Journeys in science: glycobiology and other paths.

My scientific journeys began at Oxford nearly 50 years ago. My paths have taken me from magnetic resonance through enzyme systems to antibodies, which led directly to glycobiology. Oxford University's first industrial grant helped the development of the technology for isolating and sequencing oligosaccharides from glycoproteins. This technology was disseminated through a spin-off company, Oxford GlycoSystems, and by the establishment of the Glycobiology Institute. The technology gave rise to the concept of glycoforms, which allow diversification of a protein's properties. Iminosugars, which are glucosidase inhibitors, can interfere with the initial steps of glycan processing on proteins and inhibit three-dimensional folding of glycoproteins. Glucosidase targets for therapy include viral envelope glycoproteins. Clinical trials of an iminosugar as an antiviral for dengue virus are under way. Another iminosugar activity, inhibition of glycolipid synthesis, resulted in a drug for Gaucher disease, which was approved worldwide in 2002. The success of the company and the institute allowed me to undertake several initiatives, in the United Kingdom and abroad, that might help the paths of future generations of scientists.

pH-Responsive Trihydroxylated Piperidines Rescue The Glucocerebrosidase Activity in Human Fibroblasts Bearing The Neuronopathic Gaucher-Related L444P/L444P Mutations in GBA1 Gene.

Engineering bioactive iminosugars with pH-responsive groups is an emerging approach to develop pharmacological chaperones (PCs) able to improve lysosomal trafficking and enzymatic activity rescue of mutated enzymes. The use of inexpensive l-malic acid allowed introduction of orthoester units into the lipophilic chain of an enantiomerically pure iminosugar affording only two diastereoisomers contrary to previous related studies. The iminosugar was prepared stereoselectively from the chiral pool (d-mannose) and chosen as the lead bioactive compound, to develop novel candidates for restoring the lysosomal enzyme glucocerebrosidase (GCase) activity. The stability of orthoester-appended iminosugars was studied by 1 H NMR spectroscopy both in neutral and acidic environments, and the loss of inhibitory activity with time in acid medium was demonstrated on cell lysates. Moreover, the ability to rescue GCase activity in the lysosomes as the result of a chaperoning effect was explored. A remarkable pharmacological chaperone activity was measured in fibroblasts hosting the homozygous L444P/L444P mutation, a cell line resistant to most PCs, besides the more commonly responding N370S mutation.

Deconstructing Best-in-Class Neoglycoclusters as a Tool for Dissecting Key Multivalent Processes in Glycosidase Inhibition.

Multivalency represents an appealing option to modulate selectivity in enzyme inhibition and transform moderate glycosidase inhibitors into highly potent ones. The rational design of multivalent inhibitors is however challenging because global affinity enhancement relies on several interconnected local mechanistic events, whose relative impact is unknown. So far, the largest multivalent effects ever reported for a non-polymeric glycosidase inhibitor have been obtained with cyclopeptoid-based inhibitors of Jack bean α-mannosidase (JBα-man). Here, we report a structure-activity relationship (SAR) study based on the top-down deconstruction of best-in-class multivalent inhibitors. This approach provides a valuable tool to understand the complex interdependent mechanisms underpinning the inhibitory multivalent effect. Combining SAR experiments, binding stoichiometry assessments, thermodynamic modelling and atomistic simulations allowed us to establish the significant contribution of statistical rebinding mechanisms and the importance of several key parameters, including inhitope accessibility, topological restrictions, and electrostatic interactions. Our findings indicate that strong chelate-binding, resulting from the formation of a cross-linked complex between a multivalent inhibitor and two dimeric JBα-man molecules, is not a sufficient condition to reach high levels of affinity enhancements. The deconstruction approach thus offers unique opportunities to better understand multivalent binding and provides important guidelines for the design of potent and selective multiheaded inhibitors.

Synthesis and Biological Evaluation of New Dihydrofuro[3,2-b]piperidine Derivatives as Potent α-Glucosidase Inhibitors.

Inhibition of glycoside hydrolases has widespread application in the treatment of diabetes. Based on our previous findings, a series of dihydrofuro[3,2-b]piperidine derivatives was designed and synthesized from D- and L-arabinose. Compounds 32 (IC50 = 0.07 µM) and 28 (IC50 = 0.5 µM) showed significantly stronger inhibitory potency against α-glucosidase than positive control acarbose. The study of the structure-activity relationship of these compounds provides a new clue for the development of new α-glucosidase inhibitors.

Harnessing polyhydroxylated pyrrolidines as a stabilizer of acid alpha-glucosidase (GAA) to enhance the efficacy of enzyme replacement therapy in Pompe disease.

To discover small molecules as acid alpha-glucosidase (GAA) stabilizers for potential benefits of the exogenous enzyme treatment toward Pompe disease cells, we started from the initial screening of the unique chemical space, consisting of sixteen stereoisomers of 2-aminomethyl polyhydroxylated pyrrolidines (ADMDPs) to find out two primary stabilizers 17 and 18. Further external or internal structural modifications of 17 and 18 were performed to increase structural diversity, followed by the protein thermal shift study to evaluate the GAA stabilizing ability. Fortunately, pyrrolidine 21, possessing an l-arabino-typed configuration pattern, was identified as a specific potent rh-GAA stabilizer, enabling the suppression of rh-GAA protein denaturation. In a cell-based Pompe model, co-administration of 21 with rh-GAA protein significantly improved enzymatic activity (up to 5-fold) compared to administration of enzyme alone. Potentially, pyrrolidine 21 enables the direct increase of ERT (enzyme replacement therapy) efficacy in cellulo and in vivo.

Fluorescence sensing and glycosidase inhibition effect of multivalent glycosidase inhibitors based on Naphthalimide-deoxynojirimycin conjugates.

Synthetic glycoconjugates as chemical probes have been widely developed for the detection of glycosidase enzymes. However, the binding interactions between iminosugar derivatives and glycosidases were limited, especially for the binding interactions between multivalent glycosidase inhibitors and α-glycosidases. In this paper, three naphthalimide-DNJ conjugates were synthesized. Furthermore, the binding interactions and glycosidase inhibition effects of them were investigated. It was found that the strong binding interactions of multivalent glycosidase inhibitors with enzymes were related to the efficient inhibitory activity against glycosidase. Moreover, the lengths of the chain between DNJ moieties and the triazole ring for the naphthalimide-DNJ conjugates influenced the self-assembly properties, binding interactions and glycosidase inhibition activities with multisource glycosidases. Compound 13 with six carbons between the DNJ moiety and triazole ring showed the stronger binding interactions and better glycosidase inhibition activities against α-mannosidase (jack bean) and α-glucosidase (aspergillus niger). In addition, compound 13 showed an effective PBG inhibition effect in mice with 51.18 % decrease in blood glucose at 30 min. This result opens a way for detection of multivalent glycosidase inhibition effect by a fluorescent sensing method.

Influence of Side Chain Conformation on the Activity of Glycosidase Inhibitors.

Substrate side chain conformation impacts reactivity during glycosylation and glycoside hydrolysis and is restricted by many glycosidases and glycosyltransferases during catalysis. We show that the side chains of gluco and manno iminosugars can be restricted to predominant conformations by strategic installation of a methyl group. Glycosidase inhibition studies reveal that iminosugars with the gauche,gauche side chain conformations are 6- to 10-fold more potent than isosteric compounds with the gauche,trans conformation; a manno-configured iminosugar with the gauche,gauche conformation is a 27-fold better inhibitor than 1-deoxymannojirimycin. The results are discussed in terms of the energetic benefits of preorganization, particularly when in synergy with favorable hydrophobic interactions. The demonstration that inhibitor side chain preorganization can favorably impact glycosidase inhibition paves the way for improved inhibitor design through conformational preorganization.

Synthesis, α-mannosidase inhibition studies and molecular modeling of 1,4-imino-á´ -lyxitols and their C-5-altered N-arylalkyl derivatives.

A synthesis of 1,4-imino-ᴅ-lyxitols and their N-arylalkyl derivatives altered at C-5 is reported. Their inhibitory activity and selectivity toward four GH38 α-mannosidases (two Golgi types: GMIIb from Drosophila melanogaster and AMAN-2 from Caenorhabditis elegans, and two lysosomal types: LManII from Drosophila melanogaster and JBMan from Canavalia ensiformis) were investigated. 6-Deoxy-DIM was found to be the most potent inhibitor of AMAN-2 (K i = 0.19 µM), whose amino acid sequence and 3D structure of the active site are almost identical to the human α-mannosidase II (GMII). Although 6-deoxy-DIM was 3.5 times more potent toward AMAN-2 than DIM, their selectivity profiles were almost the same. N-Arylalkylation of 6-deoxy-DIM resulted only in a partial improvement as the selectivity was enhanced at the expense of potency. Structural and physicochemical properties of the corresponding inhibitor:enzyme complexes were analyzed by molecular modeling.

Iminosugar Glucosidase Inhibitors Reduce Hepatic Inflammation in Hepatitis A Virus-Infected Ifnar1-/- Mice.

Iminosugar compounds are monosaccharide mimetics with broad but generally weak antiviral activities related to inhibition of enzymes involved in glycobiology. Miglustat (N-butyl-1-deoxynojirimycin), which is approved for the treatment of lipid storage diseases in humans, and UV-4 [N-(9-methoxynonyl)-1-deoxynojirimycin] inhibit the replication of hepatitis A virus (HAV) in cell culture (50% inhibitory concentrations [IC50s] of 32.13 µM and 8.05 µM, respectively) by blocking the synthesis of gangliosides essential for HAV cell entry. We used a murine model of hepatitis A and targeted mass spectrometry to assess the capacity of these compounds to deplete hepatic gangliosides and modify the course of HAV infection in vivo. Miglustat, given by gavage to Ifnar1-/- mice (4,800 mg/kg of body weight/day) depleted hepatic gangliosides by 69 to 75% but caused substantial gastrointestinal toxicity and failed to prevent viral infection. UV-4, similarly administered in high doses (400 mg/kg/day), was well tolerated but depleted hepatic gangliosides by only 20% after 14 days. UV-4 depletion of gangliosides varied by class. Several GM2 species were paradoxically increased, likely due to inhibition of ß-glucosidases that degrade gangliosides. Both compounds enhanced, rather than reduced, virus replication. Nonetheless, both iminosugars had surprising anti-inflammatory effects, blocking the accumulation of inflammatory cells within the liver. UV-4 treatment also resulted in a decrease in serum alanine aminotransferase (ALT) elevations associated with acute hepatitis A. These anti-inflammatory effects may result from iminosugar inhibition of cellular α-glucosidases, leading to impaired maturation of glycan moieties of chemokine and cytokine receptors, and point to the potential importance of paracrine signaling in the pathogenesis of acute hepatitis A. IMPORTANCE Hepatitis A virus (HAV) is a common cause of viral hepatitis. Iminosugar compounds block its replication in cultured cells by inhibiting the synthesis of gangliosides required for HAV cell entry but have not been tested for their ability to prevent or treat hepatitis A in vivo. We show that high doses of the iminosugars miglustat and UV-4 fail to deplete gangliosides sufficiently to block HAV infection in mice lacking a key interferon receptor. These compounds nonetheless have striking anti-inflammatory effects on the HAV-infected liver, reducing the severity of hepatitis despite enhancing chemokine and cytokine expression resulting from hepatocyte-intrinsic antiviral responses. We propose that iminosugar inhibition of cellular α-glucosidases impairs the maturation of glycan moieties of chemokine and cytokine receptors required for effective signaling. These data highlight the potential importance of paracrine signaling pathways in the inflammatory response to HAV and add to our understanding of HAV pathogenesis in mice.

sp2-Iminosugars targeting human lysosomal ß-hexosaminidase as pharmacological chaperone candidates for late-onset Tay-Sachs disease.

The late-onset form of Tay-Sachs disease displays when the activity levels of human ß-hexosaminidase A (HexA) fall below 10% of normal, due to mutations that destabilise the native folded form of the enzyme and impair its trafficking to the lysosome. Competitive inhibitors of HexA can rescue disease-causative mutant HexA, bearing potential as pharmacological chaperones, but often also inhibit the enzyme O-glucosaminidase (GlcNAcase; OGA), a serious drawback for translation into the clinic. We have designed sp2-iminosugar glycomimetics related to GalNAc that feature a neutral piperidine-derived thiourea or a basic piperidine-thiazolidine bicyclic core and behave as selective nanomolar competitive inhibitors of human Hex A at pH 7 with a ten-fold lower inhibitory potency at pH 5, a good indication for pharmacological chaperoning. They increased the levels of lysosomal HexA activity in Tay-Sachs patient fibroblasts having the G269S mutation, the highest prevalent in late-onset Tay-Sachs disease.

Investigational New Drug Enabling Nonclinical Safety Pharmacology Assessment of the Iminosugar UV-4, a Broad-Spectrum Host-Targeted Antiviral Agent.

UV-4 (N-(9-methoxynonyl)-1-deoxynojirimycin) is a broad-spectrum antiviral drug candidate with demonstrated activity in vitro and in vivo against multiple, diverse viruses. Nonclinical safety pharmacology studies were conducted to support the filing of an Investigational New Drug (IND) application. Preliminary in vitro pharmacology testing evaluating potential for binding to "off-target" receptors and enzymes indicated no significant liability for advanced development of UV-4. The safety pharmacology of UV-4 was evaluated in the in vitro human ether-à-go-go-related gene (hERG) assay, in a central nervous system (CNS) study in the mouse (modified Irwin test), in a respiratory safety study in conscious mice using whole body plethysmography, and in a cardiovascular safety study in conscious, radiotelemetry-instrumented beagle dogs. There were no observed adverse treatment-related effects following administration of UV-4 as the hydrochloride salt in the hERG potassium channel assay, on respiratory function, in the CNS study, or in the cardiovascular assessment. Treatment-related cardiovascular effect of decreased arterial pulse pressure after 50 or 200 mg of UV-4/kg was the only change outside the normal range, and all hemodynamic parameters returned to control levels by the end of the telemetry recording period. These nonclinical safety pharmacology assessments support the evaluation of this host-targeted broad-spectrum antiviral drug candidate in clinical studies.

C5-Iminosugar modification of casein kinase 1δ lead 3-(4-fluorophenyl)-5-isopropyl-4-(pyridin-4-yl)isoxazole promotes enhanced inhibitor affinity and selectivity.

The quest for isoform-selective and specific ATP-competitive protein kinase inhibitors is of great interest, as inhibitors with these qualities will come with reduced toxicity and improved efficacy. However, creating such inhibitors is very challenging due to the high molecular similarity of kinases ATP active sites. To achieve selectivity for our casein kinase (CK) 1 inhibitor series, we elected to endow our previous CK1δ-hit, 3-(4-fluorophenyl)-5-isopropyl-4-(pyridin-4-yl)isoxazole (1), with chiral iminosugar scaffolds. These scaffolds were attached to C5 of the isoxazole ring, a position deemed favorable to facilitate binding interactions with the ribose pocket/solvent-open area of the ATP binding pocket of CK1δ. Here, we describe the synthesis of analogs of 1 ((-)-/(+)-34, (-)-/(+)-48), which were prepared in 13 steps from enantiomerically pure ethyl (3R,4S)- and ethyl (3S,4R)-1-benzyl-4-[(tert-butyldimethylsilyl)oxy]-5-oxopyrrolidine-3-carboxylate ((-)-11 and (+)-11), respectively. The synthesis involved the coupling of Weinreb amide-activated chiral pyrrolidine scaffolds with 4- and 2-fluoro-4-picoline and reaction of the resulting 4-picolyl ketone intermediates ((-)-/(+)-40 and (-)-/(+)-44) with 4-fluoro-N-hydroxybenzenecarboximidoyl chloride to form the desired isoxazole ring. The activity of the compounds against human CK1δ, -ε, and -α was assessed in recently optimized in vitro assays. Compound (-)-34 was the most active compound with IC50 values (CK1δ/ε) of 1/8 µM and displayed enhanced selectivity toward CK1δ.

Multivalent Pyrrolidine Iminosugars: Synthesis and Biological Relevance.

Recently, the strategy of multivalency has been widely employed to design glycosidase inhibitors, as glycomimetic clusters often induce marked enzyme inhibition relative to monovalent analogs. Polyhydroxylated pyrrolidines, one of the most studied classes of iminosugars, are an attractive moiety due to their potent and specific inhibition of glycosidases and glycosyltransferases, which are associated with many crucial biological processes. The development of multivalent pyrrolidine derivatives as glycosidase inhibitors has resulted in several promising compounds that stand out. Herein, we comprehensively summarized the different synthetic approaches to the preparation of multivalent pyrrolidine clusters, from total synthesis of divalent iminosugars to complex architectures bearing twelve pyrrolidine motifs. Enzyme inhibitory properties and multivalent effects of these synthesized iminosugars were further discussed, especially for some less studied therapeutically relevant enzymes. We envision that this comprehensive review will help extend the applications of multivalent pyrrolidine iminosugars in future studies.

Iminosugar Amino Acid idoBR1 Reduces Inflammatory Responses in Microglia.

(1) Background. Inflammation is reported to be a key factor in neurodegeneration. The microglia are immune cells present in the central nervous system; their activation results in the release of inflammatory cytokines and is thought to be related to aging and neurodegenerative disorders, such as Alzheimer's disease. (2) Methods. A mouse BV-2 microglia cell line was activated using LPS and the anti-inflammatory cucumber-derived iminosugar amino acid idoBR1, (2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid, was used alongside dexamethasone as the control to determine whether it could reduce the inflammatory responses. (3) Results. A dose-dependent reduction in the LPS-induced production of the proinflammatory factors TNFα, IL-6, and nitric oxide and the transcription factor NF-κB was found. (4) Conclusions. Further investigations of the anti-inflammatory effects of idoBR1 in other models of neurodegenerative diseases are warranted.

The iminosugars celgosivir, castanospermine and UV-4 inhibit SARS-CoV-2 replication.

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.

Pathogen-induced inflammation is attenuated by the iminosugar MON-DNJ via modulation of the unfolded protein response.

Sepsis is a life-threatening condition involving a dysregulated immune response to infectious agents that cause injury to host tissues and organs. Current treatments are limited to early administration of antibiotics and supportive care. While appealing, the strategy of targeted inhibition of individual molecules in the inflammatory cascade has not proved beneficial. Non-targeted, systemic immunosuppression with steroids has shown limited efficacy and raises concern for secondary infection. Iminosugars are a class of small molecule glycomimetics with distinct inhibition profiles for glycan processing enzymes based on stereochemistry. Inhibition of host endoplasmic reticulum resident glycoprotein processing enzymes has demonstrated efficacy as a broad-spectrum antiviral strategy, but limited consideration has been given to the effects on host glycoprotein production and consequent disruption of signalling cascades. This work demonstrates that iminosugars inhibit dengue virus, bacterial lipopolysaccharide and fungal antigen-stimulated cytokine responses in human macrophages. In spite of decreased inflammatory mediator production, viral replication is suppressed in the presence of iminosugar. Transcriptome analysis reveals the key interaction of pathogen-induced endoplasmic reticulum stress, the resulting unfolded protein response and inflammation. Our work shows that iminosugars modulate these interactions. Based on these findings, we propose a new therapeutic role for iminosugars as treatment for sepsis-related inflammatory disorders associated with excess cytokine secretion.

Seven-Step Synthesis of All-Nitrogenated Sugar Derivatives Using Sequential Overman Rearrangements.

All-nitrogenated sugars (ANSs), in which all hydroxy groups in a carbohydrate are replaced with amino groups, are anticipated to be privileged structures with useful biological activities. However, ANS synthesis has been challenging due to the difficulty in the installation of multi-amino groups. We report herein the development of a concise synthetic route to peracetylated ANSs in seven steps from commercially available monosaccharides. The key to success is the use of the sequential Overman rearrangement, which enables formal simultaneous substitution of four or five hydroxy groups in monosaccharides with amino groups. A variety of ANSs are available through the same reaction sequence starting from different initial monosaccharides by chirality transfer of secondary alcohols. Transformations of the resulting peracetylated ANSs such as glycosylation and deacetylation are also demonstrated. Biological studies reveal that ANS-modified cholesterol show cytotoxicity against human cancer cell lines, whereas each ANS and cholesterol have no cytotoxicity.

N,O-Dialkyl deoxynojirimycin derivatives as CERT START domain ligands.

Dysregulation of the ceramide transport protein CERT is associated to diseases such as cancer. In search for new CERT START domain ligands, N-dodecyl-deoxynojirimycin (N-dodecyl-DNJ) iminosugar was found to display, as a ceramide mimic, significant protein recognition. To reinforce the lipophilic interactions and strengthen this protein binding, a docking study was carried out in order to select the optimal position on which to introduce an additional O-alkyl chain on N-dodecyl-DNJ. Analysis of the calculated poses for three different regioisomers indicated an optimal calculated interaction pattern for N,O3-didodecyl-DNJ. The two most promising regioisomers were prepared by a divergent route and their binding to the CERT START domain was evaluated with fluorescence intensity (FLINT) binding assay. N,O3-didodecyl-DNJ was confirmed to be a new binder prototype with level of protein recognition in the FLINT assay comparable to the best known ligands from the alkylated HPA-12 series. This work opens promising perspectives for the development of new inhibitors of CERT-mediated ceramide trafficking.

Kinetic analysis of inhibition of α-glucosidase by leaf powder from Morus australis and its component iminosugars.

Mulberry leaves contain iminosugars, such as 1-deoxynojirimycin (1-DNJ), fagomine, and 2-O-α-D-galactopyranosyl deoxynojirimycin (GAL-DNJ) that inhibit α-glucosidase. In this study, we quantified iminosugars in Morus australis leaves and made the kinetic analysis in the hydrolysis of maltose by α-glucosidase. By LC-MS/MS, the concentrations of 1-DNJ, fagomine, and GAL-DNJ in the powdered leaves were 4.0, 0.46, and 2.5 mg/g, respectively, and those in the roasted ones were 1.0, 0.24, and 0.73 mg/g, respectively, suggesting that the roasting process degraded iminosugars. Steady-state kinetic analysis revealed that the powdered and roasted leaves exhibited competitive inhibition. At pH 6.0 at 37ºC, the IC50 values of the extracts from the boiled powdered or roasted leaves were 0.36 and 1.1 mg/mL, respectively. At the same condition, the IC50 values of 1-DNJ, fagomine, and GAL-DNJ were 0.70 µg/mL, 0.18 mg/mL, and 2.9 mg/mL, respectively. These results suggested that in M. australis, 1-DNJ is a major inhibitor of α-glucosidase. ABBREVIATIONS: 1-DNJ: 1-deoxynojirimycin; GAL-DNJ: 2-O-α-D-galactopyranosyl-DNJ.

Characterizing the selectivity of ER α-glucosidase inhibitors.

The endoplasmic reticulum (ER) contains both α-glucosidases and α-mannosidases which process the N-linked oligosaccharides of newly synthesized glycoproteins and thereby facilitate polypeptide folding and glycoprotein quality control. By acting as structural mimetics, iminosugars can selectively inhibit these ER localized α-glycosidases, preventing N-glycan trimming and providing a molecular basis for their therapeutic applications. In this study, we investigate the effects of a panel of nine iminosugars on the actions of ER luminal α-glucosidase I and α-glucosidase II. Using ER microsomes to recapitulate authentic protein N-glycosylation and oligosaccharide processing, we identify five iminosugars that selectively inhibit N-glycan trimming. Comparison of their inhibitory activities in ER microsomes against their effects on purified ER α-glucosidase II, suggests that 3,7a-diepi-alexine acts as a selective inhibitor of ER α-glucosidase I. The other active iminosugars all inhibit α-glucosidase II and, having identified 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) as the most effective of these compounds, we use in silico modeling to understand the molecular basis for this enhanced activity. Taken together, our work identifies the C-3 substituted pyrrolizidines casuarine and 3,7a-diepi-alexine as promising "second-generation" iminosugar inhibitors.