Exploring the effect of chirality on the therapeutic potential of N-alkyl-deoxyiminosugars: anti-inflammatory response to Pseudomonas aeruginosa infections for application in CF lung disease.

In the frame of a research program aimed to explore the relationship between chirality of iminosugars and their therapeutic potential, herein we report the synthesis of N-akyl l-deoxyiminosugars and the evaluation of the anti-inflammatory properties of selected candidates for the treatment of Pseudomonas aeruginosa infections in Cystic Fibrosis (CF) lung disease. Target glycomimetics were prepared by the shortest and most convenient approach reported to date, relying on the use of the well-known PS-TPP/I2 reagent system to prepare reactive alkoxyalkyl iodides, acting as key intermediates. Iminosugars ent-1-3 demonstrated to efficiently reduce the inflammatory response induced by P. aeruginosa in CuFi cells, either alone or in synergistic combination with their d-enantiomers, by selectively inhibiting NLGase. Surprisingly, the evaluation in murine models of lung disease showed that the amount of ent-1 required to reduce the recruitment of neutrophils was 40-fold lower than that of the corresponding d-enantiomer. The remarkably low dosage of the l-iminosugar, combined with its inability to act as inhibitor for most glycosidases, is expected to limit the onset of undesired effects, which are typically associated with the administration of its d-counterpart. Biological results herein obtained place ent-1 and congeners among the earliest examples of l-iminosugars acting as anti-inflammatory agents for therapeutic applications in Cystic Fibrosis.

Design of a New α-1-C-Alkyl-DAB Derivative Acting as a Pharmacological Chaperone for ß-Glucocerebrosidase Using Ligand Docking and Molecular Dynamics Simulation.

Some point mutations in ß-glucocerebrosidase cause either improper folding or instability of this protein, resulting in Gaucher disease. Pharmacological chaperones bind to the mutant enzyme and stabilize this enzyme; thus, pharmacological chaperone therapy was proposed as a potential treatment for Gaucher disease. The binding affinities of α-1-C-alkyl 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) derivatives, which act as pharmacological chaperones for ß-glucocerebrosidase, abruptly increased upon elongation of their alkyl chain. In this study, the primary causes of such an increase in binding affinity were analyzed using protein⁻ligand docking and molecular dynamics simulations. We found that the activity cliff between α-1-C-heptyl-DAB and α-1-C-octyl-DAB was due to the shape and size of the hydrophobic binding site accommodating the alkyl chains, and that the interaction with this hydrophobic site controlled the binding affinity of the ligands well. Furthermore, based on the aromatic/hydrophobic properties of the binding site, a 7-(tetralin-2-yl)-heptyl-DAB compound was designed and synthesized. This compound had significantly enhanced activity. The design strategy in consideration of aromatic interactions in the hydrophobic pocket was useful for generating effective pharmacological chaperones for the treatment of Gaucher disease.

Probing the Inhibitor versus Chaperone Properties of sp²-Iminosugars towards Human ß-Glucocerebrosidase: A Picomolar Chaperone for Gaucher Disease.

A series of sp²-iminosugar glycomimetics differing in the reducing or nonreducing character, the configurational pattern (d-gluco or l-ido), the architecture of the glycone skeleton, and the nature of the nonglycone substituent has been synthesized and assayed for their inhibition properties towards commercial glycosidases. On the basis of their affinity and selectivity towards GH1 ß-glucosidases, reducing and nonreducing bicyclic derivatives having a hydroxylation profile of structural complementarity with d-glucose and incorporating an N'-octyl-isourea or -isothiourea segment were selected for further evaluation of their inhibitory/chaperoning potential against human glucocerebrosidase (GCase). The 1-deoxynojirimycin (DNJ)-related nonreducing conjugates behaved as stronger GCase inhibitors than the reducing counterparts and exhibited potent chaperoning capabilities in Gaucher fibroblasts hosting the neuronopathic G188S/G183W mutation, the isothiourea derivative being indeed one of the most efficient chaperone candidates reported up to date (70% activity enhancement at 20 pM). At their optimal concentration, the four selected compounds promoted mutant GCase activity enhancements over 3-fold; yet, the inhibitor/chaperoning balance became unfavorable at much lower concentration for nonreducing as compared to reducing derivatives.

Selective glycosidase inhibitors: A patent review (2012-present).

In the recent decades, the interest on glycosidases has dramatically increased, mainly because these enzymes play a vital role in many biological processes. Based on the biological potential associated to these enzymes, several glycosidase inhibitors have been developed. In this review, the most important inhibitors targeting these enzymes, including the disaccharides, iminosugars, monocyclic iminosugars, bicyclic iminosugars, thiosugars and carbasugars will be discussed and special attention will be given to the ones that are currently used clinically. This review summarizes and characterizes the current knowledge regarding the classes of glycosidase inhibitors that have therapeutic potential in a wide range of diseases. It highlights the patents, relevant research and patent applications filed in the past years in the field. Since the glycosidase inhibitors are involved in several chronic diseases and possibly pandemic, the pharmaceutical research towards developing new generations of these molecules is very important to public health. Most of the glycosidase inhibitors mimics the structures of monosaccharides or oligosaccharides and are well accepted by the organisms since they benefit from privileged drug-like properties. Disaccharides, iminosugars, carbasugars and thiosugars derivatives are the most popular inhibitors among the glycosidase inhibitors.

Iminosugar antivirals: the therapeutic sweet spot.

Many viruses require the host endoplasmic reticulum protein-folding machinery in order to correctly fold one or more of their glycoproteins. Iminosugars with glucose stereochemistry target the glucosidases which are key for entry into the glycoprotein folding cycle. Viral glycoproteins are thus prevented from interacting with the protein-folding machinery leading to misfolding and an antiviral effect against a wide range of different viral families. As iminosugars target host enzymes, they should be refractory to mutations in the virus. Iminosugars therefore have great potential for development as broad-spectrum antiviral therapeutics. We outline the mechanism giving rise to the antiviral activity of iminosugars, the current progress in the development of iminosugar antivirals and future prospects for this field.

Assessment of the potential for host-targeted iminosugars UV-4 and UV-5 activity against filovirus infections in vitro and in vivo.

Iminosugars are host-directed antivirals with broad-spectrum activity. The iminosugar, N-butyl-deoxynojirimycin (NB-DNJ or Miglustat®), is used in humans for treatment of Gaucher's disease and has mild antiviral properties. More potent analogs of NB-DNJ have been generated and have demonstrated activity against a variety of viruses including flaviviruses, influenza, herpesviruses and filoviruses. In the current study, a panel of analogs based on NB-DNJ was analyzed for activity against Ebola (EBOV) and Marburg viruses (MARV). The antiviral activity of NB-DNJ (UV-1), UV-2, UV-3, UV-4 and UV-5 against both EBOV and MARV was demonstrated in Vero cells. Subsequent studies to examine the activity of UV-4 and UV-5 using rodent models of EBOV and MARV were performed. In vivo efficacy studies provided inconsistent data following treatment with iminosugars using filovirus mouse models. A tolerability study in nonhuman primates demonstrated that UV-4 could be administered at much higher dose levels than rodents. Since UV-4 was active in vitro, had been demonstrated to be active against influenza and dengue in vivo, and was being tested in a Phase 1 clinical trial, a small proof-of-concept nonhuman primate trial was performed to determine whether this antiviral candidate could provide clinical benefit to EBOV-infected individuals. Administration of UV-4B did not provide a clinical or survival benefit to macaques infected with EBOV-Makona; however, dosing of animals was not optimal in this study. Efficacy may be improved by thrice daily dosing (e.g. by nasogastric tube feeding) to match the efficacious dosing regimens demonstrated against dengue and influenza viruses.

Bioevaluation of sixteen ADMDP stereoisomers toward alpha-galactosidase A: Development of a new pharmacological chaperone for the treatment of Fabry disease and potential enhancement of enzyme replacement therapy efficiency.

A unique molecular library consisting of all sixteen synthetic ADMDP (1-aminodeoxy-DMDP) stereoisomers has been prepared and evaluated for inhibitory activity against α-Gal A, and ability to impart thermal stabilization of this enzyme. The results of this testing led us to develop a novel pharmacological chaperone for the treatment of Fabry disease. 3-Epimer ADMDP was found to be an effective pharmacological chaperone, able to rescue α-Gal A activity in the lymphoblast of the N215S Fabry patient-derived cell line, without impairment of cellular ß-galactosidase activity. When 3-epimer ADMDP was administered with rh-α-Gal A (enzyme replacement therapy) for the treatment of Fabry patient-derived cell lines, improvements in the efficacy of rh-α-Gal A was observed, which suggests this small molecule can also provide clinical benefit of enzyme replacement therapy in Fabry disease.

Carbohydrate CuAAC click chemistry for therapy and diagnosis.

Carbohydrates are important as signaling molecules and for cellular recognition events, therefore offering scope for the development of carbohydrate-mimetic diagnostics and drug candidates. As a consequence, the construction of carbohydrate-based bioactive compounds and sensors has become an active research area. While the advent of click chemistry has greatly accelerated the progress of medicinal chemistry and chemical biology, recent literature has seen an extensive use of such approaches to construct functionally diverse carbohydrate derivatives. Here we summarize some of the progress, covering the period 2010 to mid-2015, in Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition CuAAC "click chemistry" of carbohydrate derivatives, in the context of potential therapeutic and diagnostic tool development.

An iminosugar-based heparanase inhibitor heparastatin (SF4) suppresses infiltration of neutrophils and monocytes into inflamed dorsal air pouches.

Local infiltration of inflammatory cells is regulated by a number of biological steps during which the cells likely penetrate through subendothelial basement membranes that contain heparan sulfate proteoglycans. In the present study, we examined whether administration of heparastatin (SF4), an iminosugar-based inhibitor of heparanase, could suppress local inflammation and degradation of heparan sulfate proteoglycans in basement membranes. In a carrageenan- or formyl peptide-induced dorsal air pouch inflammation model, the number of infiltrated neutrophils and monocytes was significantly lower in mice after topical administration of heparastatin (SF4). The concentration of chemokines MIP-2 and KC in pouch exudates of drug-treated mice was similar to control. In a zymosan-induced peritonitis model, the number of infiltrated cells was not altered in drug-treated mice. To further test how heparastatin (SF4) influences transmigration of inflammatory neutrophils, its suppressive effect on migration and matrix degradation was examined in vitro. In the presence of heparastatin (SF4), the number of neutrophils that infiltrated across a Matrigel-coated polycarbonate membrane was significantly lower, while the number of neutrophils passing through an uncoated membrane was not altered. Lysate of bone marrow-derived neutrophils released sulfate-radiolabeled macromolecules from basement membrane-like extracellular matrix, which was suppressed by heparastatin (SF4). Heparan sulfate degradation activity was almost completely abolished after incubation of lysate with protein G-conjugated anti-heparanase monoclonal antibody, strongly suggesting that the activity was due to heparanase-mediated degradation. Taken together, in a dorsal air pouch inflammation model heparastatin (SF4) potentially suppresses extravasation of inflammatory cells by impairing the degradation of basement membrane heparan sulfate.

A Novel Iminosugar UV-12 with Activity against the Diverse Viruses Influenza and Dengue (Novel Iminosugar Antiviral for Influenza and Dengue).

Iminosugars are capable of targeting the life cycles of multiple viruses by blocking host endoplasmic reticulum α-glucosidase enzymes that are required for competent replication of a variety of enveloped, glycosylated viruses. Iminosugars as a class are approved for use in humans with diseases such as diabetes and Gaucher's disease, providing evidence for safety of this class of compounds. The in vitro antiviral activity of iminosugars has been described in several publications with a subset of these demonstrating in vivo activity against flaviviruses, herpesviruses, retroviruses and filoviruses. Although there is compelling non-clinical in vivo evidence of antiviral efficacy, the efficacy of iminosugars as antivirals has yet to be demonstrated in humans. In the current study, we report a novel iminosugar, UV-12, which has efficacy against dengue and influenza in mouse models. UV-12 exhibits drug-like properties including oral bioavailability and good safety profile in mice and guinea pigs. UV-12 is an example of an iminosugar with activity against multiple virus families that should be investigated in further safety and efficacy studies and demonstrates potential value of this drug class as antiviral therapeutics.

Iminosugar derivative WGN-26 suppresses acute allograft rejection via inhibiting the IFN-γ/p-STAT1/T-bet signaling pathway.

This study aimed to investigate the effect of the iminosugar derivative WGN-26 on suppressing acute allograft rejection and to explore the underlying mechanisms. The results demonstrated that WGN-26 (12, 6 and 3mg/kg) significantly prolonged the skin allograft survival time in a dose-dependent manner and minimized the pathological changes. The minimum lethal dose was 320 mg/kg. By exploring the potential cellular and molecular mechanisms, we found that WGN-26 dose-dependently inhibited T lymphocyte proliferation, as determined through the single mixed lymphocyte reaction (sMLR) or the ConA-induced T cell proliferation assay in allograft recipients. The FCM results indicated that WGN-26 particularly reduced the percentage of CD3(+)CD4(+) T cells in allograft recipients. After treatment with WGN-26, the secretion of IFN-γ in allograft recipients was lowered, whereas the IL-4 and IL-17 levels remained stable. Furthermore, we found that WGN-26 inhibited the phosphorylation of STAT1 and accelerated the degradation of T-bet protein in allograft recipients. This study provides the first report that the iminosugar derivative WGN-26 dose-dependently prolongs skin allograft survival and that the possible mechanism is mediated by inhibiting CD4(+) T cell proliferation and suppressing the IFN-γ/p-STAT1/T-bet signaling pathway.

Iminosugar-based galactoside mimics as inhibitors of galactocerebrosidase: SAR studies and comparison with other lysosomal galactosidases.

Several families of iminosugar-based galactoside mimics were designed, synthesized, and evaluated as galactocerebrosidase (GALC) inhibitors. They were also tested as inhibitors of lysosomal ß- and α-galactosidases in order to find new potent and selective pharmacological chaperones for treatment of the lysosomal storage disorder, Krabbe disease. Whereas 1-C-alkyl imino-L-arabinitols are totally inactive toward the three enzymes, 1-C-alkyl imino-D-galactitols were found to be active only toward α-galactosidase A. Finally, 1-N-iminosugars provided the best results, as 4-epi-isofagomine was found to be a good inhibitor of both lysosomal ß-galactosidase and GALC. Further elaboration of this structure is required to achieve selectivity between these two galactosidases.

A systematic investigation of iminosugar click clusters as pharmacological chaperones for the treatment of Gaucher disease.

A series of 18 mono- to 14-valent iminosugars with different ligands, scaffolds, and alkyl spacer lengths have been synthesized and evaluated as inhibitors and pharmacological chaperones of ß-glucocerebrosidase (GCase). Small but significant multivalent effects in GCase inhibition have been observed for two iminosugar clusters. Our study provides strong confirmation that compounds that display the best affinity for GCase are not necessarily the best chaperones. The best chaperoning effect observed for a deprotected iminosugar cluster has been obtained with a tetravalent 1-deoxynojirimycin (DNJ) analogue (3.3-fold increase at 10 µM). In addition, our study provides the first evidence of the high potential of prodrugs for the development of potent pharmacological chaperones. Acetylation of a trivalent DNJ derivative, to give the corresponding acetate prodrug, leads to a pharmacological chaperone that produces higher enzyme activity increases (3.0-fold instead of 2.4-fold) at a cellular concentration (1 µM) reduced by one order of magnitude.

Rescue of functional CFTR channels in cystic fibrosis: a dramatic multivalent effect using iminosugar cluster-based correctors.

Cystic fibrosis is caused by a mutation in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. N-butyl 1-deoxynojirimycin (N-Bu DNJ), a clinical candidate for the treatment of cystic fibrosis, is able to act as a CFTR corrector by overcoming the processing defect of the mutant protein. To explore the potential of multivalency on CFTR correction activity, a library of twelve DNJ click clusters with valencies ranging from 3 to 14 were synthesized. Significantly, the trivalent analogues were found to be up to 225-fold more potent than N-Bu DNJ and up to 1000-fold more potent than the corresponding monovalent models. These results provide the first description of a multivalent effect for correcting protein folding defects in cells and should have application for the treatment of a number of protein folding disorders. Preliminary mechanistic studies indicated that CFTR correction activity enhancement was not due to a multivalent effect in ER-glucosidase inhibition or to a different mode of action of the multivalent iminosugars.

[Synthetic medicinal chemistry of the biomolecular components mimics].

We are studying the medicinal synthetic chemistry of biomolecular component mimics such as carbohydrates, nucleosides, amino acids, and peptides. In this review, the synthesis and biological activities of iminosugars as carbohydrate mimics are discussed. Glycosidases and glycosyltransferases are involved in a wide range of anabolic and catabolic process, including digestion, the lysosomal catabolism of glycoconjugates, glycoprotein biosynthesis. Hence, modifying or blocking these processes in vivo using inhibitors is a topic of great interest from the therapeutic point of view. Iminosugars are sugars in which the endocyclic oxygen is replaced by a basic nitrogen atom. They are regarded as transition state mimics in certain types of enzyme reactions. This makes the field of iminosugars as carbohydrate mimics an exciting area of research. We synthesized all of the stereoisomers of polyhydroxypiperidines such as fagomine, 1-deoxynojirimycine, and isofagomine. In addition, their both enantiomers, as substrates for a variety of glycosidases were evaluated. Secondly, the asymmetric synthesis of α-1-C-alkyl-arabinoiminofuranoses was achieved by asymmetric allylic alkylation, RCM, and Negishi cross coupling as key reactions. Surprisingly, the L-forms showed a quite potent inhibitory activity toward rat intestinal maltase, while the activities of the D-forms were much weaker. Some of the prepared L-forms showed potent inhibitory activities towards intestinal maltase, with IC50 values comparable to those of commercial drugs such as acarbose, voglibose, and miglitol, which are used in the treatment of type 2 diabetes. Among them, the inhibitory activity towards intestinal sucrase of α-1-C-L-butylarabinoiminofuranose was quite strong towards intestinal sucrase compared to the above commercial drugs.

Glucocerebrosidase inhibitors for the treatment of Gaucher disease.

Gaucher disease is a progressive lysosomal storage disorder caused by a deficiency in the activity of ß-glucocerebrosidase and is characterized by the accumulation of the glycosphingolipid glucosylceramide in the lysosomes of macrophages that leads to dysfunction in multiple organ system. An emerging strategy for the treatment of Gaucher disease is pharmacological chaperone therapy, based on the use of ß-glucocerebrosidase inhibitors that are capable of enhancing residual hydrolytic activity at subinhibitory concentrations. In this article, the most common lysosomal storage disorder, Gaucher disease, is introduced and the current therapeutic strategies based on the use of enzyme inhibitors to ameliorate this disease are discussed, with a focus on the efforts being made toward finding and optimizing novel molecules as pharmacological chaperones for Gaucher disease that offer the promise to remedy this malady.

α-1-C-butyl-1,4-dideoxy-1,4-imino-l-arabinitol as a second-generation iminosugar-based oral α-glucosidase inhibitor for improving postprandial hyperglycemia.

We report on the synthesis and the biological evaluation of a series of α-1-C-alkylated 1,4-dideoxy-1,4-imino-l-arabinitol (LAB) derivatives. The asymmetric synthesis of the derivatives was achieved by asymmetric allylic alkylation, ring-closing metathesis, and Negishi cross-coupling as key reactions. α-1-C-Butyl-LAB is a potent inhibitor of intestinal maltase, isomaltase, and sucrase, with IC50 values of 0.13, 4.7, and 0.032 µM, respectively. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis revealed that this compound differs from miglitol in that it does not influence oligosaccharide processing and the maturation of glycoproteins. A molecular docking study of maltase-glucoamylase suggested that the interaction modes and the orientations of α-1-C-butyl-LAB and miglitol are clearly different. Furthermore, α-1-C-butyl-LAB strongly suppressed postprandial hyperglycemia at an early phase, similar to miglitol in vivo. It is noteworthy that the effective dose was about 10-fold lower than that for miglitol. α-1-C-Butyl-LAB therefore represents a new class of promising compounds that can improve postprandial hyperglycemia.

Liposome-mediated delivery of iminosugars enhances efficacy against dengue virus in vivo.

A key challenge faced by promising antiviral drugs, such as iminosugars, is in vivo delivery to achieve effective levels of drug without toxicity. Four iminosugars, all deoxynojirimycin (DNJ) derivatives-N-butyl DNJ (NB-DNJ), N-nonyl DNJ, N-(9-methoxynonyl) DNJ, and N-(6'-[4″-azido-2″-nitrophenylamino]hexyl)-1-DNJ (NAP-DNJ)-potently inhibited both the percentage of cells infected with dengue virus and release of infectious virus from primary human monocyte-derived macrophages, demonstrating their efficacy in primary cells. In a lethal antibody-dependent enhancement mouse model of dengue pathogenesis, free NB-DNJ significantly enhanced survival and lowered viral load in organs and serum. Liposome-mediated delivery of NB-DNJ, in comparison with free NB-DNJ, resulted in a 3-log(10) reduction in the dose of drug sufficient to enhance animal survival. The optimizing of the effective dose in this way could liberate the therapeutic potential of many cytotoxic antivirals against both dengue virus and a wide array of other viruses.