In recent years, the function of pharmacological chaperones as a "thermodynamic stabilizer" has been attracting attention in combination therapy. The coadministration of a pharmacological chaperone and recombinant human acid α-glucosidase (rhGAA) leads to improved stability and maturation by binding to the folded state of the rhGAA and thereby promotes enzyme delivery. This study provides the first example of a strategy to design a high-affinity ligand toward lysosomal acid α-glucosidase (GAA) focusing on alkyl branches on 1-deoxynojirimycin (DNJ); 5-C-heptyl-DNJ produced a nanomolar affinity for GAA with a Ki value of 0.0047 μM, which is 13-fold more potent than DNJ. The protein thermal shift assay revealed that 10 μM 5-C-heptyl-DNJ increased the midpoint of the protein denaturation temperature (Tm) to 73.6 °C from 58.6 °C in the absence of the ligand, significantly improving the thermal stability of rhGAA. Furthermore, 5-C-heptyl-DNJ dose dependency increased intracellular GAA activities in Pompe patient's fibroblasts with the M519V mutation. The introduction of C5 alkyl branches on DNJ provides a new molecular strategy for pharmacological chaperone therapy for Pompe disease, which may lead to the development of higher-affinity and practically useful chaperones.

Pancreatic cancer is one of the most aggressive cancers with a poor prognosis. Previous studies suggested that nutrient-deprived conditions may play a critical role in pancreatic cancer cell survival and resistance to chemotherapy. We describe a novel series of azobenzene derivatives including (E)-1-(4-methyl-3-((2methyl-5-(naphthalen-1-yl)phenyl)diazenyl)phenyl)naphthalen-2-ol (9) with efficacy and selectivity in nutrient-deprived conditions. Although anticancer drug 5-fluorouracil (5-FU) was ineffective under nutrient-deprived conditions, five of our designed compounds, 9 and four other related compounds 11-14, showed anticancer activity with IC50 values ranging from 1.5 to 9.6 mu M. Interestingly, only 9 showed no cytotoxicity in normal conditions. This selectivity profile of 9 is clearly opposite to that of 5-FU. Furthermore, cell cycle analysis showed that, in contrast to S phase arrest induced by 5-FU, 9 caused G(0)/ G(1) phase arrest, which might block cancer cell growth by arresting them in quiescence. Therefore, it could be a novel and promising candidate for effective pancreatic cancer treatment under nutrientdeprived conditions. (C) 2021 Elsevier Ltd. All rights reserved.

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.

Background: Previous work had shown the ability of an aqueous leaf and stem extract of Cissus quandrangularis (300mg of CQR, CQR-300) to improve components of metabolic syndrome (MS) in overweight individuals. Objective: This small pilot study aimed to confirm the efficacy of CQR-300 in reducing the percentage body fat measured using two different methodsbioelectrical impedance assay versus dual-energy X-ray absorptiometry (DEXA). Design: The study was an 8-week double-blind, placebo-controlled pilot trial on 67 individuals who were requested by a dietary counselor to maintain their normal exercise and dietary routines. Participants were randomly divided into two groups, placebo (32 participants) and the CQR-300 group (35 participants), and received 300mg of corn starch or CQR-300 daily. Methods: Body fat was measured by bioelectrical impedance using a TANITA impedance meter and by DEXA, with blood samples taken at baseline and at 8 weeks for the measurement of lipid parameters. Results: After 8 weeks of treatment, participants of the placebo group showed a 1.05% decrease in body fat as determined by bioelectrical impedance analysis, but no difference using DEXA. In the same time period, the CQR-300 group had an 8.9% and 12.8% decreases in the body fat as measured by impedance and DEXA, respectively. These values were significantly (p<0.05) lower than the placebo. Compared with the placebo, the CQR-300 group demonstrated significant (p<0.05) decreases in the waist and hip circumferences, systolic and diastolic blood pressures, total cholesterol, triglycerides, fasting blood glucose, as well as leptin levels. On the contrary, there were significant (p<0.05) increases in HDL-cholesterol and adiponectin levels. Conclusion:CQR-300 administered as a single 300mg dose daily was effective in reducing body fat as well as improving blood parameters associated with MS.

ETHNOPHARMACOLOGY RELEVANCE:Maytenus ilicifolia is a Celastracea plant used in traditional medicine to alleviate digestive tract inflammatory disorders. AIM OF THE STUDY:We investigated anti-inflammatory properties of M. ilicifolia crude extract towards Caco-2 cell line, as a model of Toll-like Receptor 2 (TLR-2) inflammatory pathway. MATERIALS AND METHODS:Toxicity was assessed following culture of Caco-2 with M. ilicifolia, using apparent cell permeability and trans-epithelial electric resistance. Anti-inflammatory properties of M. ilicifolia were assessed through IL-8 secretion and TLR-2 associated gene expression of Caco-2 cells with or without an LTA challenge. RESULTS:M. ilicifolia was not toxic to Caco-2 cells. M. ilicifolia down-regulated TLR2 expression with and without LTA challenge but had no effect on other genes. Following LTA challenge of Caco-2 cells, 100 and 200µg/mL M. ilicifolia abrogated IL-8 secretion. CONCLUSIONS:We provide preliminary data for some M. ilicifolia anti-inflammatory properties. Further research must establish the full extent and mode of action on particular inflammatory pathways.

The antiprotozoal effect of saponins is transitory, as when saponins are deglycosylated to sapogenins by rumen microorganisms they become inactive. We hypothesised that the combination of saponins with glycosidase-inhibiting iminosugars might potentially increase the effectiveness of saponins over time by preventing their deglycosylation in the rumen. Alternatively, modifying the structure of the saponins by substituting the sugar moiety with other small polar residues might maintain their activity as the sugar substitute would not be enzymatically cleaved. The aim of this in vitro study was to evaluate the acute antiprotozoal effect and the stability of this effect over a 24 h incubation period using ivy saponins, a stevia extract rich in iminosugars, ivy saponins with stevia extract, and a chemically modified ivy saponin, hederagenin bis-succinate (HBS). The effects on fermentation parameters and rumen bacterial communities were also studied. Ivy saponins with stevia and HBS had a greater antiprotozoal effect than ivy saponins, and this effect was maintained after 24 h of incubation (P<0.001). The combination of ivy and stevia extracts was more effective in shifting the fermentation pattern towards higher propionate (+39%) and lower butyrate (-32%) and lower ammonia concentration (-64%) than the extracts incubated separately. HBS caused a decrease in butyrate (-45%) and an increase in propionate (+43%) molar proportions. However, the decrease in ammonia concentration (-42%) observed in the presence of HBS was less than that caused by ivy saponins, either alone or with stevia. Whereas HBS and stevia impacted on bacterial population in terms of community structure, only HBS had an effect in terms of biodiversity (P<0.05). It was concluded that ivy saponins with stevia and the modified saponin HBS had a strong antiprotozoal effect, although they differed in their effects on fermentation parameters and bacteria communities. Ivy saponins combined with an iminosugar-rich stevia extract and/or HBS should be evaluated to determine their antiprotozoal effect in vivo.

Background Two platyhelminths of biomedical and commercial significance are Schistosoma mansoni (blood fluke) and Fasciola hepatica (liver fluke). These related trematodes are responsible for the chronic neglected tropical diseases schistosomiasis and fascioliasis, respectively. As no vaccine is currently available for anti-flukicidal immunoprophylaxis, current treatment is mediated by mono-chemical chemotherapy in the form of mass drug administration (MDA) (praziquantel for schistosomiasis) or drenching (triclabendazole for fascioliasis) programmes. This overreliance on single chemotherapeutic classes has dramatically limited the number of novel chemical entities entering anthelmintic drug discovery pipelines, raising significant concerns for the future of sustainable blood and liver fluke control. Methodology/ Principle Findings Here we demonstrate that 7-keto-sempervirol, a diterpenoid isolated from Lycium chinense, has dual anthelmintic activity against related S. mansoni and F. hepatica trematodes. Using a microtiter plate-based helminth fluorescent bioassay (HFB), this activity is specific (Therapeutic index = 4.2, when compared to HepG2 cell lines) and moderately potent (LD50 = 19.1 μM) against S. mansoni schistosomula cultured in vitro. This anti-schistosomula effect translates into activity against both adult male and female schistosomes cultured in vitro where 7-keto-sempervirol negatively affects motility/behaviour, surface architecture (inducing tegumental holes, tubercle swelling and spine loss/shortening), oviposition rates and egg morphology. As assessed by the HFB and microscopic phenotypic scoring matrices, 7-keto-sempervirol also effectively kills in vitro cultured F. hepatica newly excysted juveniles (NEJs, LD50 = 17.7 μM). Scanning electron microscopy (SEM) evaluation of adult F. hepatica liver flukes co-cultured in vitro with 7-keto-sempervirol additionally demonstrates phenotypic abnormalities including breaches in tegumental integrity and spine loss. Conclusions/ Significance 7-keto-sempervirol negatively affects the viability and phenotype of two related pathogenic trematodes responsible for significant human and animal infectious diseases. This plant-derived, natural product is also active against both larval and adult developmental forms. As such, the data collectively indicate that 7-keto-sempervirol is an important starting point for anthelmintic drug development. Medicinal chemistry optimisation of more potent 7-keto-sempervirol analogues could lead to the identification of novel chemical entities useful for future combinatorial or replacement anthelmintic control.

A concise synthesis of (-)-steviamine is reported along with the synthesis of its analogues 10-nor-steviamine, 10-nor-ent-steviamine and 5-epi-ent-steviamine. These compounds were tested against twelve glycosidases (at 143 μg mL(-1) concentrations) and were found to have in general poor inhibitory activity against most enzymes. The 10-nor analogues however, showed 50-54% inhibition of α-L-rhamnosidase from Penicillium decumbens while one of these, 10-nor-steviamine, showed 51% inhibition of N-acetyl-β-D-glucosaminidase (from Jack bean) at the same concentration (760 μM).

Homochiral piperidines containing a quaternary carbon branch at C-2 of the piperidine ring may be made from readily available carbohydrate lactones containing a branched 2-C-hydroxymethyl substituent. This gives an easy access to the branched iminosugars. In particular a trihydroxypipecolic acid analogue, (3R,4R,5R)-3,4,5-trihydroxypiperidine-3-carboxylic acid, has been synthesised efficiently from the starting material d-ribose and (3S,4R,5S)-3-(hydroxymethyl)piperidine-3,4,5-triol was found to be a specific inhibitor of α-d-glucosidase from Bacillus Stearothermophilus with an IC50 value of 93μM.

Three homochiral polyhydroxylated piperidines containing a quaternary carbon branch at C-2 of the heterocyclic ring, which can be considered as branched analogues of deoxymannorijimycin (DMJ), and their corresponding lactams were synthesised from readily available and versatile carbohydrate lactones containing a 2-C-hydroxymethyl branch. The key step in the synthesis of these iminosugars, 1,5-dideoxy-2-C-hydroxymethyl-1,5-imino-D-mannitol, 1,5-dideoxy-2-C-hydroxymethyl-1,5-imino-L-gulitol and 1,5-dideoxy-2-C-hydroxymethyl-1,5-imino-D-talitol, was an efficient modified Kiliani reaction performed on the readily available ketose starting materials L-sorbose and D-fructose. The introduction of the carbon branch at C-2 resulted in loss of glycosidase inhibitory activity, which could be advantageous for therapeutic use of such compounds as chaperones.

The first stereocontrolled nitro sugar-mediated synthesis of polyhydroxylated beta-amino acids and their incorporation into peptides is described. The key steps of this approach are a Michael addition of the lithium salt of tris(phenylthio)methane (a carboxyl synthetic equivalent) to sugar nitro olefins, and the reduction of the nitro group to an amino group. Specifically, two glyceraldehyde-based beta-amino acids and a polyhydroxylated cyclohexyl beta-amino acid were prepared. The latter, which could act as a water-soluble mimic of the proline-glycine d-turn, was easily transformed into the corresponding 5-amino-6-(hydroxymethyl)cyclohexane-1,2,3,4-tetraol, a novel amino-carbasugar. However, this did not show significant glycosidase inhibiting properties.

The absolute stereochemistry of the title compound, C(9)H(15)NO(7), was determined from the use of d-glucuronolactone as the starting material. The compound crystallizes as the zwitterion. The five-membered ring adopts an envelope conformation with the -CH(2)OH-substituted C atom forming the flap. An intramolecular N-H⋯O hydrogen-bond occurs. In the crystal, the compound exists as a three-dimensional O-H⋯O intermolecular hydrogen-bonded network with each mol-ecule acting as a donor and acceptor for four hydrogen bonds.

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.

The syntheses of 4-C-Me-DAB [1,4-dideoxy-1,4-imino-4-C-methyl-d-arabinitol] from l-erythronolactone and of 4-C-Me-LAB [from d-erythronolactone] require only a single acetonide protecting group. The effect of pH on the NMR spectra of 4-C-Me-DAB [pKa of the salt around 8.4] is discussed and illustrates the need for care in the analysis of both coupling constants and chemical shift. 4-C-Me-DAB (for rat intestinal sucrase Ki 0.89 μM, IC50 0.41 μM) is a competitive—whereas 4-C-Me-LAB (for rat intestinal sucrase Ki 0.95 μM, IC50 0.66 μM) is a non-competitive—specific and potent α-glucosidase inhibitor. A rationale for the α-glucosidase inhibition by DAB, LAB, 4-C-Me-DAB, 4-C-Me-LAB and isoDAB—but not isoLAB—is provided. Both are inhibitors of endoplasmic reticulum (ER) resident α-glucosidase I and II.

We report the structure-activity relationship of a series of D-, and L-isofagomine and fagomine isomers as glycosidase inhibitors. Our study revealed that a positive charge at the anomeric position of d-isofagomines enhanced the potency toward β-glycosidases, while the epimerization at the C3 OH group drastically reduced their inhibitory potency by over three orders of magnitude. Furthermore, d-3,4-di-epi-isofagomine abolished their inhibition activities against all enzymes. L-Isofagomine was also a fairly potent inhibitor of human β-glucocerebrosidase, with an IC₅₀ value of 8.7 μM. A molecular docking study revealed that the positions and orientations of the piperidine ring of D-3-epi-isofagomine in the binding site was similar to that of D-isofagomine, while D-3-epi-isofagomine missed the hydrogen bond interactions between Asp127 and the 3-OH group and between Trp179 and the 3-OH group. Furthermore, the top 10 docking models ranked by IFDscore suggested that D-3,4-di-epi-isofagomine can not bind to β-glucocerebrosidase at a stable interaction mode. These results provide an insight into the structural requirements of isofagomine isomers for developing a new type of pharmacological chaperone for Gaucher disease.

For the purpose of this article, iminosugars are polyhydroxylated secondary and tertiary amines in which the molecules resemble monosaccharide sugars in which the ring oxygen is replaced by the nitrogen. The bicyclic structures may biologically resemble disaccharides. Very few iminosugars have been available up to now for evaluation of their pharmaceutical applications. The early compounds were discovered and selected for study due to glycosidase inhibition, which is now known to not be necessary for pharmacological activity and may cause off-target effects. Glyset® and Zavesca®, derived from the glucosidase-inhibiting natural product 1-deoxynojirimycin, are the first two examples of iminosugar drugs. Since the discovery of this first generation, many new natural products have been identified with a wide range of biological activities but few are widely available. Among the biological properties of these compounds are good oral bioavailability and very specific immune modulatory and chaperoning activity. Although the natural products from plants and microorganisms can have good specificity, modifications of the template natural products have been very successful recently in producing bioactive compounds with good profiles. The field of iminosugars continues to open up exciting new opportunities for therapeutic agent discovery and offers many new tools for precisely modifying carbohydrate structures and modulating glycosidase activity in vivo. Current efforts are directed towards a greater range of structures and a wider range of biochemical targets.

A convenient large-scale synthesis of 1-deoxynojirimyin (DNJ) from d-glucuronolactone involves introduction of azide at C-5 with retention of configuration to give 5-azido-5-deoxy-1,2-O-isopropylidene-α-d-glucofuranose as a key intermediate in an overall yield of up to 72%; the same intermediate can be transformed into DMDP [(2R,3R,4R,5R)-2,5-bis(hydroxymethyl)pyrrolidine-3,4-diol] and (3R)-3-hydroxy-l-bulgecinine [(2S,3R,4R,5R)-3,4-dihydroxy-5-hydroxymethyl-l-proline]. l-Glucuronolactone, a readily available l-sugar chiron, may similarly be used to access the enantiomers l-DNJ, l-DMDP, and (3S)-3-hydroxy-d-bulgecinine. A comparison of glycosidase inhibition by DMDP (an inhibitor of β-glucosidases and β-galactosidases) and l-DMDP (a potent and specific α-glucosidase inhibitor) with the corresponding enantiomeric hydroxybulgecinines is reported; DMDP and (3R)-3-hydroxy-l-bulgecinine show weak inhibition of glycogen phosphorylase.

(+)-Steviamine, the enantiomer of the natural (-)-steviamine, and its corresponding C5 epimer have been synthesized from the D-ribose-derived cyclic nitrone. (-)-Steviamine was found to be the first naturally occurring iminosugar that causes any inhibition of alpha-galactosaminidases.