ToP-DNJ, a Selective Inhibitor of Endoplasmic Reticulum α-Glucosidase II Exhibiting Antiflaviviral Activity

Iminosugars have therapeutic potential against a range of diseases, due to their efficacy as glycosidase inhibitors. A major challenge in the development of iminosugar drugs lies in making a compound that is selective for the glycosidase associated with a given disease. We report the synthesis of ToP-DNJ, an antiviral iminosugar–tocopherol conjugate. Tocopherol was incorporated into the design of the iminosugar in order to direct the drug to the liver and immune cells, specific tissues of interest for antiviral therapy. ToP-DNJ inhibits ER α-glucosidase II at low micromolar concentrations and selectively accumulates in the liver in vivo. In cellular assays, the drug showed efficacy exclusively in immune cells of the myeloid lineage. Taken together, these data demonstrate that inclusion of a native metabolite into an iminosugar provides selectivity with respect to target enzyme, target cell, and target tissue.


Enzyme Inhibition Assays
All rat enzymes were isolated from 18-week-old, male Wistar rats, while human lysosomal α-glucosidase was obtained in commercially available form from Sanofi Genzyme. Inhibition of isolated glucosidases was evaluated as described previously. 3 Briefly, maltase, isomaltase, sucrase, and cellobiase were used as partially purified tissue extracts. Inhibition assays for each enzyme were performed with the respective natural substrate, and release of d-glucose measured by Glucose B-test (Wako Diagnostics). Assays of rat ER α-glucosidase II and human lysosomal α-glucosidase inhibition were conducted with α-p-nitrophenylglucoside as substrate; 400 mm Na 2 CO 3 was added to quench these reactions, and the extent of hydrolysis was evaluated as absorbance at 400 nm by spectrophotometer.

Isolation and Culture of Primary Human MDMΦ
Primary human MDMΦ were generated from human monocytes isolated from buffy coats (NHS Blood and Transplant, surplus to clinical requirements) as described previously 5 and cultured in X-VIVO10 medium (Lonza) supplemented with 1% (v/v) heat-inactivated (56 • C, 30 min) autologous plasma. The use of human blood was approved by the NHS National Research Ethics Service (09/H0606/3).
After the incubation, 20 µL of MTS reagent was added to the 200 µL of media in each well..
After a further one to two hour incubation, the absorbance at 490 nm was measured on a Molecular Devices SpectraMax M5 microplate reader. The endpoint for evaluation was taken when untreated cells had an absorbance reading of approximately 1.
Absorbances were averaged across replicates, and corrected for background by subtracting the value for media-only wells. Each drug treatment was then evaluated as a percentage of untreated cells. This was used to determine the highest nontoxic concentration.

Free Oligosaccharide Assay
FOS analysis was carried out as described previously. 4 Briefly, cells were cultured in 6-well plates with 2 mL of media in each well, with each treatment carried out in triplicate. All assays were performed for 2 d. The primary MDMΦ cells (2 donors) were seeded at a density of 3 × 10 6 cells/well, while all other cell types were seeded at a density of 1 × 10 6 cells/well. At the end of the incubation, cells were washed with phosphate buffered saline (PBS, Gibco, 3 × 1 mL), suspended in 1 mL of H 2 O and subjected to freeze-thaw cycling (3 × 25 to -80 • C) to effect cell lysis. Before purifying the FOS, each sample was analyzed for protein content using the modified Bradford assay. 6;7 Charged species were removed from the sample by mixed bed ion exchange chromatography, and the obtained neutral sample dried by lyophilisation. FOS were fluorescently labelled with anthranilic acid. The product was purified by solid phase extraction using a DPA-6S column (Sigma), followed by Concanavalin A column. The labelled glycans were then analyzed by normal phase high performance liquid chromatography. Retention times and peak area were used to identify and quantify glycans, respectively, by comparison to standards of known identity and molar quantity. FOS quantities were normalized to protein levels.

HCV Antiviral Assay
Huh7.5 cells were seeded in a 96-well plate at a density of 5 × 10 3 cells/well in 100 µL growth media. After an 18 h incubation, the supernatant was removed, and the wells were

DENV Plaque Assay
The infectious DENV titres in supernatants collected were evaluated by plaque assay as described previously. 9 Briefly, LLC-MK 2 cells were seeded in confluent monolayers in a 12-well plate. Four 10-fold dilutions of each supernatant to be analyzed were prepared in minimum essential media (MEM, Gibco), supplemented with 100 µg mL −1 streptomycin, The adherent monolayers were washed once with Hank's buffered salt solution (Gibco), then inoculated with 100 µL of analyte and 100 µL of the supplemented MEM used to prepare the dilutions. The plates were rocked at 20 • C for 90 min, after which the inoculum was removed and nutrient-supplemented primary overlay (1 mL) was added to each well. The overlay was allowed to solidify at 20 • C, before returning the cells to the incubator. After 5 d, nutrient-supplemented, neutral red-containing secondary overlay (1 mL) was added to each well and allowed to solidify at 20 • C, then the cells were returned to the incubator.
After 1 d, plaques were observed and counted from the underside of the plate by eye.

Pharmacokinetics and Biodistribution Study
The study was carried out by Sai Life Science, Ltd. were euthanized by CO 2 asphyxiation, and spleen, brain, liver, kidney, heart, and lung were collected. The tissues were dipped three times in 20 mL fresh PBS, dried on blotted paper and weighed. The samples were then homogenized in ice-cold PBS and stored at -70 • C until analysis. All samples were analyzed by liquid chromatography-tandem mass spectrometry.