The amino-sugars that inhibit the processing glycosidases (see Figure 6) can alter the glycosylation of viral glycoproteins, but this does not in general affect the infectivity of the viruses [for a brief summary of this work see Fellows et al. (1989)]. There are, however, some important exceptions. Some inhibitors of processing a-glucosidase I and II do decrease the infectivity of the human immunodeficiency virus (HIV) responsible for the acquired immune deficiency syndrome (AIDS) (Gruters et al., 1987; Tyms et al, 1987; Walker et al, 1987) and other retroviruses (Ruprecht et al, 1989), including the feline equivalent of HIV (Stephens etal, 1991). Castanospermine, DNJ and DMDP all affect HIV infectivity at concentrations which are not cytotoxic to lymphocytes, whereas inhibitors of processing a-mannosidases, such as deoxymannojirimycin and swainsonine, have no effect. The cellular basis of this activity is not clearly understood. An interaction between the heavily glycosylated viral envelope protein gpl20 and the membrane glycoprotein CD4 on the surface of T-lymphocytes is essential for infection. In vitro, this interaction leads to syncytium formation by cell-to-cell fusion. This model has been used to test the cytotoxicity of amino-sugars and their efficacy in decreasing viral infectivity. A systematic survey of 47 synthetic and natural amino-sugar analogues sought to define the structural features

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Both catabolic and processing glycosidases are involved in the transformation of normal cells to cancer cells and in tumour cell invasion and migration. Many tumour cells display aberrant glycosylation due to an altered expression of glycosyltransferases (Hakomori, 1985) and it has been known for a long time that the levels of glycosidases are elevated in the sera of many patients with different tumours (Woollen and Turner, 1965). Secreted glycosidases may be involved in the degradation of the extracellular matrix in tumour cell invasion (Bernacki et al, 1985). Furthermore, the lysosomal system is highly active in transformed cells, presumably reflecting enhanced turnover of glycoproteins and other macromolecules, and possibly increased exocytosis of lysosomal hydrolases. The use of amino-sugar analogues to prevent the formation of the aberrant asparagine-linked glycans and to inhibit catabolic glycosidases is being actively pursued as a therapeutic strategy for cancer [for a review, see Olden et al. (1991)]. The addition of aminosugars to cultures of transformed cells can alter their growth and ability to cause tumours. When murine melanoma B16-F10 cells were pretreated with swainsonine or castanospermine prior to intravenous injection into mice, pulmonary colonization was decreased (>80%) (Humphries etal, 1986a, b). The treated cells had more endoglycosidase-H-sensitive and concanavalin-A binding glycans on the cell surface, indicating that the inhibitors had prevented the formation of complex glycans, which have been implicated in metastasis (Humphries and Olden, 1989). The inhibitors were not cytotoxic and did not appear to affect the tumorigenicity or adhesiveness of the treated cells. It is probable that the changes in glycosylation affect late stages in metastasis, such as arrest and aggregation in the microvasculature of the lungs. Comparable in vitro studies with other transformed cells have shown that the chemically induced changes in glycosylation can have a variety of effects on the properties of the transformed cells, including growth (Hadwiger et al., 1986), decreased rate of growth of tumours (Ostrander et al., 1988) and expression of non-glycosylated oncogene products (Desantis et al., 1987). With some cells, no effect was observed (Sargent etal., 1987), and differential effects may be observed with different cell lines and inhibitors (Spearman etal., 1991).

1985) and in vivo (Humphries etal., 1988). It causes an increase in spleen cell number, particularly natural killer (NK) cells in normal mice, but has no anti-metastatic effect in mice depleted of NK cells. It also enhances lymphocyte interleukin-2 (IL-2) receptor expression and IL-2-induced proliferation following mitogen stimulation (Bowlin and Sunkara, 1988), and induces tumoricidal activity and secretion of IL-1 in macrophages (Grzegorzewski etal., 1989). The observation that swainsonine can induce the secretion of various cytokines has led to an investigation of its effect on bone marrow cells, an important consideration in chemotherapy for cancer (White etal., 1991). Swainsonine appears to be able to mimic colony-stimulating factors in murine bone marrow. It is not known whether this is due to a direct effect on haematopoiesis or to its ability to induce the secretion of cytokines. This property, together with its general lack of cytotoxicity, suggests it may have potential use in co-administration with immunosuppressive drugs in the chemotherapy of cancer or in the treatment of immunodeficiency.

Amino-sugar glycosidase inhibitors

Future prospects Amino-sugars may also affect other biological processes involving sugars and their derivatives. There is evidence that some effects of amino-sugars are due to a direct interaction of the analogue with a protein rather than to inhibition of a glycosidase. Swainsonine appears to inhibit mouse thymocyteerythrocyte rosette formation by direct interaction with a cell surface structure such as a lectin, rather than by altering the glycosylation of cell surface glycans (Sem et al., 1991). The transport of sugars may also be affected directly in a similar way. Sugar phosphates are involved in many energy-producing and biosynthetic pathways. It would be interesting to see the effect of phosphorylated amino-sugars on the enzymes of the glycolytic pathway, pentose phosphate pathway, and the de novo synthesis of purines and pyrimidines. The incorporation of amino-sugars into nucleoside diphosphate-sugar analogues might cause the inhibition of glycosyltransferase and the biosynthesis of glycoconjugates. The usefulness of aminosugars might not be restricted to the inhibition of glycosidases as they are very versatile and stable vehicles on which to carry

functional groups for the investigation of processes involving recognition of monosaccharide derivatives.

Acknowledgements The authors would like to acknowledge their longstanding collaborations with Dr Isabelle Cenci di Bello, Dr Peter Dorhng and Dr Linda Fellows and colleagues, and thank Miss Annie Clein for her expert secretarial help.

Abbreviations DFJ, deoxyfiiconojirimycin; D1A, l,4-dideoxy-l,4-imino-L-allitol; DIM, l,4-dideoxy-l,4-imino-D-mannitol; DMDP, 2,5-dihydroxymethyl-3,4-dihydroxypyrrohdine; DMJ, deoxymannojirimycin; DNJ, l,5-dideoxy-l,5-iminoD-glucitol; IL, interleukin; NK, natural killer.

References Al Daher.S., Fleet.G., Namgoong,S.K and Winchester.B. (1989) Change in specificity of glycosidase inhibition by A'-alkylation of amino sugars. Biochem. J., 258, 613-615. Axamawaty,M.T.H., Fleet.G.W.J., Hannah, K.A., Namgoong,S.K. and Sinnott.M.L. (1990) Inhibition of the a-L-arabinoftiranosidase III of Monilinia fructigena by l,4-dideoxy-l,4-imino-L-threitol and 1,4-dideoxy1,4-imino-L-arabinitol. Biochem. J., 266, 245-249. Bernacki,R.J., Niedbala.M.J. and Korytnyk.W. (1985) Glycosidases in cancer and invasion. Cancer Metastasis Rev., 4, 81—102. Bernotas.R.C. and Ganem,B. (1990) Easy assembly of ligands for glycosidase affinity chromatography. Biochem. J., 270, 539-540. Bischoff.J. and Kornfeld.R. (1984) The effect of 1-deoxymannojirimycin on rat liver a-mannosidases. Biochem. Biophys. Res. Commun., 125, 324-331. Blaney.W.M., Simmonds,M.S.J., Evans.S.V. and Fellows,L.E. (1984) The role of the plant secondary compound 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine as a feeding inhibitor for insects. Enlomol. Exp. Appl., 36, 209-216. Bowlin,T.L. and Sunkara.P.S. (1988) Swainsonine, an inhibitor of glycoprotein processing, enhances mitogen-induced interleukin-2 production and receptor expression in human lymphocytes. Biochem. Biophys. Res. Commun., 151, 846-859. Bruce.I., Fleet,G.W.J., Cenci di Bello.l. and Winchester.B. (1989) Iminoheptitols as glycosidase inhibitors: synthesis of, and mannosidase and fucosidase inhibition by, a-homomannojirimycin and 6-epi-homomannojirimycm. Tetrahedron Lett., 30, 7257-7260. Campbell.B.C, Molyneux.R.J. and Jones.K.C. (1987) Differential inhibition by castanospermine of various insect disacchandases. J. Chem. Ecoi, 13, 1759-1770. Carpenter,N.M , Fleet.G.W.J., Cenci di Bello.I., Winchester.B., Fellows, L.E. and Nash.R.J. (1989) Synthesis of the mannosidase inhibitors swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol and of the ring contracted swainsonines, (IS, 2R, 7R, 7aR)-l,2,7-trihydroxypyrrolizidine and (IS, 2R, 7S, 7aR)-l,2,7-trihydroxypyrrolizidine. Tetrahedron Lett., 30, 7261-7264. Cenci di Bello.I., Dorling.P. and Winchester.B. (1983) The storage products in genetic and swainsonine-induced human mannosidosis. Biochem. J., 215, 693-696. Cenci di Bello,I., Dorling.P., Evans.S., Fellows.L. and Winchester.B. (1985) Inhibition of human a- and 0-D-glucosidases and a- and /3-D-mannosidases by 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine. Biochemical Society Transactions, 613th Meeting, Cardiff, pp. 1127-1128. Cenci di Bello.I., Mann.D., Nash.R. and Winchester.B. (1988) Castanospermine-induced deficiency of lysosomal /3-D-glucosidase: a model of Gaucher's disease in fibroblasts. In Salvayre.R., Douste-Blazy.L. and Gatt.S. (eds), Lipid Storage Disorders. Plenum, pp. 635-641. Cenci di Bello.I., Fleet.G., Namgoong.S.K., Tadano,K.-I. and Winchester.B. (1989) Structure-activity relationship of swainsonine. Inhibition of human a-mannosidases by swainsonine analogues. Biochem. J., 259, 855-861. Chester.M.A., Hultberg.B. and Ockerman,P.-A. (1976) The common identity of five glycosidases in human liver. Biochim. Biophys. Acta, 429, 517-526. Chinchetru.M.A., Calvo.P., Cenci di Bello.I. and Winchester.B. (1986) /3-D-galactosidase isoenzymes in different sheep organs. Comp. Biochem. Physiol, 84,623-628.

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necessary in the inhibitors for antiviral activity and the site of action in the infectious cycle (Fleet et al., 1988c; Karpas et ai, 1988). Although l,4-dideoxy-l,4-imino-D-arabinitol and N-(5-carboxymethy 1-1 -pentyl)-1,5-imino-L-fucitol decreased the cytopathic effect and yield of infectious virus, the most effective compounds were the A'-methyl, -ethyl and -butyl derivatives of deoxynojirimycin. In particular, the N-butyl derivative markedly decreased the number of infectious particles and prolonged culture of infected cells with But DNJ led to the eventual elimination of HIV from cultures. This compound is now in Stage 2 clinical trials for the treatment of HIV. The precise basis of action of But DNJ is not known, but it is presumed that the aliphatic chain enhances uptake into cells and perhaps retention at a sensitive intracellular site. This may also explain the effect of the DFJ derivative, which is not an a-glucosidase inhibitor, but does inhibit lysosomal a-fucosidase. This indicates that turnover of glycoproteins, rather than alteration of glycosylation, may be the mechanism. It would be interesting to see if adding a hydrophobic tag to other potent inhibitors of lysosomal catabolism of glycoproteins is equally effective. Castanospermine is not taken up so readily into some cells in culture (B. Winchester, unpublished results) and this may explain why it is less effective than inhibitors that are less potent a-glucosidase inhibitors in vitro. Some support for this explanation is provided by a comparison of the anti-HIV activity of a series of castanospermine analogues (Sunkara et al., 1989) and other amino-sugars and drugs (Taylor et al., 1991). 6-0-Butanoyl castanospermine was — 20 times more active than castanospermine and 50 times more active than But DNJ. If this derivative is transported to the lysosomes or another acidic intracellular compartment, the castanospermine is probably released in situ by the action of endogenous esterases. Castanospermine inhibits HIV type 1 and 2 replication synergistically with 3'-azido-3'-deoxythymidine (zidovudine) (Johnson et al., 1989) and other drugs (Montefiori et al., 1989). Inhibition of the replication of a herpes virus cytomegalovirus by castanospermine and DNJ has also been attributed to the disruption of the processing and intracellular transport of viral glycoproteins, rather than to a direct consequence of altered glycosylation (Taylor et al., 1988).

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Fleet.G.W.J., Smith.P.W., Evans.S.V. and Fellows.L.E. (1984) Design, synthesis and preliminary evaluation of a potent a-mannosidase inhibitor: 1,4-dideoxy-l,4-imino-D-mannitol. / Chem. Soc. Chem. Commun., 1240-1241. Fleet,G.W.J., Nicholas.S.J., Smith.P.W., Evans.S.V., Fellows.L.E. and Nash.R.J. (1985) Potent competitive inhibition of a-galactosidase and a-glucosidase activity by l,4-dideoxy-l,4-iminopentitols: syntheses of l,4-dideoxy-l,4-imino-D-lyxitol and of both enantiomers of 1,4-dideoxy1,4-iminoarabinitol. Tetrahedron Lett., 26, 3127-3130. Fleet.G.W.J., Ramsden.N.G., Dwek.R.A., Rademacher.T.W., Fellows.L.E., Nash.R.J., Green,D.St.C. and Winchester.B. (1988a) 6-Lactams: synthesis from D-glucose, and preliminary evaluation as a fticosidase inhibitor, of L-fuconic-6-lactam. J. Chem. Soc. Chem. Commun., 483—485. Fleet.G.W.J., Son.J.C, Green,D.St.C, Cenci di Bello.I. and Winchester.B. (1988b) Synthesis from D-mannose of l,4-dideoxy-l,4-imino-L-ribitol and of the a-mannosidase inhibitor l,4-dideoxy-l,4-imino-D-talitol. Tetrahedron, 44, 2649-2655. Fleet.G.W.J., Karpas,A., Dwek.R.A., Fellows.L.E., Tyms.A.S., Petursson, S., Namgoong.S.K., Ramsden.N.G., Smith.P.W., Son.J.C, Wilson.F.X., Witty.D.R., Jacob.G.S. and Rademacher.T.W. (1988c) Inhibition of HIV replication by aminosugar derivatives. FEBS Lett., 237, 128—132. Fleet,G., Fellows,L. and Winchester.B. (1990) Plagiarizing plants: amino sugars as a class of glycosidase inhibitors. In Chadwick, D.J. (ed.), Bioactive Compounds from Plants. Wiley, Chichester (Ciba Foundation Symposium 154), pp. 112-125. Fuhrmann,U., Bause,E., Legler.G. and Ploegh,H. (1984) Novel mannosidase inhibitor blocking conversion of high mannose to complex ohgosaccharides. Nature, 307, 755-758. Fuhrmann,U., Bause.E. and Ploegh.H. (1985) Inhibitors of oligosaccharide processing. Biochim. Biophys. Acta, 825, 95-110. Gruters.R.A., Neefjes.J.J , Tersmette,M., de Goede,R.E.Y., Tulp,A., Huisman.H.G., Miedema.F. and Ploegh.H.L. (1987) Interference with HIVmduced syncytium formation and viral infectivity by inhibitors of trimming glucosidase. Nature, 330, 74-77. Grzegorzewski.K., Newton,S.A., Akiyama.S.K., Sharrow.S., Olden,K. and White,S.L. (1989) Induction of macrophage tumoricidal activity, major histocompatibility complex class II antigen (Iak) expression, and interleukin-1 production by swainsonine. Cancer Commun., 1, 373—379. Hadwiger.A., Niemann.H., Kabisch.A., Bauer.H. and Tamura.T. (1986) Appropriate glycosylation of the fins gene product is a prerequisite for its transforming potency. EMBO J., 5, 689-694. Hakomori.S. (1985) Aberrant glycosylation in cancer cell membranes as focused on glycolipids: Overview and perspectives. Cancer Res., 45, 2405-2414. Hettkamp.H., Legler,G. and Bause.E (1984) Purification by affinity chromatography of glucosidase I, an endoplasmic reticulum hydrolase involved in the processing of asparagine-linked oligosacchandes. Eur. J. Biochem., 142, 85-90. Hino,M., Nakayama,O., Tsururru.Y., Adachi,K., Shibata.T., Terano,H., Koshaka.M., Aoki.H. and Imanaki.H. (1985) Studies of an immunomodulator, swainsonine. I Enhancement of immune response by swainsonine in vitro. J. Antibiot., 38, 926-935. Hohenschutz.L.D , Bell,E.A., Jewess,P.J., Leworthy.D P., Pryce.R.J., Arnold,E. and Clardy.J. (1981) Castanospermine, a 1,6,7,8-tetrahydroindolizidine alkaloid from seeds of Castanospermum australe. Phytochemistry; 20, 811-814. Humphries,M.J. and Olden,K. (1989) Asparagine-linked oligosaccharides and tumor metastases. Pharmacol. Ther., 44, 85—105. Humphries.M.J., Matsumoto,K., White,S.L. and Olden.K. (1986a) Oligosaccharide modification by swainsonine treatment inhibits pulmonary colonization by B16-F10 murine melanoma cells. Proc. Natl. Acad. Sci. USA, 83, 1752-1756. Humphries,M.J., Matsumoto.K., White.S.L. and Olden,K. (1986b) Inhibition of experimental metastasis by castanospermine in mice, blockage of two distinct stages of tumor colonization by oligosaccharide processing inhibitors. Cancer Res., 46, 5215-5222. Humphries.M.J., Matsumoto.K., White.S.L., Molyneux.R.J. and Olden,K. (1988) Augmentation of murine natural killer cell activity by swainsonine: a new antimetastatic immunomodulator. Cancer Res., 48, 1410-1415. Inouye.S., Tsurouka.T., Ito.T. and Niida.T. (1968) Structure and synthesis of nojirimycin. Tetrahedron, 24, 2125-2144. Ishida.N., Kumagai,K., Niida.T., Tsuruoka.T. and Yumato.H. (1967) Nojirimycin. a new antibiotic. II Isolation, characterization and biological activity. /. Antibiot., 20, 66-71. Johnson,V.A., Walker,B.D., Barlow.M.A., Paradis.T.J., Chou.T.C. and Hirsch.M.S. (1989) Synergistic inhibition of human immunodeficiency virus type 1 and type 2 replication in vitro by castanospermine and 3'-azido-3'deoxythymidine. Antimicrob. Agents Chemother., 33, 53—57.

Downloaded from http://glycob.oxfordjournals.org/ at Purdue University Libraries ADMN on July 24, 2015

Chotai.K., Jennings,C, Winchester,B. and Dorling,P. (1983) The uptake of swainsonine, a specific inhibitor of a-D-mannosidase, into normal human fibroblasts in culture. J. Cell Biol., 21, 107-117. Colegate.S.M., Dorling,P.R. and Huxtable,C.R. (1984) Synthesis of 8-deoxyswainsonine. Aust. J. Chem., 37, 1503. Cooper.T.G., Yeung.C.H., Nashan.D., Jockenhovel.F. and Nieschlag.E. (1990) Improvement in the assessment of human epididymal function by the use of inhibitors in the assay of a-glucosidase in seminal plasma. Int. J. Androi, 13, 297-305. Dale.M.P., Ensley.H.E., Kenn.K., Sastry.K.A.R. and Byers.L.D. (1985) Reversible inhibitors of j3-glucosidase. Biochemistry, 24, 3530-3539. Daniel,P.F., Evans.J.E., De Gasperi,R., Winchester.B. and Warren.C.D. (1992) A human lysosomal a(l-6)mannosidase active on the branched trimannosyl core of complex glycans. Glycobiology, 2, in press. De Gasperi.R., Al Daher,S., Winchester.B. and Warren,C.D. (1992) The substrate specificity of the bovine and feline cytosolic a-mannosidases. Biochem. J., in press. Dennis.J. (1986) Effects of swainsonine and polyinosinic:polycytidylic acid on murine tumor cell growth and metastasis. Cancer Res., 46, 5131-5136. DennisJ.W., Koch.K. and Beckner,D. (1989) Inhibition of human HT29 colon carcinoma growth in vitro and in vivo by swainsonine and human interferon-al. J. Natl. Cancer lnst., 81, 1028-1033. Desantis.R., Santer.V.V. and Glick,M C. (1987) NIH 3T3 cells transfected with human tumor DNA lose the transformed phenotype when treated with swainsonine. Biochem. Biophys. Res. Commun., 142, 348-353. Dorling,P.R., Huxtable.C.R. and Vogel.P. (1978) Lysosomal storage in Swainsona spp. Toxicosis: an induced mannosidosis. Neuropathol. Appl. Neurobwl., 4, 285-295. Dreyer,D.L., Jones,K.C. and Molyneux.R.J. (1985) Feeding deterrency of some pyrrolizidine, indolizidine, and quinolizidine alkaloids towards pea aphid (Acyrthosiphon pisum) and evidence for phloem transport of indolizidine alkaloid swainsonine. J. Chem. EcoL, 11, 1045— 1051. Elbein.A.D. (1987) Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Annu. Rev. Biochem., 56, 497-534. Elbein.A.D., Mitchell,M., Sanford.B.A., Fellows.L.E. and Evans,S.V. (1984) The pyrrolidine alkaloid, 2,5-dihydroxymethyl-3,4-dihydroxypyrrohdine, inhibits glycoprotein processing. J. Biol. Chem., 259, 12409-12413. Elbein.A.D., Szumilo.T., Sanford.B.A., Sharpless.K.B. and Adams.C. (1987) Effect of isomers of swainsonine on glycosidase activity and glycoprotein processing. Biochemistry, 26, 2502-2510. Elbein.A.D., Tropea.J.E., Mitchell.M. and Kaushal,G.P. (1990) Kifunensine, a potent inhibitor of the glycoprotein processing mannosidase I. J. Biol. Chem., 265, 15599-15605. Evans.S.V., Fellows.L.E., Shing.T.K.M. and Fleet.G.W.J. (1985) Glycosidase inhibition by plant alkaloids which are structural analogues of monosaccharides. Phytochemistry, 24, 1953-1955. Fairbanks.A.J., Fleet.G.W.J., Jones.A.H., Bruce.I , Al Daher.S., Cenci di Bello.I. and Winchester.B. (1991) Synthesis from a heptonolactone and effect on glycosidases of (lS,2R,6R,7S)-l,2,6,7-tetrahydroxypyrrolizidine. Tetrahedron, 47, 131-138. Fairbanks.A.J., Carpenter.N.C, Fleet.G.W.J., Ramsden.N.G., Cenci di Bello.I., Winchester.B.G., Al-Daher,S.S. and Nagahashi.G. (1992) Synthesis of and lack of inhibition of a rhamnosidase by both enantiomers of deoxyrhamnojirimycin and rhamnonolactam: /3-mannosidase inhibition by 6-lactams. Tetrahedron, 48, 3365-3376. Fellows,L. and Nash.R. (1990) Sugar-shaped alkaloids. Sa. Prog. Oxford-, 74. 245-255. Fellows,L., Kite,G., Nash,R., Simmonds.M. and Scofield.A. (1989) Castanospermine, swainsonine and related polyhydroxy alkaloids: structure, distribution and biological activity. In Poulton.J.E., Romeo.J.T. and Conn.E.E. (eds), Plant Nitrogen Metabolism. Plenum, pp. 395-427. Fellows,L.E., Kite.G.C, Nash.RJ., Simmonds.M.S.J. and Scofield.A.M. (1992) Distribution and biological activity of alkaloidal glycosidase inhibitors from plants. In Mengel.K. and Pilbeam.D.J. (eds), Nitrogen Metabolism of Plants. Clarendon Press, Oxford, pp. 271-284. Fleet,G. (1989) Homochiral compounds from sugars. Chemistry in Britain, 287-292. Fleet.G.W.J. (1985) An alternative proposal for the mode of inhibition of glycosidase activity by polyhydroxylaed piperidines, pyrrolidines and indolizidines: implications for the mechanism of action of some glycosidases. Tetrahedron Lett., 26, 5073-5076. Fleet.G.W.J. (1988) Amino-sugar derivatives and related compounds as glycosidase inhibitors. In Leeming.P.R. (ed.), 4th SCI-Med. Chem. Symposium. Cambridge, September 1987. Royal Society of Chemistry, London, pp. 149-162.

Amino-sugar glycosidase inhibitors Niwa.T., TsuroukaX. Goi.H., Kodama,Y., Itoh.J., Inoue.S., Yamada,Y., Niida,T., Nobe.M. and Ogawa.Y. (1984) Novel glycosidase inhibitors, nojirimycin B and D mannonic-6-lactam. /. Antibiot., 37, 1579-1586. Olden,K., Breton.P., Grzegorzewski.K., Yasuda.Y., Gause.B.L., Oredipe, O.A., Newton.S.A. and White.S.L. (1991) The potential importance of swainsonine in therapy for cancers and immunology. Pharmacol. Ther., 50, 285-290. Ostrander.G.K., Scribner.N.K. and Rohrschneider,L.R. (1988) Inhibition of v-yms-induced tumor growth in nude mice by castanospermine. Cancer Res., 48, 1091-1094. Pan,Y.T., Hori.H., Saul.R., Sanford.B.A., Molyneux.R.J. and Elbein, A.D. (1983) Castanospermine inhibits the processing of the oligosaccharide portion of the influenza viral hemagglutinin. Biochemistry, 22, 3975-3984. Pastuszak,I., Molyneux,R.J., James,L.F. and Elbein.A.D. (1990) Lentiginosine, a dihydroxyindolizidine alkaloid that inhibits amyloglucosidase. Biochemistry, 29, 1886-1891. Paulsen.H. and Matzke.M. (1988) Synthese des a-L-fucosidase-inhibitors l,5-didesoxy-l,5-imino-L-fucit und einiger seiner derivate. Leibigs Ann. Chem., 1121-1126. Rhinehart.G.L., Robinson.K.M., Payne.A.J., Wheately.M.E., Fisher.J.L., Liu,P.S. and Cheng,W. (1987) Castanospermine blocks the hyperglycemic response to carbohydrates in vivo: a result of intestinal disaccharidase inhibition. Life Sci., 41, 2325-2331. Rhinehart.B.L., Robinson.K M., King.C.H. and Liu.P.S. (1990) Castanospermine-glucosides as selective disaccharidase inhibitors. Biochem. Pharmacol., 39, 1537-1543. Rhinehart.B.L., Begovic.M.E. and Robinson,K.M. (1991) Quantitative relationship of lysosomal glycogen accumulation to lysosomal a-glucosidase inhibition in castanospermine-treated rats. Biochem. Pharmacol., 41, 223-228. Robinson.K.M., Heineke.E.W. and Begovic.M.E. (1990) Quantitative relationship between intestinal sucrase inhibition and reduction of the glycemic response to sucrose in rats. J. Nutr., 120, 105-111. Ruprecht.R.M., Mullaney,S., Andersen,J. and Bronson,R. (1989) In vivo analysis of castanospermine, a candidate antiretroviral agent. J. Acquir. Immune Defic. Syndr., 2, 149—157. Samulitis.B.K., Goda.T., Lee.S.M. and Koldovski,O. (1987) Inhibitory mechanism of acarbose and 1 -deoxynojirimycin derivatives on carbohydrases in rat small intestine. Drugs Exp. Clin. Res., 13, 517—524. Sargent,N.S.E., Price.I.E. and Dorling,D.L. (1987) Effects of altering surface glycoprotein composition on metastatic colonization potential of mammary tumour cells. Br. J. Cancer, 55, 21-28. Saul.R., Chambers,J.P., Molyneux.R.J. and Elbein.A.D. (1983) Castanospermine, a tetrahydroxylated alkaloid that inhibits 0-glucosidase and |3-glucocerebrosidase. Arch. Biochem. Biophys., 221, 593—597. Saul,R., Molyneux.R.J. and Elbein.A.D. (1984) Studies on the mechanism of castanospermine inhibition of a- and ^-glucosidases. Arch. Biochem. Biophys., 230, 668-675. Saul.R., Ghidoni,JJ., Molyneux.R.J. and Elbein.A.D. (1985) Castanospermine inhibits a-glucosidase activities and alters glycogen distribution in animals. Proc. Natl. Acad. Sci. USA, 82, 93-97. Schwarz.R.T. (1991) Manipulation of the biosynthesis of protein-modifying glycoconjugates by the use of specific inhibitors. Behring Inst. Mitt., 89, 198-208. Schweden.J. and Bause,E. (1989) Characterization of trimming Man,-mannosidase from pig liver. Purification of a catalytically active fragment and evidence for the transmembrane nature of the intact 65 kDa enzyme. Biochem. J., 264, 347-355. Schweden.J., Legler,G. and Bause,E. (1986) Purification and characterization of a neutral processing mannosidase from calf liver acting on (Man)9(GlcNAc)2 oligosaccharides. Eur. J. Biochem., 157, 563-570. Scofield,A.M., Fellows.L.E., Nash.R.J. and Fleet.G.W.J. (1986) Inhibition of mammalian digestive disaccharidases by polyhydroxy alkaloids. Life Sci., 39, 645-650. Scudder.P., Neville.D C.A., Butters.T.D., Fleet.G.W.J., Dwek.R.A., Rademacher.T.W. and Jacob,G.S. (1990) The isolation by ligand affinity chromatography of a novel form of a-L-fucosidase from almond. J. Biol. Chem., 265, 16472-16477. Sem,A., Sambhara.S., Chadwick.B.S. and Miller.R.G. (1991) Dependence of mouse thymocyte-erythrocyte rosette formation on complete identity at class-I-MHC. J. Cell. Physiol., 148, 485-192. Spearman.M.A., Damen.J.E., Kolodka.T., Greenberg.A.H., Jamieson.J.C. and Wright.J.A. (1991) Differential effects of glycoprotein processing inhibition on experimental metastasis formation by T24-H-ras transformed fibroblasts. Cancer Lett., 57, 7-13. Stephens.E.B., Monck.E., Reppas.K. and Butfiloski.E.J. (1991) Processing of the glycoprotein of feline immunodeficiency virus: effect of inhibitors of glycosylation. J. Virol., 65, 1114-1123.

209

Downloaded from http://glycob.oxfordjournals.org/ at Purdue University Libraries ADMN on July 24, 2015

Kajimoto.T., Liu,K.K.-C, Pederson,R.L., Zhong,Z., Ichikawa,Y., Porco, J.A.,Jr and Wong,C.-H. (1991) Enzyme-catalyzed aldol condensation for asymmetric synthesis of azasugars—synthesis, evaluation, and modelling of glycosidase inhibitors. J. Am. Chem. Soc, 113, 6187-6196. Kang,M.S. and Elbein,A.D. (1983) Mechanism of inhibition of jack bean a-mannosidase by swainsonine. Plant Physiol., 71, 551-554 Karpas,A., Fleet,G.W.J., Dwek.R.A., Petursson.S., Namgoong.S.K., Ramsden.N.G., Jacob.G.S. and Rademacher.T.W. (1988) Aminosugar derivatives as potential anti-human immunodeficiency virus agents. Proc. Nail. Acad. Sci. USA, 85, 9229-9233. Kaushal,G.P., Pan,Y.T., Tropea.J.E., Mitchell,M., Liu.P. and Elbein.A.D. (1988) Selective inhibition of glycoprotein-processing enzymes. Differential inhibition of glucosidases I and II in cell culture. J. Biol. Chem., 263, 17278-17283. Kaushal.G.P., Szumilo.T., Pastuszak,I. and Elbein.A.D. (1990) Purification to homogeneity and properties of mannosidase II from mung bean seedlings. Biochemistry, 29, 2168-2176. Kinast.G. and Schnedel, M. (1981) A four step synthesis of 1-deoxynojirimycin with a biotransformation as cardinal step. Angew. Chem. Int. Ed., 20, 805-806. Kino,T., Inamura.N., Nakahara,K., Kiyoto.S., Goto,T., Terano,H., Kohsaka.N , Aoki,H. and Imanaka.H. (1985) Studies on the immunomodulator, swainsonine. n Effect of swainsonine on mouse lmmunodeficient system and experimental munne tumour. J. Antibiot., 37, 936-940. Kite,G.C, Fellows.L.E., Fleet.G.W.J , Liu.P.S., Scofield.A.M. and Smith,N.G. (1988) a-Homonojirimycin (2,6-dideoxy-2,6-imino-D-glyceroL-guloheptitol) from Omphalea diandra L.: isolation and glycosidase inhibition. Tetrahedron Lett., 29, 6483-6486. Legler.G. (1990) Glycoside hydrolases: mechanistic information from studies with reversible and irreversible inhibitors. Adv. Carbohydr. Chem. Biochem., 48, 319-384. Legler.G. and Julich,E. (1984) Synthesis of 5-amino-5-deoxy-Dmannopyranose and l,5-dideoxy-l,5-imino-D-mannitol, and inhibition of aand /3-D-mannosidases. Carbohydr. Res., 128, 61—72. Lembcke,B , Folsch,U.R. and Creutzfeldt,W. (1985) Effect of 1 -desoxynojirimycin derivatives on small intestinal disaccharidase activities and on active transport in vitro. Digestion, 31, 120-127. Liu.P.S. (1987) Total synthesis of 2,6-dideoxy-2,6-imino-7-0-/3-D-glucopyranosyl-D-glycero-L-gulo-heptitol hydrochloride—a potent inhibitor of a-glucosidases. J. Org. Chem., 52, Al\l-A12\. Liu,P.S., Kang.M.S. and Sunkara,P.S. (1991) A potent inhibitor of /3-/V-acetylglucosamimdases: 6-acetamido-6-deoxycastanospermine. Tetrahedron Lett., 32, 719-720. McDowell,W and Schwarz,R.T. (1988) Dissecting glycoprotein biosynthesis by the use of specific inhibitors. Biochimie, 70, 1535—1549. Miyake,Y. and Ebata,M. (1988) The structure of a (3-galactosidase inhibitor, |3-galactostatin and its derivatives. Agric. Biol. Chem., 52, 661—666. Molyneux,R.J. (1990) 7-deoxy-6-epi-castanospermine, a trihydroxyindolizidine alkaloid glycosidase inhibitor from Castanospermum australe. J. Nat. Prod., 53, 609-614. Molyneux.R.J. and James,L.F. (1982) Loco intoxication: Indolizidine alkaloids of spotted locoweed (Astragalus lentiginosus). Science, 216, 190-191. Molyneux.R.J., Roitman,J.N., Dunnheim.G., Szumilo.T. and Elbein,A.D. (1986) 6-epicastanospermine, a novel indolizidine alkaloid that inhibits a-glucosidase. Arch. Biochem. Biophys., 251, 450-457. Molyneux.R.J., Benson.M., Wong.R.Y., Tropea.J.E. and Elbein,A.D. (1988) Australine, a novel pyrrolizidine alkaloid glucosidase inhibitor from Castanospermum australe. J. Nat. Prod., 51, 1198-1206. Montefiori.D.C, Robinson.W.E.Jr and Mitchell.W.M. (1989) In vitro evaluation of mismatched double-stranded RNA (ampligen) for combination therapy in the treatment of acquired immunodeficiency syndrome. AIDS Res Hum. Retroviruses, 5, 193-203. Murao.S. and Miyata.S. (1980) Isolation and characterization of a new trehalase inhibitor, S-GI. Agric. Biol. Chem., 44, 219-221. Nash.R.J., Fellows,L.E., Dring,J.V., Fleet.G.W.J., Derome.A.E., Hamer.T.A., Scofield.A.M. and Watkin.D.J. (1988) Isolation from Alexa leiopetala and X-ray crystal structure of alexine, (1/?, 2R, 3R, 75, 8S)-3-hydroxymethyl-l,2,7-trihydroxypyrrolizidine, a unique pyrrolizidine alkaloid. Tetrahedron Lett., 29, 2487-2490. Nash.R.J., Fellows.L.E., Dring,J.V., Fleet.G.W.J., Girdhar.A., Ramsden, N.G., Peach,J.M., Hegarty.M.P. and Scofield.A.M. (1990) Two alexines [3-hydroxymethyl-l,2,7-trihydroxypyrrolizidines] from Castanospermum australe. Phytochemistry, 29, 111-114. Newton.S.A., White.S., Humphries.M.J. and Olden.K. (1989) Swainsonine inhibition of spontaneous metastasis. J. Natl. Cancer Inst., 81, 1024-1028.

B.Winchester and G.W.J.Fleet

Received on March 20, 1992; accepted on March 27, 1992

210

Downloaded from http://glycob.oxfordjournals.org/ at Purdue University Libraries ADMN on July 24, 2015

Sunkara,P.S., Taylor.D.L., Kang.M.S., Bowlin.T.L., Liu.P.S., Tyms.A.S. and Sjoerdsma.A. (1989) Anti-HFV activity of castanospermine analogues. Lancet, May 27th, 1206. Taylor.R.H., Barker.H.M., Bowey,E.A. and Canfield.J.E. (1986) Regulation of the absorption of dietary carbohydrate in man by two new glucosidase inhibitors. Gut, 27, 1471-1478. Taylor,D.L., Fellows,L.E., Farrar,G.H., Nash.R.J., Taylor-Robinson.D., Mobberley.M.A., Ryder,T.A., Jeffries.D.J. and Tyms.A.S. (1988) Loss of cytomegalovirus infectivity after treatment with castanospermine or related plant alkaloids correlates with aberrant glycoprotein synthesis. Antiviral Res., 10, 11-26. Taylor,D.L., Sunkara.P.S., Liu.P.S., Kang.M.S., Bowlin,T.L. and Tyms, A.S. (1991) 6-0-butanoylcastanospermine (MDL 28,574) inhibits glycoprotein processing and the growth of HIVs. AIDS, 5, 693-698. Tropea.J.E., Molyneux.R.J., Kaushal.G.P., Pan,Y.T., Mitchell,M. and Elbein.A.D. (1989) Australine, a pyrrolizidine alkaloid that inhibits amyloglucosidase and glycoprotein processing. Biochemistry, 28, 2027-2034. Tulsiani.D.R.P., Harris.T.M. and Touster,O. (1982) Swainsonine inhibits the biosynthesis of complex glycoproteins by inhibition of Golgi mannosidase II. J. Biol. Chem., 257, 7936-7939. Tyms.A.S., Berrie.E.M., Ryder.T.A., Nash,R.J., Hegarty,M.P., Taylor, D.L., Mobberley.M.A., Davis,J.M., Bell.E.A., Jeffries,D.J., TaylorRobinson.D. and Fellows,L.E. (1987) Castanospermine and other plant alkaloid inhibitors of glucosidase activity block the growth of HIV. Lancet, 11, October 31, 1025-1026. Walker.B.D., Kowalski,M., Goh.W.C, Kozarsky.K., Krieger,M., Rosen.C, Rohrschneider.L., Haseltine.W.A. and Sodroski.J. (1987) Inhibition of human immunodeficiency virus syncytium formation and virus replication by castanospermine. Proc. Nail. Acad. Sci. USA, 84, 8120-8124. Welter.A , Jadot.J., Dardenne,G., Marlier,M. and Casimir,J. (1976) 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine dans les feuiles de Derris elliptica. Phytochemistry, 15, 747-749. White.S.L., Nagai.T., Akiyama,S.K., Reeves,E.J., Grzegorzewski.K. and Olden,K. (1991) Swainsonine stimulation of the proliferation and colony forming activity of murine bone marrow. Cancer Commun., 3, 83-91. Winchester.B. (1984) Role of a-D-mannosidases in the biosynthesis and catabolism of glycoproteins. Biochemical Society Transactions, 605th Meeting, Strathclyde, pp. 522-524. Winchester,B., Barker,C, Baines.S., Jacob.G.S., Namgoong.S.K. and Fleet,G. (1990) Inhibition of a-L-fucosidase by derivatives of deoxyfuconojirimycin and deoxymannojirimycin. Biochem. J., 265, 277-282. Wisselaar.H.A., van Dongen,J.M. and Reuser,A.J.J. (1989) Effects of /V-hydroxyethyl-1-deoxynojirimycin (BAY m 1099) on the activity of neutraland acid a-glucosidases in human fibroblasts and HepG2 cells. Clin. Chim. Ada, 182, 41-52. Woollen.J.W. and Turner.P. (1965) Plasma A'-acetyl-/3-glucosaminidase and glucuronidase in health and disease. Clin. Chim. Acta, 12, 671-683. Woynarowska.B., Wikiel,H., Sharma.M., Carpenter,N., Fleet.G.W.J. and Bernacki.R.J. (1992) Inhibition of human ovarian carcinoma (HOC) cell- and hexosaminidase-mediated degradation of extracellular matrix (ECM) by sugar analogs. Anticancer Res., 12, 161-166. Yagel.S., Kerbel.R., Lala.P., Eldar-Gera.T. and Dennis,J.W. (1990) Basement membrane invasion by first trimester human trophoblast: requirement for branched complex-type Asn-linked oligosaccharides. Clin. Exp. Metastasis, 8, 305-317. Yagi,M., Kuono.T , Aoyagi,Y. and Murai.H. (1976) The structure of moranoline, a piperidine alkaloid from Moms species. Nippon Nogei Kagaku Kaishi, 50, 571-572.