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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic poses an unprecedented challenge for health care and the global economy. Repurposing drugs that have shown promise in inhibiting other viral infections could allow for more rapid dispensation of urgently needed therapeutics. The Spike protein of SARS-CoV-2 is extensively glycosylated with 22 occupied N glycan sites and is required for viral entry. In other glycosylated viral proteins, glycosylation is required for interaction with calnexin and chaperone-mediated folding in the endoplasmic reticulum, and prevention of this interaction leads to unfolded viral proteins and thus inhibits viral replication. As such, we investigated two iminosugars, celgosivir, a prodrug of castanospermine, and UV-4, or N-(9-methoxynonyl)-1-deoxynojirimycin, a deoxynojirimycin derivative. Iminosugars are known inhibitors of the α-glucosidase I and II enzymes and were effective at inhibiting authentic SARS-CoV-2 viral replication in a cell culture system. Celgosivir prevented SARS-CoV-2-induced cell death and reduced viral replication and Spike protein levels in a dose-dependent manner in culture with Vero E6 cells. Castanospermine, the active form of celgosivir, was also able to inhibit SARS-CoV-2, confirming the canonical castanospermine mechanism of action of celgosivir. The monocyclic UV-4 also prevented SARS-CoV-2-induced death and reduced viral replication after 24 h of treatment, although the reduction in viral copies was lost after 48 h. Our findings suggest that iminosugars should be urgently investigated as potential SARS-CoV-2 inhibitors.

We previously cloned a lepidopteran insect cell cDNA that encodes a class II alpha-mannosidase that is localized in the Golgi apparatus but is cobalt-dependent, has a neutral pH optimum, hydrolyzes Man(5)GlcNAc(2) to Man(3)GlcNAc(2), and cannot hydrolyze GlcNAcMan(5)GlcNAc(2). This enzyme was designated SfManIII to distinguish it from Golgi alpha-mannosidase II and indicate its derivation from the fall armyworm Spodoptera frugiperda. In the present study, we prepared a polyclonal antibody and used it to study the biosynthesis and processing of SfManIII. The results showed that Sf9 cells produce at least three different forms of SfManIII. SfManIII is initially synthesized as a precursor glycoprotein, which is slowly converted to two smaller end products with at least some endoglycosidase H-resistant N-glycans. The smallest form of SfManIII is the only one of these two products that accumulates in the extracellular fraction. Tunicamycin blocked the production of SfManIII activity and the secretion of SfManIII protein and activity. Castanospermine blocked production of the larger SfManIII product, retarded production of the smaller, increased intracellular SfManIII activity, and decreased extracellular SfManIII activity. Together, these results indicate that SfManIII is initially synthesized as a high-mannose glycoprotein precursor, its N-glycans are trimmed as it is transported to the Golgi apparatus, and a subpopulation, which appears to be proteolytically cleaved, is secreted in enzymatically active form. N-glycosylation is required for the production of active SfManIII, and N-glycosylation and N-glycan trimming are both required for the efficient secretion of an active form of this protein.

The alpha1,3/4-fucosyltransferases are involved in the synthesis of fucosylated cell surface glycoconjugates. Human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, and -VI) contain two conserved C-terminal N-glycosylation sites (hFucTIII: Asn154 and Asn185; hFucTV: Asn167 and Asn198; and hFucTVI: Asn153 and Asn184). In the present study, we have analyzed the functional role of these potential N-glycosylation sites, laying the main emphasis on the sites in hFucTIII. Tunicamycin treatment completely abolished hFucTIII enzyme activity while castanospermine treatment diminished hFucTIII enzyme activity to approximately 40% of the activity of the native enzyme. To further analyze the role of the conserved N-glycosylation sites in hFucTIII, -V, and -VI, we made a series of mutant genomic DNAs in which the asparagine residues in the potential C-terminal N-glycosylation sites were replaced by glutamine. Subsequently, the hFucTIII, -V, and -VI wild type and the mutants were expressed in COS-7 cells. All the mutants exhibited lower enzyme activity than the wild type and elimination of individual sites had different effects on the activity. The mutations did not affect the protein level of the mutants in the cells, but reduced the molecular mass as predicted. Kinetic analysis of hFucTIII revealed that lack of glycosylation at Asn185 did not change the Km values for the oligosaccharide acceptor and the nucleotide sugar donor. The present study demonstrates that hFucTIII, -V, and -VI require N-glycosylation at the two conserved C-terminal N-glycosylation sites for expression of full enzyme activity.

The most frequent type of N-glycan synthesized by lepidopteran Sf9 cells appears to be fucosylated Man3GlcNAc2,and this has been a limitation for a large scale production and utilization of therapeutic glycoproteins in cultured insect cells. The current knowledge of the protein glycosylation pathway derived from structural studies on recombinant glyco-proteins expressed by using baculovirus vectors. In this work we provide more direct evidence for the sequential events occurring in the processing of endogenous N-glycoproteins of noninfected Sf9 cells. By metabolic labeling with radioactive mannose, we characterized the glycan structures which accumulated in the presence of processing inhibitors (castanospermine and swainsonine) and in the presence of an intracellular trafficking inhibitor (monensin). We thus demonstrated that from the glycan precursor Glc3Man9GlcNAc2 to GlcNAcMan5(Fuc)GlcNAc2 intermediate, the processing pathway in Sf9 cells paralleled the one demonstrated in mammalian cells. By using monensin, we demonstrated the formation of Man3(Fuc)GlcNAc2 from GlcNAcMan3(Fuc)GlcNAc2, a reaction which has not been described in mammalian cells. Our results support the idea that the hexosaminidase activity is of physiological relevance to the glycosylation pathway and is Golgi located.

Several hundred molecules of enzyme reaction products were detected in a single spheroplast from yeast cells incubated with a tetramethylrhodamine (TMR) labeled triglucoside, alpha-d-Glc(1-->2)alpha-d-Glc(1-->3)alpha-d-Glc-O(CH2)8CONHCH2- CH2NH- COTMR. Product detection was accomplished using capillary electrophoresis and laser induced fluorescence following the introduction of a single spheroplast into the separation capillary. The in vivo enzymatic hydrolysis of the TMR-trisaccharide involves at least two enzymes, limited by processing alpha-glucosidase I, producing TMR-disaccharide, TMR-monosaccharide, and the free TMR-linking arm. Hydrolysis was reduced by preincubation of the cells with the processing enzyme inhibitor castanospermine. Confocal laser scanning microscopy studies confirmed the uptake and internalization of fluorescent substrate. This single cell analysis methodology can be applied for the in vivo assay of any enzyme with a fluorescent substrate.

Endo-alpha-D-mannosidase is an enzyme involved in N-linked oligosaccharide processing which through its capacity to cleave the internal linkage between the glucose-substituted mannose and the remainder of the polymannose carbohydrate unit can provide an alternate pathway for achieving deglucosylation and thereby make possible the continued formation of complex oligosaccharides during a glucosidase blockade. In view of the important role which has been attributed to glucose on nascent glycoproteins as a regulator of a number of biological events, we chose to further define the in vivo action of endomannosidase by focusing on the well characterized VSV envelope glycoprotein (G protein) which can be formed by the large array of cell lines susceptible to infection by this pathogen. Through an assessment of the extent to which the G protein was converted to an endo-beta-N-acetylglucosaminidase (endo H)-resistant form during a castanospermine imposed glucosidase blockade, we found that utilization of the endomannosidase-mediated deglucosylation route was clearly host cell specific, ranging from greater than 90% in HepG2 and PtK1 cells to complete absence in CHO, MDCK, and MDBK cells, with intermediate values in BHK, BW5147.3, LLC-PK1, BRL, and NRK cell lines. In some of the latter group the electrophoretic pattern after endo H treatment suggested that only one of the two N-linked oligosaccharides of the G protein was processed by endomannosidase. In the presence of the specific endomannosidase inhibitor, Glcalpha1-->3(1-deoxy)mannojirimycin, the conversion of the G protein into an endo H-resistant form was completely arrested. While the lack of G protein processing by CHO cells was consistent with the absence of in vitro measured endomannosidase activity in this cell line, the failure of MDBK and MDCK cells to convert the G protein into an endo H-resistant form was surprising since these cell lines have substantial levels of the enzyme. Similarly, we observed that influenza virus hemagglutinin was not processed in castanospermine-treated MDCK cells. Our findings suggest that studies which rely on glucosidase inhibition to explore the function of glucose in controlling such critical biological phenomena as intracellular movement or quality control should be carried out in cell lines in which the glycoprotein under study is not a substrate for endomannosidase action.

Oral treatment of mice, cutaneously infected with herpes simplex virus type 1 (HSV-1) (strain SC16), with the alpha-glucosidase 1 inhibitor 6-O-butanoyl castanospermine (MDL 28,574) produced a significant delay in lesion development and reduced the amount of virus recovered from the brain. Virus load in the brains of mice, whose treatment started 2 days prior to infection, was reduced approximately 100-fold when compared to untreated controls. Treatment initiated at the time of infection, while less effective than pre-treatment, nevertheless reduced virus recovery from the brain by 10-fold. Consistent with its antiviral activity, orally administered MDL 28,574 was rapidly incorporated by brain tissue and mice fed with compound over extended periods maintained relatively high levels of drug at this site.

The concanavalin A staining of cellular glycoproteins and the direct analysis of their sugar chains released by hydrazinolysis revealed that the processing of N-linked sugar chains of some glycoproteins is suppressed by exposure of mouse monocytoid cells P388D1 to dimethyl sulphoxide, which can induce Fc receptor-mediated phagocytosis. To elucidate the significance of altered glycosylation in inducing phagocytosis, the effects of exposure of the cells to processing inhibitors (swainsonine and castanospermine) were examined and it was found that the cells are induced to acquire an ability to ingest IgG-coated sheep red blood cells, depending on the dose of the inhibitors and incubation time. Analysis of the N-linked sugar chains liberated from cellular glycoproteins by hydrazinolysis confirmed that the processing of the sugar chains is suppressed by the two inhibitors as expected. Since no significant alteration was induced in protein synthesis and DNA synthesis after exposure to the inhibitors, it is suggested that the altered glycosylation of cellular glycoproteins may have some direct role in the induction of Fc receptor-mediated phagocytosis. The inhibitors did not affect the binding of the IgG-coated red blood cells to Fc receptors on the cells, non-specific phagocytosis of latex beads, and the contents of lysosomal enzymes, beta-glucuronidase and acid phosphatase. These results suggest that the glycosylation status of cellular glycoproteins influences some specific processes involved in the ingestion of the ligands bound to Fc receptors.

Retinal pigment epithelial cells selectively phagocytize rod outer segments by a process that may be mediated by specific cell surface receptors. Since many receptors are glycoproteins, we have studied the effect of tunicamycin, an inhibitor of N-linked oligosaccharide synthesis, and of castanospermine and swainsonine, which are inhibitors of oligosaccharide processing, on the ability of cultured retinal pigment epithelial cells to phagocytize rod outer segment. Tunicamycin inhibits the glycosylation of newly synthesized glycoproteins by 85-90%; concomitantly, the phagocytosis of rod outer segments is inhibited by 70-80%. The effect of tunicamycin is to initially reduce rod outer segments binding, and therefore the subsequent ingestion of rod outer segments. SDS-PAGE analysis and autoradiography of [35S]methionine labelled extracts of tunicamycin-treated cells, demonstrates the disappearance of a number of glycoprotein bands, and the appearance of a number of protein bands of lower Mr. Kinetic analysis of the disappearance and reappearance of specific glycoproteins suggests that the lower Mr bands are the non-glycosylated forms of the higher Mr bands. By contrast, castanospermine and swainsonine have no effect on the ability of retinal pigment epithelial cells to phagocytize rod outer segments, or on the SDS-PAGE pattern of treated cells, although they were shown to inhibit oligosaccharide processing as expected. These results support the hypothesis that rod outer segment phagocytosis by retinal pigment epithelial cells is mediated by specific glycoprotein receptors. N-Glycosylation of these receptors is required for their function, or for their insertion into the plasma membrane, whereas processing of the N-linked oligosaccharide chains of these receptors is not crucial for rod outer segment phagocytosis by retinal pigment epithelial cells.

Glucosidase II is an ER heterodimeric enzyme that cleaves sequentially the two innermost alpha-1,3-linked glucose residues from N-linked oligosaccharides on nascent glycoproteins. This processing allows the binding and release of monoglucosylated (Glc(1)Man(9)GlcNAc(2)) glycoproteins with calnexin and calreticulin, the lectin-like chaperones of the endoplasmic reticulum. We have isolated two cDNA isoforms of the human alpha subunit (alpha1 and alpha2) differing by a 66 bp stretch, and a cDNA for the corresponding beta subunit. The alpha1 and alpha2 forms have distinct mobilities on SDS-PAGE and are expressed in most of the cell lines we have tested, but were absent from the glucosidase II-deficient cell line PHA(R) 2.7. Using COS7 cells, the coexpression of the beta subunit with the catalytic alpha subunit was found to be essential for enzymatic activity, solubilization, and/or stability, and ER retention of the alpha/beta complex. Transfected cell extracts expressing either alpha1 or alpha2 forms with the beta subunit showed similar activities, while mutating( )the nucleophile (D542N) predicted from the glycoside hydrolase Family 31 active site consensus sequence abolished enzymatic activity. In order to compare the kinetic parameters of both alpha1/beta and alpha2/beta forms of human glucosidase II the protein was expressed with the baculovirus expression system. Expression of the human alpha or beta subunit alone led to the formation of active human/insect heteroenzymes, demonstrating functional complementation by the endogenous insect glucosidase II subunits. The activity of both forms of recombinant human glucosidase II was examined with a p-nitrophenyl alpha-D-glucopyranoside substrate, and a two binding site kinetic model for this substrate was shown. The K(M1-2) values and apparent K(i1-2 )for deoxynojirimycin and castanospermine were determined and found to be identical for both isoforms suggesting they have similar catalysis and inhibition characteristics. The substrate specificities of both isoforms using the physiological oligosaccharides were assessed and found to be similar.

Glycopeptides are exported from the endoplasmic reticulum to the cytosol of eukaryotic membranes in an ATP- and cytosol-requiring process (Romisch and Ali, 1997, Proc. Natl. Acad. Sci. USA,94, 6730-6734). Oligosaccharides of the polymannose-type are also exported from the endoplasmic reticulum of mammalian cells to the cytosol in an ATP-dependent fashion. These findings raise the strong possibility that the two substrate classes are transported by the same mechanism but the precise identity of the trans-location machinery for each substrate class has not been fully defined. Here we have investigated the mechanism by which a glycopeptide is exported from rat liver microsomes, and compare this to the export of free polymannose oligosaccharides. Using EGTA and the endoplasmic reticulum calcium mobilizing agents thapsigargicin and calcium ionophores A23187 and ionomycin, we show that glycopeptides, in contrast to oligosaccharides, are exported by a calcium-independent mechanism. On the other hand, Mg(2+)is required in the assay for the transport of glycopeptide from mammalian microsomes which is in common with oligosaccharide export. Deoxynojirimycin and castanospermine, inhibitors of ER glucosidases, when added to rat liver microsomes prior to loading with peptide that bears an N -glycosylation sequon, had no effect on the release of glucosylated glycopeptides from membranes, indicating that removal of the alpha-glucose units from the oligomannose glycan structure of the glycopeptide is not required for export. In contrast to oligosaccharides, where transport is efficiently inhibited, mannosides were without effect or only weak inhibitors of glycopeptide export. Taken together, these data suggest that glycopeptides are exported by a distinct mechanism from oligosaccharides of the polymannose-type and that the peptide moiety is an important structural determinant for glycopeptide export and capable of directing translocation of substrates to a specific transport pathway.

The involvement of N-glycans in the folding of influenza virus hemagglutinin (HA) was analyzed in two CHO-derived glycosylation mutants exhibiting a thermosensitive defect for secretion of human placental alkaline phosphatase. Truncated Man(5)GlcNAc(2)oligosaccharides with one or three glucose residues are attached to proteins of the MadIA214 and B3F7AP2-1 mutant cells, respectively. Newly synthesized proteins retained in these cells carry a Man(4)trimmed glycan generated by a mannosidase different from the ER mannosidases I and II and suggesting a recycling through the Golgi complex. The glucosidase inhibitor castanospermine affects the binding of HA folding intermediates to the lectin-like chaperone calnexin in B3F7AP2-1 but not in MadIA214 cells. We demonstrated that calnexin interacts in vivo with truncated Man(5)derivatives. In MadIA214 cells, this is only possible when Man(5)GlcNAc(2)on protein becomes reglucosylated. The pattern of intermediates seen during the folding of HA in the MadIA214 and B3F7AP2-1 mutant cell lines is different than in control cells. We also observed a variable occupancy of the seven glycosylation-sites. However, even under conditions that restore glycosylation of all sites, the folding intermediates of HA in the mutant cells still remain heterogeneous. Our results demonstrate that addition of truncated N-glycans interferes extensively with the folding of newly synthesized proteins in vivo.

The polyhydroxylated nortropane alkaloids called calystegines occur in many plants of the Convolvulaceae, Solanaceae, and Moraceae families. Certain of these alkaloids exhibit potent inhibitory activities against glycosidases and the recently demonstrated occurrence of calystegines in the leaves, skins, and sprouts of potatoes (Solanum tuberosum), and in the leaves of the eggplant (S. melongena), has raised concerns regarding the safety of these vegetables in the human diet. We have surveyed the occurrence of calystegines in edible fruits and vegetables of the families Convolvulaceae, Solanaceae, and Moraceae by GC-MS. Calystegines A3, B1, B2, and C1 were detected in all the edible fruits and vegetables tested; sweet and chili peppers, potatoes, eggplants, tomatoes, Physalis fruits, sweet potatoes, and mulberries. Calystegines B1 and C1 were potent competitive inhibitors of the bovine, human, and rat beta-glucosidase activities, with Ki values of 150, 10, and 1.9 microM, respectively for B1 and 15, 1.5, and 1 microM, respectively, for C1. Calystegine B2 was a strong competitive inhibitor of the alpha-galactosidase activity in all the livers. Human beta-xylosidase was inhibited by all four nortropanes, with calystegine C1 having a Ki of 0.13 microM. Calystegines A3 and B2 selectively inhibited the rat liver beta-glucosidase activity. The potent inhibition of mammalian beta-glucosidase and alpha-galactosidase activities in vitro raises the possibility of toxicity in humans consuming large amounts of plants that contain these compounds.

UDP-N-acetylglucosamine: beta-D-mannoside beta-1,4N-acetylglucosaminyltransferase III (GnT-III, EC 2.4.1.144) is a glycoprotein involved in the biosynthesis of N-linked oligosaccharides. Rat GnT-III contains three potential N-glycosylation sites, which have been predicted to be Asn243, Asn261, and Asn399. To study the roles of N-glycosylation in the GnT-III function, rat GnT-III was expressed in COS-1 cells under tunicamycin or castanospermine treatment. The tunicamycin-treated GnT-III, which was not N-glycosylated, had almost no activity. The castanospermine-treated GnT-III was not localized in the Golgi, but glucosylation did not affect its activity. To clarify the role of individual N-glycosylations, we obtained a series of mutant cDNAs in which some or all of the potential glycosylation sites were eliminated by site-directed mutagenesis, and expressed them in COS-1 cells. All the mutants exhibited lower enzyme activity than the wild-type, but deglycosylation at individual sites had different effects on the enzyme activity. The deglycosylation at Asn243 or Asn261 was more effective on the activity than that at Asn399. The enzyme activity decreased as the number of glycosylation sites decreased. The null glycosylation mutant had no activity, corresponding to the case of tunicamycin-treated wild-type GnT-III. Kinetic analysis revealed that the deglycosylation at Asn243 or Asn261 resulted in slightly lower affinity for the donor substrate, but the other mutation did not significantly change the K(m) value for either the donor or acceptor. None of the mutant GnT-IIIs showed perinuclear localization or Golgi retention, that was observed for the wild-type protein. This is the first demonstration that the glycosyltransferase localized in the Golgi apparatus requires N-glycosylation for its activity and retention.

Endo-alpha-D-mannosidase is a processing enzyme which in contrast to other glycosidases involved in the trimming of N-linked oligosaccharides of glycoproteins acts at an internal position by cleaving the linkage between the glucose-substituted mannose and the internal portion of the polymannose unit and thereby provides an alternate deglucosylating pathway. In order to evaluate at what stage in evolution this unusual enzyme first emerged, we have carried out a phylogenetic survey of its distribution among a broad group of eukaryotes ranging from unicellular organisms to highly developed animals and plants, all of which are known to have the capacity to N-glycosylate proteins and subsequently trim the nascent glucosylated polymannose oligosaccharides. It became evident from enzyme assays and in vivo studies that endomannosidase is limited in its distribution to members of the chordate phylum, including placental and marsupial mammals, birds, reptiles, amphibians, and fish, with the single except of the Mollusca in which it was detected in three distinct classes. The enzyme's absence in all other invertebrates examined as well as in yeast, various protozoa and higher plants, stands in contrast to glucosidase II and alpha 1,2-mannosidase which were found to be present in all eukaryotes studied. The observation that endomannosidase activity was not present in insects was confirmed by radiolabeling experiments with Sf9 cells in culture. These cells, which are widely employed for the expression of mammalian genes, were in distinction to mouse cells unable to circumvent a castanospermine (CST)-induced glucosidase blockade. Moreover we observed that Tetrahymenae, which synthesize glycoproteins with truncated N-linked oligosaccharides, could not process these beyond the Glc3Man5GlcNAc2 stage in the presence of CST. The late appearance of endomannosidase during evolution suggests a need for an alternate deglucosylation route in higher animals which parallels the development of elaborate complex N-linked oligosaccharides. Such carbohydrate units are believed to carry out vital biological functions and deglucosylation is a prerequisite to the further processing steps required for their formation.

We have previously shown (Sunkara et al., 1989; Taylor et al., 1991) that 6-o-butanoyl castanospermine (BuCast) was a 30-50-fold better inhibitor of HIV syncytia formation than castanospermine (Cast). Radiolabeled Cast and BuCast were used to study the uptake and metabolism of these compounds in cultured cells and in mice. BuCast was preferentially taken up by cells compared to Cast. Approximately 30-50-fold higher radioactivity was found in cells treated with BuCast compared to cells treated with Cast during the initial 4-6 h of labeling. HPLC analysis showed that once BuCast was taken up by cells, it was rapidly converted to Cast. Mice given oral doses of BuCast had 5-10-fold higher levels of Cast in the plasma and tissues as compared to Cast treated mice. However, when the compounds were given i.v., the levels of plasma and tissue radioactivity obtained from Cast of BuCast were equivalent suggesting rapid conversion of BuCast to Cast in the blood. In mice orally treated with BuCast, HPLC analysis suggested that only Cast was found in the plasma and tissues. With multiple dosing of mice, additive results were obtained, suggesting that multiple doses may be used to obtain higher concentrations of the compound in the target cells. These data suggest that the lipophilic properties of butanoyl side chain on the Cast ring makes BuCast significantly better absorbed, and this may help to alleviate some of the gut toxicity associated with Cast treatment.

The antiviral clinical candidate 6-O-butanoyl castanospermine (MDL 28,574), an alpha-glucosidase 1 inhibitor, was examined for its effect on elementary parameters of immune function. It did not affect the mitogenic response of uninfected human mononuclear leukocytes or the detection of a range of cell surface markers, with the exception of the integrin LFA-1 (CD18/CD11a), which was reduced, after cell growth in vitro. The detection of LFA-1 was also reduced on both human and murine cells after oral administration of the compound to xenochimaeric or normal mice, respectively. Altered LFA-1 expression or function may contribute to reduced cell adhesion and the observed reduction in the in vitro allogeneic response by uninfected cells, as well as the previously described prevention of cell conjugate and HIV-induced syncytium formation.

We have used a simple and efficient procedure for the synthesis of N-5-carboxypentyl-1-deoxynojirimycin, an affinity ligand for alpha-glucosidase I (Bernotas, R. C. and Ganem, B., Biochem. J., 270, 539-540, 1990). The affinity gel was used to purify alpha-glucosidase I in one step from crude extract. In subsequent steps, partially purified alpha-glucosidase II was obtained. We have synthesized several analogues of castanospermine and studied their inhibition of alpha-glucosidase I in vitro using purified alpha-glucosidase I and in vivo in cultured B16F10 cells. Although the castanospermine analogues were significantly less active against the purified enzyme (IC50 approximately 1-23 micrograms/ml) as compared to castanospermine (IC50 = 0.02 microgram/ml), several compounds had up to 30-fold higher activity than castanospermine against alpha-glucosidase I in B16F10 cells, based on the accumulation of G3M7-9N2 oligosaccharide-containing glycoproteins. These results suggest that these analogues with lipophilic side chains cross the membrane barrier more efficiently than castanospermine. Once inside the cell, they may be converted to their active metabolite, castanospermine, by cellular esterases to give enzyme inhibition.