Structural essentials for ß-N-acetylhexosaminidase inhibition by amides of prolines, pipecolic and azetidine carboxylic acids.

This paper explores the computer modelling aided design and synthesis of ß-N-acetylhexosaminidase inhibitors along with their applicability to human disease treatment through biological evaluation in both an enzymatic and cellular setting. We investigated the importance of individual stereocenters, variations in structure-activity relationships along with factors influencing cell penetration. To achieve these goals we modified nitrogen heterocycles in terms of ring size, side chains present and ring nitrogen derivatization. By reducing the inhibitor interactions with the active site down to the essentials we were able to determine that besides the established 2S,3R trans-relationship, the presence and stereochemistry of the CH2OH side chain is of crucial importance for activity. In terms of cellular penetration, N-butyl side chains favour cellar uptake, while hydroxy- and carboxy-group bearing sidechains on the ring nitrogen retarded cellular penetration. Furthermore we show an early proof of principle study that ß-N-acetylhexosaminidase inhibitors can be applicable to use in a potential anti-invasive anti-cancer strategy.

N- and C-alkylation of seven-membered iminosugars generates potent glucocerebrosidase inhibitors and F508del-CFTR correctors.

The glycosidase inhibitory properties of synthetic C-alkyl and N-alkyl six-membered iminosugars have been extensively studied leading to therapeutic candidates. The related seven-membered iminocyclitols have been less examined despite the report of promising structures. Using an in house ring enlargement/C-alkylation as well as cross-metathesis methodologies as the key steps, we have undertaken the synthesis and biological evaluation of a library of fourteen 2C- and eight N-alkyl tetrahydroxylated azepanes starting from an easily available glucopyranose-derived azidolactol. Four, six, nine and twelve carbon atom alkyl chains have been introduced. The study of two distinct D-gluco and L-ido stereochemistries for the tetrol pattern as well as R and S configurations for the C-2 carbon bearing the C-alkyl chain is reported. We observed that C-alkylation of the L-ido tetrahydroxylated azepane converts it from an α-L-fucosidase to a ß-glucosidase and ß-galactosidase inhibitor while N-alkylation of the D-gluco iminosugar significantly improves its inhibition profile leading to potent ß-glucosidase, ß-galactosidase, α-L-rhamnosidase and ß-glucuronidase inhibitors whatever the stereochemistry of the alkyl chain. Interestingly, the N-alkyl chain length usually parallels the azepane inhibitor potency as exemplified by the identification of a potent glucocerebrosidase inhibitor (Ki 1 µM) bearing a twelve carbon atom chain. Additionally, several C-alkyl azepanes demonstrated promising F508del-CFTR correction unlike the parent tetrahydroxyazepanes. None of the C-alkyl and N-alkyl azepanes did inhibit ER α-glucosidases I or II.

3,4-O-Isopropyl-idene-2-C-methyl-d-galactonolactone.

X-ray crystallography unequivocally confirmed the stereochemistry of the 2-C-methyl group in the title mol-ecule, C(10)H(16)O(6), in which the 1,5-lactone ring exists in a boat conformation. The use of d-galactose in the synthesis determined the absolute stereochemistry. The crystal exists as O-H⋯O hydrogen-bonded layers in the ab plane, with each mol-ecule acting as a donor and acceptor for two hydrogen bonds.

1-(1-Carboxy-methyl-1,4-anhydro-2,3-O-isopropyl-idene-α-d-erythrofuranos-yl)thymine.

X-Ray crystallography unequivocally determined the stereochemistry of the thymine base in the title compound, C(14)H(18)N(2)O(7). The absolute stereochemistry was determined from the use of d-ribose as the starting material. There are two independent mol-ecules in the asymmetric unit (Z' = 2) which exist as N-H⋯O hydrogen-bonded pairs in the crystal structure.

(1R,2R,3S,6aS,7R,8R,9S,12aS)-1,2,3,7,8,9-Hexahydroxy-perhydro-dipyrido[1,2-a:1',2'-d]pyrazine-6,12-dione.

The crystal structure of the title compound, C(12)H(18)N(2)O(8), exists as O-H⋯O hydrogen-bonded layers of mol-ecules running parallel to the ab plane. Each mol-ecule is a donor and acceptor for six hydrogen bonds. The absolute stereochemistry was determined by the use of d-glucuronolactone as the starting material.

6-Azido-3-O-benzyl-6-de-oxy-N,N-diethyl-1,2-O-isopropyl-idene-d-glycero-α-d-gluco-heptofuran-uronamide.

Reaction of 3-O-benzyl-1,2-O-isopropyl-idene-α-xylo-pentodialdo-1,4-furan-ose with N,N-diethyl-2-(dimethyl-sulfuranil-idene)acetamide gave stereoselectively an ep-oxy-amide, which was regioselectively opened by NaN(3) in dimethyl formamide to give the title compound, C(21)H(30)N(4)O(6). X-ray crystallography confirmed the relative stereochemistry of the title compound and the absolute configuration was determined by the use of d-glucose as the starting material. There are two mol-ecules in the asymmetric unit (Z' = 2). The crystal structure consists of two types of chains of O-H⋯O hydrogen-bonded mol-ecules running parallel to the b axis, with each mol-ecule acting as a donor and acceptor of one hydrogen bond.

Demonstration of a fibrillar component in the cell wall of the yeast Saccharomyces cerevisiae and its chemical nature.

The ultrastructure of isolated cell walls of Saccharomyces cerevisiae from the log and stationary phases of growth was studied after treatment with the following enzymes: purified endo-beta-(1 --> 3)-glucanase and endo-beta-(1 --> 6)-glucanase produced by Bacillus circulans; purified exo-beta-glucanase and endo-beta-(1 --> 3)-glucanase produced by Schizosaccharomyces versatilis; commercial Pronase. While exo-beta-glucanase from S. versatilis had no electron microscopically detectable effect on the walls, Pronase removed part of the external amorphous wall material disclosing an amorphous wall layer in which fibrils were indistinctly visible. Amorphous wall material was completely removed by the effect of either endo-beta-(1 --> 3)- or endo-beta-(1 --> 6)-glucanase of B. circulans or by a mixture of the two enzymes. As a result of these treatments a continuous fibrillar component appeared, composed of densely interwoven microfibrils resisting further action by both of the B. circulans enzymes. The fibrillar wall component was also demonstrated in untreated cell walls by electron microscopy after negative staining. Because of the complete disappearance of the fibrils following treatment with the S. versatilis endo-beta-(1 --> 3)-glucanase it can be concluded that this fibrillar component is composed of beta-(1 --> 3)-linked glucan. Bud scars were the only wall structures resistant to the effect of the latter enzyme.

Glucanases in Schizosaccharomyces. Isolation and properties of an exo-beta-glucanase from the cell extracts and culture fluid of Schizosaccharomyces japonicus var. versatilis.

(11 Cell extracts and extracellular culture fluids of species of the yeast genus Schizosaccharomyces exhibited exo-beta-(1 leads to 3)- and exo-beta-(1 leads to 6)-glucanase (EC 3.2.1.-) activities. (2) Using a combination of Sephadex G-100 and DEAE-cellulose chromatography, the exo-beta-(1 leads to 3)-glucanases from the cell extracts and culture fluid of Schizosaccharomyces japonicus var. versatilis were purified extensively. The enzymes from either location exhibited similar purification and other properties. (3) The purified enzymes hydrolysed the beta-(1 leads to 6)-glucosidic linkage in addition to the beta-(1 leads to 3) linkage. Heat denaturation, inhibition and electrophoretic studies indicated that both hydrolytic activities were properties of a single protein. Laminarin and pustulan hydrolysis followed Michaelis-Menten kinetics. The Km and V for laminarin hydrolysis were 6.25 mg/ml and 350 mumol of glucose released/min/mg protein, and for pustulan they were 166 mg/ml and 52 mumol of glucose released/min/mg protein. (4) The exo-beta-glucanase was assigned a molecular weight of 43 000. (5) the purified enzyme failed to hydrolyse isolated cell walls from either baker's yeast or Schizosaccharomyces pombe or to induce protoplast formation from intact cells of S. japonicus var. versatilis or Saccharomyces cerevisiae.

N-acetyl-beta-D-glucopyranosylamine: a potent T-state inhibitor of glycogen phosphorylase. A comparison with alpha-D-glucose.

Structure-based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to alpha-D-glucose (Ki = 1.7 mM). The best inhibitor in the class of N-acyl derivatives of beta-D-glucopyranosylamine, N-acetyl-beta-D-glucopyranosylamine (1-GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1-GlcNAc acts as a competitive inhibitor for both the b (Ki = 32 microM) and the a (Ki = 35 microM) forms of the enzyme with respect to glucose 1-phosphate and in synergism with caffeine, mimicking the binding of glucose. Sedimentation velocity experiments demonstrated that 1-GlcNAc was able to induce dissociation of tetrameric phosphorylase a and stabilization of the dimeric T-state conformation. Co-crystals of the phosphorylase b-1-GlcNAc-IMP complex were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the complex structure has been refined to give a crystallographic R factor of 18.1%, for data between 8 and 2.3 A resolution. 1-GlcNAc binds tightly at the catalytic site of T-state phosphorylase b at approximately the same position as that of alpha-D-glucose. The ligand can be accommodated in the catalytic site with very little change in the protein structure and stabilizes the T-state conformation of the 280s loop by making several favorable contacts to Asn 284 of this loop. Structural comparisons show that the T-state phosphorylase b-1-GlcNAc-IMP complex structure is overall similar to the T-state phosphorylase b-alpha-D-glucose complex structure. The structure of the 1-GlcNAc complex provides a rational for the biochemical properties of the inhibitor.

Effects of commonly used cryoprotectants on glycogen phosphorylase activity and structure.

The effects of a number of cryoprotectants on the kinetic and structural properties of glycogen phosphorylase b have been investigated. Kinetic studies showed that glycerol, one of the most commonly used cryoprotectants in X-ray crystallographic studies, is a competitive inhibitor with respect to substrate glucose-1-P with an apparent Ki value of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that glycerol binds at the catalytic site and competes with glucose analogues that bind at the catalytic site, thus preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulfoxide (DMSO) activated glycogen phosphorylase b to different extents, by stabilizing its most active conformation, while sucrose acted as a noncompetitive inhibitor and ethylene glycol as an uncompetitive inhibitor with respect to glucose-1-P. A parallel experimental investigation by X-ray crystallography showed that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding studies with glycogen phosphorylase.

The structure of a glycogen phosphorylase glucopyranose spirohydantoin complex at 1.8 A resolution and 100 K: the role of the water structure and its contribution to binding.

A glucopyranose spirohydantoin (a pyranose analogue of the potent herbicide, hydantocidin) has been identified as the highest affinity glucose analogue inhibitor of glycogen phosphorylase b (GPb). In order to elucidate the structural features that contribute to the binding, the structures of GPb in the native T state conformation and in complex with glucopyranose spirohydantoin have been determined at 100 K to 2.0 A and 1.8 A resolution, respectively, and refined to crystallographic R values of 0.197 (R[free] 0.248) and 0.182 (R[free] 0.229), respectively. The low temperature structure of GPb is almost identical to that of the previously determined room temperature structure, apart from a decrease in overall atomic temperature factors ((B) room temperature GPb = 34.9 A2; (B) 100 K GPb = 23.4 A2). The glucopyranose spirohydantoin inhibitor (Ki = 3.0 microM) binds at the catalytic site and induces small changes in two key regions of the protein: the 280s loop (residues 281-286) that results in a decrease in mobility of this region, and the 380s loop (residues 377-385) that undergoes more significant shifts in order to optimize contact to the ligand. The hydantoin group, that is responsible for increasing the affinity of the glucose compound by a factor of 10(3), makes only one hydrogen bond to the protein, from one of its NH groups to the main chain oxygen of His377. The other polar groups of the hydantoin group form hydrogen bonds to five water molecules. These waters are involved in extensive networks of hydrogen bonds and appear to be an integral part of the protein structure. Analysis of the water structure at the catalytic site of the native enzyme, shows that five waters are displaced by ligand binding and that there is a significant decrease in mobility of the remaining waters on formation of the GPb-hydantoin complex. The ability of the inhibitor to exploit existing waters, to displace waters and to recruit new waters appears to be important for the high affinity of the inhibitor.

Inhibition of HIV replication by amino-sugar derivatives.

The plant alkaloids castanospermine, dihydroxymethyldihydroxypyrrolidine and deoxynojirimycin have recently been shown to have potential anti-HIV activity [(1987) Proc. Natl. Acad. Sci. USA 84, 8120-8124; (1987) Nature 330, 74-77; (1987) Lancet i, 1025-1026]. They are thought to act by inhibiting alpha-glucosidase I, an enzyme involved in the processing of N-linked oligosaccharides on glycoproteins. We report here the relative efficacy of a spectrum of amino-sugar derivatives as inhibition of HIV cytopathicity. Several alpha-glucosidase inhibitors and alpha-fucosidase inhibitors were found to be active at concentrations which were non-cytotoxic.

Homonojirimycin isomers and N-alkylated homonojirimycins: structural and conformational basis of inhibition of glycosidases.

A series of natural epimers of alpha-homonojirimycin and its N-alkylated derivatives have been prepared to investigate the contribution of the different chiral centers and conformation of the specificity and potency of inhibition of glycosidases. These epimers and N-alkylated derivatives are alpha-homonojirimycin (1), beta-homonojirimycin (2), alpha-homomannojirimycin (3), beta-homomannojirimycin (4), alpha-3,4-di-epi-homonojirimycin (5), beta-4,5-di-epi-homonojirimycin (6), N-methyl-alpha-homonojirimycin (7), and N-butyl-alpha-homonojirimycin (8). Compound 1 was a potent inhibitor of a range of alpha-glucosidases with IC50 values of 1 to 0.01 microM. Compounds 2, 3, and 4 were surprisingly inactive as inhibitors of beta-glucosidase and alpha- and beta-mannosidases but were moderately good as inhibitors of rice and some mammalian alpha-glucosidases. Compound 4 was active in the micromolar range toward all alpha-glucosidases tested. Furthermore, compound 4, which superimposes well on beta-l-fucose, was a 10-fold more effective inhibitor of alpha-l-fucosidase than 1-deoxymannojirimycin (12) and 3, with a Ki value of 0.45 microM. Only compounds 5 and 6 showed inhibitory activity toward alpha- and beta-galactosidases (6with an IC50 value of 6.4 microM against alpha-galactosidase). The high-resolution structure of 1 has been determined by X-ray diffraction and showed a chair conformation with the C1 OH (corresponding to the C6 OH in 1-deoxynojirimycin) predominantly equatorial to the piperidine ring in the crystal structure. This preferred (C1 OH equatorial) conformation was also corroborated by 1H NMR coupling constants. The coupling constants for 7 suggest the axial orientation of the C1 OH, while in 8 the C1 OH axial conformation was not observed. The C1 OH axial conformation appears to be responsible for more potent inhibition toward processing alpha-glucosidase I than alpha-glucosidase II. It has been assumed that the anti-HIV activity of alkaloidal glycosidase inhibitors results from the inhibition of processing alpha-glucosidase I, but 1, 7, and 8 were inactive against HIV-1 replication at 500 microg/mL as measured by inhibition of virus-induced cytopathogenicity in MT-4 cells. In contrast, the EC50 value for N-butyl-1-deoxynojirimycin (11), which also inhibits processing alpha-glucosidase I, was 37 microg/mL. Compound 7 has been shown to be a better inhibitor of alpha-glucosidase I than 1 and 8 both in vitro and in the cell culture system. These data imply that inhibition of HIV by glycosidase inhibitors can be due to factors other than simply inhibition of processing alpha-glucosidase I.

Polyhydroxylated alkaloids -- natural occurrence and therapeutic applications.

Over one hundred polyhydroxylated alkaloids have been isolated from plants and micro-organisms. These alkaloids can be potent and highly selective glycosidase inhibitors and are arousing great interest as tools to study cellular recognition and as potential therapeutic agents. However, only three of the natural products so far have been widely studied for therapeutic potential due largely to the limited commercial availability of the other compounds.

Polyhydroxylated pyrrolidine and piperidine alkaloids from Adenophora triphylla var. japonica (Campanulaceae).

Adenophora triphylla var. japonica (Campanulaceae) yielded two new alkaloids, the 6-C-butyl derivative of 2R,5R-bis(hydroxymethyl)-3R,4R-dihydroxypyrrolidine (DMDP) and alpha-1-C-ethyl-fagomine, together with the known alkaloids 1,4-dideoxy-1,4-imino-D-arabinitol, 1-deoxynojirimycin, and 1-deoxymannojirimycin. 6-C-Butyl-DMDP showed inhibitory activity toward almond beta-glucosidase (IC50 = 68 microM), whereas alpha-1-C-ethyl-fagomine inhibited bovine liver beta-galactosidase (IC50 = 29 microM).

Inhibition of mammalian digestive disaccharidases by polyhydroxy alkaloids.

Several polyhydroxy alkaloids, including the eight presently known to occur in plants, have been compared as inhibitors of mouse gut digestive disaccharidases. The indolizidine castanospermine inhibited all activities tested, but others showed a selectivity which could be of value in studies of carbohydrate digestion and errors of metabolism.

Inhibition of human ovarian carcinoma cell- and hexosaminidase- mediated degradation of extracellular matrix by sugar analogs.

Human ovarian carcinoma (HOC) cell beta-N-acetylglucosaminidase (beta-NAG, EC 3.2.1.30) was found to be present in three isoenzymatic forms. All three forms were capable of degrading ECM. Therefore, inhibitors of beta-NAG were sought as potential anti-invasive agents. Two sugar analogs, 2-acetamido-2-deoxy-1,5-gluconolactone (CD80110) and 2-acetamido-1,5-imino-1,2,5-trideoxy-D-glucitol (CD 86022), were evaluated for their ability to inhibit 1) human ovarian carcinoma beta-NAG isoenzyme activities, 2) degradation of radiolabeled ECM mediated by HOC cells and beta-NAG, and 3) cell growth. Both compounds were found to be competitive inhibitors of beta-NAG isoenzyme activities, with Ki values similar for all isoenzymes (2-8 microM). CD 80110 and CD 86022 inhibited HOC cell-mediated degradation of [3H]glucosamine labeled ECM without notable effect on cell growth. Enzyme-mediated degradation of ECM was also inhibited by both sugar analogs. Analysis of degradation products after cell- or enzyme-mediated digestion of ECM revealed a decrease in the amount of both free aminosugars and high molecular material caused by inhibitors. These results support a role for beta-NAG in degradation of extracellular matrix components and suggest the usefulness of hexosaminidase inhibitors as potential antiinvasive agents.

The microbial ecology of tape ketan fermentation.

The growth of fungi, yeasts and bacteria was followed during the fermentation of tape ketan. The tape was prepared using samples of Indonesian ragi as inoculum. Fermentation was characterized by the dominant growth of Amylomyces rouxii and Candida pelliculosa (10(5)-10(7) cfu/g), and a lesser contribution from Saccharomyces cerevisiae. Hansenula anomala grew to a limited extent during some fermentations. Bacteria of the genera Bacillus and Acetobacter contributed also to the fermentation, producing populations up to 10(5) cfu/g. Ragi was the main source of microorganisms involved in the fermentation. Tape fermented by inoculating pure cultures of Amylomyces rouxii and the yeasts, either individually or as mixtures, was not of typical quality, indicating the importance of bacteria in the overall fermentation.

The occurrence and growth of yeasts in Camembert and blue-veined cheeses.

Yeast populations greater than 10(6) cfu/g were found in approximately 54% and 36%, respectively in surface samples of retail Camembert (85 samples) and Blue-veined (45 samples) cheeses. The most predominant species isolated were Debaryomyces hansenii, Candida catenulata, C. lipolytica, C. kefyr, C. intermedia, Saccharomyces cerevisiae, Cryptococcus albidus and Kluyveromyces marxianus. The salt concentration of the surface samples of the cheeses varied between 2.5-5.5% (w/w) (Camembert) and 7.5-8.3 (Blue-veined), depending upon brand, and influenced the yeast ecology, especially the presence of S. cerevisiae. Yeasts grew to populations of 10(6)-10(8) cfu/g when cheeses were stored at either 25 degrees C or 10 degrees C. These populations decreased on continued storage at 25 degrees C, but such decreases were not so evident on storage at 10 degrees C. The properties of yeasts influencing their occurrence and growth in cheese were: fermentation/assimilation of lactose; production of extracellular lipolytic and proteolytic enzymes, utilisation of lactic and citric acids; and growth at 10 degrees C.

Growth of yeasts in milk and associated changes to milk composition.

The growth of several yeast species in milk containing added sodium chloride (0-15%, w/v) at 25 degrees C and 10 degrees C was examined in conjunction with yeast metabolism of milk constituents. Depending on conditions, all yeasts grew to maximum populations of 10(7)-10(8) cfu/ml. Kluyveromyces marxianus gave strong utilisation of lactose and weak metabolism of citrate, protein and fat with the production of ethanol, glycerol, lactic acid and propionic acid. As measured by the production of free amino acids and free fatty acids, Candida lipolytica and Candida catenulata gave strong proteolytic and lipolytic reactions, the specificities of which appeared to be influenced by temperature and the presence of NaCl. These species also metabolised organic acids. Although giving strong growth responses, Debaryomyces hansenii and Saccharomyces cerevisiae did not metabolise lactose and gave only very weak lipolytic and proteolytic reactions. Citrate was metabolised by D. hansenii but not by S. cerevisiae. Both species produced small amounts of ethanol, glycerol and lactic acid.