Structure of a GH51 α-L-arabinofuranosidase from Meripilus giganteus: conserved substrate recognition from bacteria to fungi.

α-L-Arabinofuranosidases from glycoside hydrolase family 51 use a stereochemically retaining hydrolytic mechanism to liberate nonreducing terminal α-L-arabinofuranose residues from plant polysaccharides such as arabinoxylan and arabinan. To date, more than ten fungal GH51 α-L-arabinofuranosidases have been functionally characterized, yet no structure of a fungal GH51 enzyme has been solved. In contrast, seven bacterial GH51 enzyme structures, with low sequence similarity to the fungal GH51 enzymes, have been determined. Here, the crystallization and structural characterization of MgGH51, an industrially relevant GH51 α-L-arabinofuranosidase cloned from Meripilus giganteus, are reported. Three crystal forms were grown in different crystallization conditions. The unliganded structure was solved using sulfur SAD data collected from a single crystal using the I23 in vacuo diffraction beamline at Diamond Light Source. Crystal soaks with arabinose, 1,4-dideoxy-1,4-imino-L-arabinitol and two cyclophellitol-derived arabinose mimics reveal a conserved catalytic site and conformational itinerary between fungal and bacterial GH51 α-L-arabinofuranosidases.

A common strategy towards the synthesis of 1,4-dideoxy-1,4-imino-l-xylitol, deacetyl (+)-anisomycin and amino-substituted piperidine iminosugars.

A strategy towards the synthesis of three different target molecules, namely 1,4-dideoxy-1,4-imino-l-xylitol, deacetyl (+)-anisomycin and amino-substituted piperidine iminosugars, molecules of potential biological and medicinal significance, is reported from a common amino-vicinal diol intermediate derived from tri-O-benzyl-d-glucal. Construction of the key pyrrolidine ring present in 1,4-dideoxy-1,4-imino-l-xylitol and (+)-anisomycin was a consequence of thermodynamically driven concomitant intramolecular nucleophilic addition reaction of the amino group to the resultant aldehyde obtained by oxidative cleavage of the amino-vicinal diol. Alternatively, double nucleophilic substitution on an amino-diol, after mesylation, with various amines delivered amino-substituted piperidine iminosugars in good yields.

Synthesis and glycosidase inhibition of N-substituted derivatives of 1,4-dideoxy-1,4-imino-d-mannitol (DIM).

N-Substituted derivatives of 1,4-dideoxy-1,4-imino-d-mannitol (DIM), the pyrrolidine core of swainsonine, have been synthesized efficiently and stereoselectively from d-mannose with 2,3:5,6-di-O-isopropylidene DIM (10) as a key intermediate. These N-substituted derivatives include N-alkylated, N-alkenylated, N-hydroxyalkylated and N-aralkylated DIMs with the carbon number of the alkyl chain ranging from one to nine. The obtained 33 N-substituted DIM derivatives were assayed against various glycosidases, which allowed a systematic evaluation of their glycosidase inhibition profiles. Though N-substitution of DIM decreased their α-mannosidase inhibitory activities, some of the derivatives showed significant inhibition of other glycosidases.

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.

Efficient Synthesis of UDP-Furanoses via 4,5-Dicyanoimidazole(DCI)-Promoted Coupling of Furanosyl-1-Phosphates with Uridine Phosphoropiperidate.

A P(V)-N activation method based on nucleoside phosphoropiperidate/DCI system has been developed for improved synthesis of diverse UDP-furanoses. The reaction conditions including temperature, amount of activator, and reaction time were optimized to alleviate the degradation of UDP-furanoses to cyclic phosphates. In addition, an efficient and facile phosphoramidite route was employed for the preparation of furanosyl-1-phosphates.

Voruciclib, a Potent CDK4/6 Inhibitor, Antagonizes ABCB1 and ABCG2-Mediated Multi-Drug Resistance in Cancer Cells.

BACKGROUND/AIMS: The overexpression of ATP-Binding Cassette (ABC) transporters has known to be one of the major obstacles impeding the success of chemotherapy in drug resistant cancers. In this study, we evaluated voruciclib, a CDK 4/6 inhibitor, for its chemo-sensitizing activity in ABCB1- and ABCG2- overexpressing cells. METHODS: Cytotoxicity and reversal effect of voruciclib was determined by MTT assay. The intracellular accumulation and efflux of ABCB1 and ABCG2 substrates were measured by scintillation counter. The effects on expression and intracellular localization of ABCB1 and ABCG2 proteins were determined by Western blotting and immunofluorescence, respectively. Vanadate-sensitive ATPase assay was done to determine the effect of voruciclib on the ATPase activity of ABCB1 and ABCG2. Flow cytometric analysis was done to determine the effect of voruciclib on apoptosis of ABCB1 and ABCG2-overexpressing cells and docking analysis was done to determine the interaction of voruciclib with ABCB1 and ACBG2 protein. RESULTS: Voruciclib significantly potentiated the effect of paclitaxel and doxorubicin in ABCB1-overexpressing cells, as well as mitoxantrone and SN-38 in ABCG2-overexpressing cells. Voruciclib moderately sensitized ABCC10- overexpressing cells to paclitaxel, whereas it did not alter the cytotoxicity of substrates of ABCC1. Furthermore, voruciclib increased the intracellular accumulation and decreased the efflux of substrate anti-cancer drugs from ABCB1- or ABCG2-overexpressing cells. However, voruciclib did not alter the expression or the sub-cellular localization of ABCB1 or ABCG2. Voruciclib stimulated the ATPase activity of both ABCB1 and ABCG2 in a concentration-dependent manner. Lastly, voruciclib exhibited a drug-induced apoptotic effect in ABCB1- or ABCG2- overexpressing cells. CONCLUSION: Voruciclib is currently a phase I clinical trial drug. Our findings strongly support its potential use in combination with conventional anti-cancer drugs for cancer chemotherapy.

Inhibitory properties of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) derivatives acting on glycogen metabolising enzymes.

Glycogen synthase (GS) and glycogen phosphorylase (GP) are the key enzymes that control, respectively, the synthesis and degradation of glycogen, a multi-branched glucose polymer that serves as a form of energy storage in bacteria, fungi and animals. An abnormal glycogen metabolism is associated with several human diseases. Thus, GS and GP constitute adequate pharmacological targets to modulate cellular glycogen levels by means of their selective inhibition. The compound 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) is a known potent inhibitor of GP. We studied the inhibitory effect of DAB, its enantiomer LAB, and 29 DAB derivatives on the activity of rat muscle glycogen phosphorylase (RMGP) and E. coli glycogen synthase (EcGS). The isoform 4 of sucrose synthase (SuSy4) from Solanum tuberosum L. was also included in the study for comparative purposes. Although these three enzymes possess highly conserved catalytic site architectures, the DAB derivatives analysed showed extremely diverse inhibitory potential. Subtle changes in the positions of crucial residues in their active sites are sufficient to discriminate among the structural differences of the tested inhibitors. For the two Leloir-type enzymes, EcGS and SuSy4, which use sugar nucleotides as donors, the inhibitory potency of the compounds analysed was synergistically enhanced by more than three orders of magnitude in the presence of ADP and UDP, respectively. Our results are consistent with a model in which these compounds bind to the subsite in the active centre of the enzymes that is normally occupied by the glucosyl residue which is transferred between donor and acceptor substrates. The ability to selectively inhibit the catalytic activity of the key enzymes of the glycogen metabolism may represent a new approach for the treatment of disorders of the glycogen metabolism.

Nectrisine Biosynthesis Genes in Thelonectria discophora SANK 18292: Identification and Functional Analysis.

The fungus Thelonectria discophora SANK 18292 produces the iminosugar nectrisine, which has a nitrogen-containing heterocyclic 5-membered ring and acts as a glycosidase inhibitor. In our previous study, an oxidase (designated NecC) that converts 4-amino-4-deoxyarabinitol to nectrisine was purified from T. discophora cultures. However, the genes required for nectrisine biosynthesis remained unclear. In this study, the nectrisine biosynthetic gene cluster in T. discophora was identified from the contiguous genome sequence around the necC gene. Gene disruption and complementation studies and heterologous expression of the gene showed that necA, necB, and necC could be involved in nectrisine biosynthesis, during which amination, dephosphorylation, and oxidation occur. It was also demonstrated that nectrisine could be produced by recombinant Escherichia coli coexpressing the necA, necB, and necC genes. These findings provide the foundation to develop a bacterial production system for nectrisine or its intermediates through genetic engineering. IMPORTANCE: Iminosugars might have great therapeutic potential for treatment of many diseases. However, information on the genes for their biosynthesis is limited. In this study, we report the identification of genes required for biosynthesis of the iminosugar nectrisine in Thelonectria discophora SANK 18292, which was verified by disruption, complementation, and heterologous expression of the genes involved. We also demonstrate heterologous production of nectrisine by recombinant E. coli, toward developing an efficient production system for nectrisine or its intermediates through genetic engineering.

Transition State Structure and Inhibition of Rv0091, a 5'-Deoxyadenosine/5'-methylthioadenosine Nucleosidase from Mycobacterium tuberculosis.

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a bacterial enzyme that catalyzes the hydrolysis of the N-ribosidic bond in 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH). MTAN activity has been linked to quorum sensing pathways, polyamine biosynthesis, and adenine salvage. Previously, the coding sequence of Rv0091 was annotated as a putative MTAN in Mycobacterium tuberculosis. Rv0091 was expressed in Escherichia coli, purified to homogeneity, and shown to be a homodimer, consistent with MTANs from other microorganisms. Substrate specificity for Rv0091 gave a preference for 5'-deoxyadenosine relative to MTA or SAH. Intrinsic kinetic isotope effects (KIEs) for the hydrolysis of [1'-(3)H], [1'-(14)C], [5'-(3)H2], [9-(15)N], and [7-(15)N]MTA were determined to be 1.207, 1.038, 0.998, 1.021, and 0.998, respectively. A model for the transition state structure of Rv0091 was determined by matching KIE values predicted via quantum chemical calculations to the intrinsic KIEs. The transition state shows a substantial loss of C1'-N9 bond order, well-developed oxocarbenium character of the ribosyl ring, and weak participation of the water nucleophile. Electrostatic potential surface maps for the Rv0091 transition state structure show similarity to DADMe-immucillin transition state analogues. DADMe-immucillin transition state analogues showed strong inhibition of Rv0091, with the most potent inhibitor (5'-hexylthio-DADMe-immucillinA) displaying a Ki value of 87 pM.

Gem-difluoromethylated and trifluoromethylated derivatives of DMDP-related iminosugars: synthesis and glycosidase inhibition.

Gem-difluoromethylated and trifluoromethylated derivatives of DMDP-related iminosugars have been synthesized from cyclic nitrones 12, 13, 18, ent-18 or 23 and nitrone-derived aldehydes 20 or ent-20. The fluorinated iminosugars were assayed against various glycosidases, and ent-8 showed moderate but selective α-l-rhamnosidase inhibition. Difluoro or trifluoro units influenced the inhibitory activities of iminosugars in a more complex manner than single fluoro substitution. This may be correlated with their highly hydrophobic character and strong electron-withdrawing effect.

Design, synthesis and bioactivity evaluation of Galf mimics as antitubercular agents.

A series of novel Galf mimics has been synthesized and characterized by IR, (1)H NMR, (13)C NMR, mass spectral and element analysis. All the newly prepared compounds were screened for their antitubercular activities. Bioactivity assays manifested that most of Galf mimics exhibited good antitubercular activities. Especially compound 4d and 4e displayed remarkable antitubercular efficacies, which were comparable to ethambutol.

Evaluation of fluoropyruvate as nucleophile in reactions catalysed by N-acetyl neuraminic acid lyase variants: scope, limitations and stereoselectivity.

The catalysis of reactions involving fluoropyruvate as donor by N-acetyl neuraminic acid lyase (NAL) variants was investigated. Under kinetic control, the wild-type enzyme catalysed the reaction between fluoropyruvate and N-acetyl mannosamine to give a 90 : 10 ratio of the (3R,4R)- and (3S,4R)-configured products; after extended reaction times, equilibration occurred to give a 30 : 70 mixture of these products. The efficiency and stereoselectivity of reactions of a range of substrates catalysed by the E192N, E192N/T167V/S208V and E192N/T167G NAL variants were also studied. Using fluoropyruvate and (2R,3S)- or (2S,3R)-2,3-dihydroxy-4-oxo-N,N-dipropylbutanamide as substrates, it was possible to obtain three of the four possible diastereomeric products; for each product, the ratio of anomeric and pyranose/furanose forms was determined. The crystal structure of S. aureus NAL in complex with fluoropyruvate was determined, assisting rationalisation of the stereochemical outcome of C-C bond formation.

Design and Synthesis of Labystegines, Hybrid Iminosugars from LAB and Calystegine, as Inhibitors of Intestinal α-Glucosidases: Binding Conformation and Interaction for ntSI.

This paper identifies the required configuration and orientation of α-glucosidase inhibitors, miglitol, α-1-C-butyl-DNJ, and α-1-C-butyl-LAB for binding to ntSI (isomaltase). Molecular dynamics (MD) calculations suggested that the flexibility around the keyhole of ntSI is lower than that of ctSI (sucrase). Furthermore, a molecular-docking study revealed that a specific binding orientation with a CH-π interaction (Trp370 and Phe648) is a requirement for achieving a strong affinity with ntSI. On the basis of these results, a new class of nortropane-type iminosugars, labystegines, hybrid iminosugars of LAB and calystegine, have been designed and synthesized efficiently from sugar-derived cyclic nitrones with intramolecular 1,3-dipolar cycloaddition or samarium iodide catalyzed reductive coupling reaction as the key step. Biological evaluation showed that our newly designed 3(S)-hydroxy labystegine (6a) inherited the selectivity against intestinal α-glucosidases from LAB, and its inhibition potency was 10 times better than that of miglitol. Labystegine, therefore, represents a promising new class of nortropane-type iminosugar for improving postprandial hyperglycemia.

Biosynthesis of nectrisine in Thelonectria discophora SANK 18292.

Nectrisine, an iminosugar with a heterocyclic nitrogen-containing 5-membered ring, acts as a glycosidase inhibitor. Thelonectria discophora SANK 18292, a fungus, was identified as a nectrisine producer from its microbial library in our screening for nectrisine producing microorganisms. Biosynthesis of nectrisine produced by the fungus was studied using stable isotope tracer techniques. Incorporation of (13)C-labeled d-ribose and d-xylose into nectrisine was confirmed by mass spectrometry and (13)C NMR spectroscopy, which suggested that these were its precursors. Chromatographic separation of the hot water extract from the culture broth afforded not only nectrisine, but also substantial amounts of 4-amino-4-deoxyarabinitol. Incubation of the latter with the crude enzyme of the fungus at room temp. caused an increase in levels of nectrisine together with a decrease in amounts of the administered potential precursor suggesting that it is a biosynthetic intermediate. From these results, a biosynthetic pathway to nectrisine is proposed via d-xylulose 5-phosphate and 4-amino-4-deoxyarabinitol by the pentose phosphate pathway.

Casuarine stereoisomers from achiral substrates: chemoenzymatic synthesis and inhibitory properties.

A straightforward chemoenzymatic synthesis of four uncovered casuarine stereoisomers is described. The strategy consists of L-fuculose-1-phosphate aldolase F131A-variant-catalyzed aldol addition of dihydroxyacetone phosphate to aldehyde derivatives of 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and its enantiomer (LAB) and subsequent one-pot catalytic deprotection-reductive amination. DAB and LAB were obtained from dihydroxyacetone and aminoethanol using D-fructose-6-phosphate aldolase and L-rhamnulose-1-phosphate aldolase catalysts, respectively. The new ent-3-epi-casuarine is a strong inhibitor of α-d-glucosidase from rice and of rat intestinal sucrase.

Conformational studies of gas-phase ribose and 2-deoxyribose by density functional, second order PT and multi-level method calculations: the pyranoses, furanoses, and open-chain structures.

We present an extensive computational study of a complex conformational isomerism of two gas phase pentoses of biological and potential astrobiological importance, d-ribose and 2-deoxy-d-ribose. Both cyclic (α- and ß-pyranoses, α- and ß-furanoses) and open-chain isomers have been probed using second order Møller-Plesset perturbation theory (MP2), M06-2X density functional, and multi-level G4 methods. This study revealed a multitude of existing minima structures. Numerous furanose conformers found are described with the Altona and Sundaralingam pseudorotation parameters. In agreement with the recent gas-phase microwave (MW) investigation of Cocinero et al., the calculated free ribose isomers of lowest energy are the two ß-pyranoses with the (1)C4 and (4)C1 ring chair conformations. Both ß-pyranoses lie within 0.9kJ/mol in terms of ΔG(298K) (G4), thus challenge the computational methods used to predict the ribose global minimum. The calculated most favoured ribofuranose is the α-anomer having the twist (2)T1 ring conformation, put 10.4kJ/mol higher in ΔG than the global minimum. By contrast with d-ribose, the lowest energy 2-deoxy-d-ribose is the α-pyranose, with the most stable 2-deoxy-d-furanose (the α-anomer) being only 6.2kJ/mol higher in free energy. For both pentoses, the most favoured open-chain isomers are significantly higher in energy than the low-lying cyclic forms. A good overall agreement is observed between the M06-2X and MP2 results in terms of both the existing low-energy minima structures and intramolecular H-bonding geometrical parameters. The natural orbital analysis confirms the occuring of the endo- and exo-anomeric effects and maximization of intramolecular H-bonding in the lowest-lying pyranoses and furanoses of both sugars.

Chemo-enzymatic synthesis and glycosidase inhibitory properties of DAB and LAB derivatives.

A chemo-enzymatic strategy for the preparation of 2-aminomethyl derivatives of (2R,3R,4R)-2-(hydroxymethyl)pyrrolidine-3,4-diol (also called 1,4-dideoxy-1,4-imino-D-arabinitol, DAB) and its enantiomer LAB is presented. The synthesis is based on the enzymatic preparation of DAB and LAB followed by the chemical modification of their hydroxymethyl functionality to afford diverse 2-aminomethyl derivatives. This strategy leads to novel aromatic, aminoalcohol and 2-oxopiperazine DAB and LAB derivatives. The compounds were preliminarily explored as inhibitors of a panel of commercial glycosidases, rat intestinal disaccharidases and against Mycobacterium tuberculosis, the causative agent of tuberculosis. It was found that the inhibitory profile of the new products differed considerably from the parent DAB and LAB. Furthermore, some of them were active inhibiting the growth of M. tuberculosis.

Synthesis from D-altrose of (5R,6R,7R,8S)-5,7-dihydroxy-8-hydroxymethylconidine and 2,4-dideoxy-2,4-imino-D-glucitol, azetidine analogues of swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol.

Ring closure of a 3,5-di-O-triflate derived from D-altrose with benzylamine allowed the formation of both monocyclic and bicyclic azetidine analogues of swainsonine.

Syntheses and biological activities of 1,4-iminoalditol derivatives as alpha-L-fucosidase inhibitors.

A review dealing with 1,4-iminoalditol (hydroxylated pyrrolidine) derivatives as inhibitors of alpha-L-fucosidases including the different synthetic approaches for their preparation as well as their inhibitory properties is presented.

A versatile approach to N-alkylated 1,4-dideoxy-1,4-imino-D-arabinitols and 1,4-dideoxy-1,4-imino-L-xylitols.

A versatile and concise synthesis of N-alkylated 1,4-dideoxy-1,4-imino-D-arabinitol and 1,4-dideoxy-1,4-imino-L-xylitol derivatives is described. These were prepared using pseudohemiketal lactams as key intermediates, which in turn were obtained from sucrose. The key intermediates were prepared by a diastereospecific tandem reaction which facilitated the introduction of various substituents on the nitrogen atom of the iminosugars.