Alginate-Based Carriers Loaded with Mulberry (Morus alba L.) Leaf Extract: A Promising Strategy for Prolonging 1-Deoxynojirimicyn (DNJ) Systemic Activity for the Nutraceutical Management of Hyperglycemic Conditions.

The iminosugar 1-deoxynojirimicyn (DNJ) contained in mulberry leaves has displayed systemic beneficial effects against disorders of carbohydrate metabolism. Nevertheless, its effect is impaired by the short half-life. Alginate-based carriers were developed to encapsulate a DNJ-rich mulberry extract: Ca-alginate beads, obtained by external gelation, and spray-dried alginate microparticles (SDMs). Mean size and distribution, morphology, drug loading, encapsulation efficiency, experimental yield, and release characteristics were determined for the two formulations. Ca-alginate beads and SDMs exhibited an encapsulation efficiency of about 54% and 98%, respectively, and a DNJ loading in the range of 0.43-0.63 µg/mg. The in vitro release study demonstrated the carriers' capability in controlling the DNJ release in acid and basic conditions (<50% in 5 h), due to electrostatic interactions, which were demonstrated by 1H-NMR relaxometry studies. Thus, alginate-based particles proved to be promising strategies for producing food supplements containing mulberry leaf extracts for the management of hyperglycemic state.

Structure-Based Design of Potent Iminosugar Inhibitors of Endoplasmic Reticulum α-Glucosidase I with Anti-SARS-CoV-2 Activity.

Enveloped viruses depend on the host endoplasmic reticulum (ER) quality control (QC) machinery for proper glycoprotein folding. The endoplasmic reticulum quality control (ERQC) enzyme α-glucosidase I (α-GluI) is an attractive target for developing broad-spectrum antivirals. We synthesized 28 inhibitors designed to interact with all four subsites of the α-GluI active site. These inhibitors are derivatives of the iminosugars 1-deoxynojirimycin (1-DNJ) and valiolamine. Crystal structures of ER α-GluI bound to 25 1-DNJ and three valiolamine derivatives revealed the basis for inhibitory potency. We established the structure-activity relationship (SAR) and used the Site Identification by Ligand Competitive Saturation (SILCS) method to develop a model for predicting α-GluI inhibition. We screened the compounds against SARS-CoV-2 in vitro to identify those with greater antiviral activity than the benchmark α-glucosidase inhibitor UV-4. These host-targeting compounds are candidates for investigation in animal models of SARS-CoV-2 and for testing against other viruses that rely on ERQC for correct glycoprotein folding.

Synthesis of 1-Deoxymannojirimycin from d-Fructose using the Mitsunobu Reaction.

Three different Mitsunobu reactions have been investigated for the synthesis of 1-deoxymannojirimycin (1-DMJ) from d-fructose. The highest yielding and most practical synthesis can be undertaken on a 10 g scale with minimal chromatography. In the key step, N,O-di-Boc-hydroxylamine reacts with methyl 1,3-isopropylidene-α-d-fructofuranose under Mitsunobu conditions to give 14. Acidic hydrolysis affords nitrone 15, which reduces quantitatively via catalytic hydrogenolysis to afford 1-DMJ (4) in 55% overall yield from d-fructose (cf. 37% for azide route and 29% for nosyl route).

Introduction of C-alkyl branches to L-iminosugars changes their active site binding orientation.

L-ido-Deoxynojirimycin (L-ido-DNJ) itself showed no affinity for human lysosomal acid α-glucosidase (GAA), whereas 5-C-methyl-L-ido-DNJ showed a strong affinity for GAA, comparable to the glucose analog DNJ, with a Ki value of 0.060 µM. This excellent affinity for GAA and enzyme stabilization was observed only when methyl and ethyl groups were introduced. Docking simulation analysis revealed that the alkyl chains of 5-C-alkyl-L-ido-DNJs were stored in three different pockets, depending on their length, thereby the molecular orientation was changed. Comparison of the binding poses of DNJ and 5-C-methyl-L-ido-DNJ showed that they formed a common ionic interaction with Asp404, Asp518, and Asp616, but both the binding orientation and the distance between the ligand and each amino acid residue were different. 5-C-Methyl-L-ido-DNJ dose-dependently increased intracellular GAA activity in Pompe patient fibroblasts with the M519V mutation and also promoted enzyme transport to lysosomes. This study provides the first example of a strategy to design high-affinity ligands by introducing alkyl branches into rare sugars and L-sugar-type iminosugars to change the orientation of binding.

Hybrid Multivalent Jack Bean α-Mannosidase Inhibitors: The First Example of Gold Nanoparticles Decorated with Deoxynojirimycin Inhitopes.

Among carbohydrate-processing enzymes, Jack bean α-mannosidase (JBα-man) is the glycosidase with the best responsiveness to the multivalent presentation of iminosugar inhitopes. We report, in this work, the preparation of water dispersible gold nanoparticles simultaneously coated with the iminosugar deoxynojirimycin (DNJ) inhitope and simple monosaccharides (ß-d-gluco- or α-d-mannosides). The display of DNJ at the gold surface has been modulated (i) by using an amphiphilic linker longer than the aliphatic chain used for the monosaccharides and (ii) by presenting the inhitope, not only in monomeric form, but also in a trimeric fashion through combination of a dendron approach with glyconanotechnology. The latter strategy resulted in a strong enhancement of the inhibitory activity towards JBα-man, with a Ki in the nanomolar range (Ki = 84 nM), i.e., more than three orders of magnitude higher than the monovalent reference compound.

Resolving interactions of miglitol with normal and glycated human serum albumin by multivariate methods.

This article reports results of one of our projects related to the investigation of interactions of miglitol (MIG) with normal human serum albumin (HSA) and glycated HSA (GHSA) with the help of recording spectroscopic and electrochemical data. The experimental data were analyzed by conventional and chemometric methods to extract useful information for comprehensive justifications of the interactions of the MIG with HSA and GHSA. Hard- and soft-modeling chemometric methods were used to extract quantitative and qualitative information. Then, molecular docking techniques were used to further investigation of the binding of the MIG with HSA and GHSA and the extracted results were compatible with those obtained by experimental methods. Finally, according to the binding of the BV with HSA and GHSA, second-order differential pulse voltammetric data were recorded and calibrated with three-way calibration methods for exploiting second-order advantage for determination of the GHSA in the presence of the HSA to develop a novel chemometrics assisted-electroanalytical method for diagnostic and monitoring of diabetic.

Alpha-amylase as molecular target for treatment of diabetes mellitus: A comprehensive review.

The alpha (α)-amylase is a calcium metalloenzyme that aids digestion by breaking down polysaccharide molecules into smaller ones such as glucose and maltose. In addition, the enzyme causes postprandial hyperglycaemia and blood glucose levels to rise. α-Amylase is a well-known therapeutic target for the treatment and maintenance of postprandial blood glucose elevations. Various enzymatic inhibitors, such as acarbose, miglitol and voglibose, have been found to be effective in targeting this enzyme, prompting researchers to express an interest in developing potent alpha-amylase inhibitor molecules. The review mainly focused on designing different derivatives of drug molecules such as benzofuran hydrazone, indole hydrazone, spiroindolone, benzotriazoles, 1,3-diaryl-3-(arylamino) propan-1-one, oxadiazole and flavonoids along with their target-receptor interactions, IC50 values and other biological activities.

Development and evaluation of 1-deoxynojirimycin sustained-release delivery system: In vitro and in vivo characterization studies.

We aimed to establish a 1-Deoxynojirimycin (DNJ) sustained-release delivery system to improve the hypoglycemic effect of DNJ. We used a transdermal diffusion meter in an in vitro orthogonal experiment to determine the optimal composition of the DNJ sustained-release transdermal system. Based on the in vitro analysis results, a sustained-release patch was prepared, and its pharmacokinetics and other properties were determined in vivo. The results showed that 30% hydroxypropyl methylcellulose (K100M ), 14% carboxymethyl cellulose sodium and 26% oleic acid-azone compound as the matrix material, drug excipient, and penetration enhancer, respectively, produced an optimal DNJ sustained-release delivery system. In vitro release tests showed that the system slowly released DNJ within 12 hr, conforming to the Higuchi equation. In vivo experiments showed that the prepared patch had good hypoglycemic activity and continuously released DNJ within 10 hr. In vivo pharmacokinetic study results showed that compared to conventional patches, the prepared patch exhibited significantly different maximum concentration (Cmax ), time to achieve Cmax (Tmax ), and area under the curve from 0 to time t (AUC[0-t] ) as well as improved pharmacokinetics. In conclusion, the prepared DNJ patch has high stability, a sustained-release effect, and relatively good pharmacokinetics and is a safe dosage form that does not cause skin irritation.

[Synthesis of L-Iminofuranoses and Their Biological Evaluations].

Iminosugars are one of the compounds that mimic the structure of monosaccharides. Such sugar mimics have the ability to effectively and specifically inhibit various glycosidases and glycosyltransferases. After studying iminopyranose, miglitol, which has α-glucosidase inhibitory activity, was approved and used in the clinical treatment of diabetes. This study focused on l-iminofuranose derivatives to develop new anti-diabetic drug. As a result, it was found that l-iminofuranose having an alkyl group at C1 position show potent α-glucosidase inhibitory activity. Further structural-activity relationship studies were conducted, and interesting findings were obtained. This paper describes the details of those research developments.

Tuning the activity of iminosugars: novel N-alkylated deoxynojirimycin derivatives as strong BuChE inhibitors.

We have designed unprecedented cholinesterase inhibitors based on 1-deoxynojirimycin as potential anti-Alzheimer's agents. Compounds are comprised of three key structural motifs: the iminosugar, for interaction with cholinesterase catalytic anionic site (CAS); a hydrocarbon tether with variable lengths, and a fragment derived from 2-phenylethanol for promoting interactions with peripheral anionic site (PAS). Title compounds exhibited good selectivity towards BuChE, strongly depending on the substitution pattern and the length of the tether. The lead compounds were found to be strong mixed inhibitors of BuChE (IC50 = 1.8 and 1.9 µM). The presumptive binding mode of the lead compound was analysed using molecular docking simulations, revealing H-bond interactions with the catalytic subsite (His438) and CAS (Trp82 and Glu197) and van der Waals interactions with PAS (Thr284, Pro285, Asn289). They also lacked significant antiproliferative activity against tumour and non-tumour cells at 100 µM, making them promising new agents for tackling Alzheimer's disease through the cholinergic approach.

Comparative Transcriptome Analysis of Key Reductase Genes Involved in the 1-Deoxynojirimycin Biosynthetic Pathway in Mulberry Leaves and Cloning, Prokaryotic Expression, and Functional Analysis of MaSDR1 and MaSDR2.

The alkaloid 1-deoxynojirimycin (DNJ) is the main bioactive ingredient in the hypoglycemic action of mulberry leaves (Morus alba L.). Our previous research clarified the upstream pathway from lysine to Δ1-piperideine in the biosynthesis of DNJ in mulberry leaves, but the pathway and related reductase genes from Δ1-piperideine to piperidine are still unclear. Here, a comparative transcriptome was used to analyze the transcriptome data of two samples (July and November) of mulberry leaves with significant differences in the content of DNJ and screen-related reductase genes. Results showed that expression levels of MaSDR1 and MaSDR2 were significantly and positively correlated with the content of DNJ (P < 0.05) in different seasons. MaSDR1 (GenBank accession no. MT989445) and MaSDR2 (GenBank accession no. MT989446) were successfully cloned and used for prokaryotic expression and functional analysis in vitro. MaSDR1 and MaSDR2 could catalyze the reaction of Δ1-piperideine with the coenzyme NADPH to generate piperidine. The kinetic parameters of MaSDR1 and MaSDR2 indicated that MaSDR2 had a higher binding ability to Δ1-piperideine than MaSDR1. This study provided insights into the biosynthesis of DNJ in mulberry leaves.

Misfolding of Lysosomal α-Galactosidase a in a Fly Model and Its Alleviation by the Pharmacological Chaperone Migalastat.

Fabry disease, an X-linked recessive lysosomal disease, results from mutations in the GLA gene encoding lysosomal α-galactosidase A (α-Gal A). Due to these mutations, there is accumulation of globotriaosylceramide (GL-3) in plasma and in a wide range of cells throughout the body. Like other lysosomal enzymes, α-Gal A is synthesized on endoplasmic reticulum (ER) bound polyribosomes, and upon entry into the ER it undergoes glycosylation and folding. It was previously suggested that α-Gal A variants are recognized as misfolded in the ER and undergo ER-associated degradation (ERAD). In the present study, we used Drosophila melanogaster to model misfolding of α-Gal A mutants. We did so by creating transgenic flies expressing mutant α-Gal A variants and assessing development of ER stress, activation of the ER stress response and their relief with a known α-Gal A chaperone, migalastat. Our results showed that the A156V and the A285D α-Gal A mutants underwent ER retention, which led to activation of unfolded protein response (UPR) and ERAD. UPR could be alleviated by migalastat. When expressed in the fly's dopaminergic cells, misfolding of α-Gal A and UPR activation led to death of these cells and to a shorter life span, which could be improved, in a mutation-dependent manner, by migalastat.

Glycoside Hydrolases Restrict the Side Chain Conformation of Their Substrates To Gain Additional Transition State Stabilization.

Carbohydrate side chain conformation confers a significant influence on reactivity during glycosylation and anomeric bond hydrolysis due to stabilization of the oxocarbenium-like transition state. By analysis of 513 pyranoside-bound glycoside hydrolase (GH) crystal structures, we determine that most glucosidases and ß-mannosidases preferentially bind their substrates in the most reactive gauche,gauche (gg) conformation, thereby maximizing stabilization of the corresponding oxocarbenium ion-like transition state during hydrolysis. α-Galactoside hydrolases mostly show a preference for the second most activating gauche,trans (gt) conformation to avoid the energy penalty that would arise from imposing the gg conformation on galacto-configured ligands. These preferences stand in stark contrast to the side chain populations observed for these sugars both in free solution and bound to nonhydrolytic proteins, where for the most part a much greater diversity of side chain conformations is observed. Analysis of sequences of GH-ligand complexes reveals that side chain restriction begins with the enzyme-substrate complex and persists through the transition state until release of the hydrolysis product, despite changes in ring conformation along the reaction coordinate. This work will inform the design of new generations of glycosidase inhibitors with restricted side chains that confer higher selectivity and/or affinity.

Dietary 5,6,7-Trihydroxy-flavonoid Aglycones and 1-Deoxynojirimycin Synergistically Inhibit the Recombinant Maltase-Glucoamylase Subunit of α-Glucosidase and Lower Postprandial Blood Glucose.

1-Deoxynojirimycin (1-DNJ) is the major effective component of mulberry leaves, exhibiting inhibitory activity against α-glucosidase. However, due to the low content of 1-DNJ in mulberry products, its level cannot meet the lowest dose to exhibit its activity. In this study, a combination of dietary 5,6,7-trihydroxy-flavonoid aglycones with 1-DNJ showed synergistic inhibitory activity against maltase of mice α-glucosidase and recombinant C- and N-termini of maltase-glucoamylase (MGAM) and baicalein with 1-DNJ exhibited the strongest synergistic effect. The synergistic effect of the combination was also confirmed by the maltose tolerance test in vivo. Enzyme kinetics, molecular docking, fluorescence spectrum, and circular dichroism spectrometry studies indicated that the major mechanism of the synergism is that baicalein was a positive allosteric inhibitor and bound to the noncompetitive site of MGAM, causing an increase of the binding affinity of 1-DNJ to MGAM. Our results might provide a theoretical basis for the design of dietary supplements containing mulberry products.

Strategy for Designing Selective Lysosomal Acid α-Glucosidase Inhibitors: Binding Orientation and Influence on Selectivity.

Deoxynojirimycin (DNJ) is the archetypal iminosugar, in which the configuration of the hydroxyl groups in the piperidine ring truly mimic those of d-glucopyranose; DNJ and derivatives have beneficial effects as therapeutic agents, such as anti-diabetic and antiviral agents, and pharmacological chaperones for genetic disorders, because they have been shown to inhibit α-glucosidases from various sources. However, attempts to design a better molecule based solely on structural similarity cannot produce selectivity between α-glucosidases that are localized in multiple organs and tissues, because the differences of each sugar-recognition site are very subtle. In this study, we provide the first example of a design strategy for selective lysosomal acid α-glucosidase (GAA) inhibitors focusing on the alkyl chain storage site. Our design of α-1-C-heptyl-1,4-dideoxy-1,4-imino-l-arabinitol (LAB) produced a potent inhibitor of the GAA, with an IC50 value of 0.44 µM. It displayed a remarkable selectivity toward GAA (selectivity index value of 168.2). A molecular dynamic simulation study revealed that the ligand-binding conformation stability gradually improved with increasing length of the α-1-C-alkyl chain. It is noteworthy that α-1-C-heptyl-LAB formed clearly different interactions from DNJ and had favored hydrophobic interactions with Trp481, Phe525, and Met519 at the alkyl chain storage pocket of GAA. Moreover, a molecular docking study revealed that endoplasmic reticulum (ER) α-glucosidase II does not have enough space to accommodate these alkyl chains. Therefore, the design strategy focusing on the shape and acceptability of long alkyl chain at each α-glucosidase may lead to the creation of more selective and practically useful inhibitors.

Intestinal Absorption and Tissue Distribution of Aza-Sugars from Mulberry Leaves and Evaluation of Their Transport by Sugar Transporters.

Mulberry leaves are rich in aza-sugars, particularly 1-deoxynojirimycin (DNJ), fagomine, and 2-O-α-d-galactopyranosyl-1-deoxynojirimycin (GAL-DNJ), which have antidiabetes and antiobesity properties. To help us understand the mechanisms of action of aza-sugars, pharmacokinetic studies are necessary. Therefore, in this study, we evaluated and compared the absorption and organ distribution of these aza-sugars in rats. Following oral intake, DNJ exhibited the highest plasma concentration followed by fagomine and GAL-DNJ. Meanwhile, similar amounts of DNJ and fagomine were present in organs, while GAL-DNJ was hardly detected, suggesting the diversity in absorption and distribution characteristics of these aza-sugars. We then investigated the role of the sodium-glucose cotransporter and the glucose transporter (GLUT) in the transport of aza-sugars and found that both are involved in DNJ transport, while transport of fagomine is solely facilitated by the GLUT. These findings provide insight into the bioavailability and bioactive mechanisms of these aza-sugars.

Synthesis and Therapeutic Applications of Iminosugars in Cystic Fibrosis.

Iminosugars are sugar analogues endowed with a high pharmacological potential. The wide range of biological activities exhibited by these glycomimetics associated with their excellent drug profile make them attractive therapeutic candidates for several medical interventions. The ability of iminosugars to act as inhibitors or enhancers of carbohydrate-processing enzymes suggests their potential use as therapeutics for the treatment of cystic fibrosis (CF). Herein we review the most relevant advances in the field, paying attention to both the chemical synthesis of the iminosugars and their biological evaluations, resulting from in vitro and in vivo assays. Starting from the example of the marketed drug NBDNJ (N-butyl deoxynojirimycin), a variety of iminosugars have exhibited the capacity to rescue the trafficking of F508del-CFTR (deletion of F508 residue in the CF transmembrane conductance regulator), either alone or in combination with other correctors. Interesting results have also been obtained when iminosugars were considered as anti-inflammatory agents in CF lung disease. The data herein reported demonstrate that iminosugars hold considerable potential to be applied for both therapeutic purposes.

The pharmacological chaperone N-n-butyl-deoxygalactonojirimycin enhances ß-galactosidase processing and activity in fibroblasts of a patient with infantile GM1-gangliosidosis.

GM1-gangliosidosis, a lysosomal storage disorder, is associated with ~ 161 missense variants in the GLB1 gene. Affected patients present with ß-galactosidase (ß-Gal) deficiency in lysosomes. Loss of function in ER-retained misfolded enzymes with missense variants is often due to subcellular mislocalization. Deoxygalactonojirimycin (DGJ) and its derivatives are pharmaceutical chaperones that directly bind to mutated ß-Gal in the ER promoting its folding and trafficking to lysosomes and thus enhancing its activity. An Emirati child has been diagnosed with infantile GM1-gangliosidosis carrying the reported p.D151Y variant. We show that p.D151Y ß-Gal in patient's fibroblasts retained < 1% residual activity due to impaired processing and trafficking. The amino acid substitution significantly affected the enzyme conformation; however, p.D151Y ß-Gal was amenable for partial rescue in the presence of glycerol or at reduced temperature where activity was enhanced with ~ 2.3 and 7 folds, respectively. The butyl (NB-DGJ) and nonyl (NN-DGJ) derivatives of DGJ chaperoning function were evaluated by measuring their IC50s and ability to stabilize the wild-type ß-Gal against thermal degradation. Although NN-DGJ showed higher affinity to ß-Gal, it did not show a significant enhancement in p.D151Y ß-Gal activity. However, NB-DGJ promoted p.D151Y ß-Gal maturation and enhanced its activity up to ~ 4.5% of control activity within 24 h which was significantly increased to ~ 10% within 6 days. NB-DGJ enhancement effect was sustained over 3 days after washing it out from culture media. We therefore conclude that NB-DGJ might be a promising therapeutic chemical chaperone in infantile GM1 amenable variants and therefore warrants further analysis for its clinical applications.

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.

Design, Synthesis, and Activity Evaluation of Novel N-benzyl Deoxynojirimycin Derivatives for Use as α-Glucosidase Inhibitors.

To obtain α-glucosidase inhibitors with high activity, 19 NB-DNJDs (N-benzyl-deoxynojirimycin derivatives) were designed and synthesized. The results indicated that the 19 NB-DNJDs displayed different inhibitory activities towards α-glucosidase in vitro. Compound 18a (1-(4-hydroxy-3-methoxybenzyl)-2-(hydroxymethyl) piperidine-3,4,5-triol) showed the highest activity, with an IC50 value of 0.207 ± 0.11 mM, followed by 18b (1-(3-bromo-4-hydroxy-5-methoxybenzyl)-2-(hydroxymethyl) piperidine-3,4,5-triol, IC50: 0.276 ± 0.13 mM). Both IC50 values of 18a and 18b were significantly lower than that of acarbose (IC50: 0.353 ± 0.09 mM). According to the structure-activity analysis, substitution of the benzyl and bromine groups on the benzene ring decreased the inhibition activity, while methoxy and hydroxyl group substitution increased the activity, especially with the hydroxyl group substitution. Molecular docking results showed that three hydrogen bonds were formed between compound 18a and amino acids in the active site of α-glucosidase. Additionally, an arene‒arene interaction was also modelled between the phenyl ring of compound 18a and Arg 315. The three hydrogen bonds and the arene‒arene interaction resulted in a low binding energy (-5.8 kcal/mol) and gave 18a a higher inhibition activity. Consequently, compound 18a is a promising candidate as a new α-glucosidase inhibitor for the treatment of type Ⅱ diabetes.