In Silico Screening, In Vitro and Cell-Based Evaluations and Target Identification Studies of Tryptamine-Based Endoplasmic Reticulum Glucosidase II Inhibitors

PS1 Poster session 1 Odd numbers
Location (hall): 
Start/end time: 
Monday, July 1, 2019 - 15:45 to 17:15

Wataru Hakamata1, Rie Hoshida1, Yuhei Takemoto1, Takako Hirano1, Toshiyuki Nishio1

1Nihon University, Kanagawa, Fujisawa-shi, Japan

Majority of secretory proteins are co-translationally labeled with asparagine-linked oligosaccharides (N-glycans). During protein synthesis and folding, N-glycans and N-glycan–processing enzymes play important roles in protein quality control. Endoplasmic reticulum (ER) glucosidase II (GII) inhibitors are N-glycan–processing enzyme inhibitors; further, they have been reported as potential therapeutics for lysosomal storage disorders. Moreover, some inhibitors from this class have been clinically applied as antitumor, antiviral, and apoptosis-inducing agents. The present study focused on the antiviral activity of the above-mentioned inhibitors, which exert their effects by disrupting N-glycan maturation in viral envelope proteins, thereby effectively inhibiting GII enzymes and considerably influencing particle assembly and viral infectivity. Because host-targeting antivirals are involved in viral infection and proliferation in human host cells, GII inhibitors offer several attractive options for addressing the appearance of drug resistance in viral strains. Owing to the extremely low mutational rate of host cells, GII inhibitors provide a broad scope of antiviral activity with very high genetic barriers to drug resistance (W. Hakamata et al, Trend. Glycosci. Glycotechnol., 30, E139-E145, 2018).

Given that α-glucosidase inhibitors were previously discovered by our group using in silico high-throughput screening techniques from a compound library containing approximately 6,000,000 commercially available drug-like small molecules (W. Hakamata et al, Bioorg. Med. Chem. Lett., 22, 62-64, 2012), virtual ligand screening was performed against the substrate binding pocket for α-glucosidase and in vitro assays of α-glucosidase. A series of AR200 compounds with a tryptamine scaffold as a non-carbohydrate mimetic inhibitor were subsequently identified as potential α-glucosidase inhibitors. To determine the specificity of these inhibitors for α-glucosidase, the AR200 compounds were performed for inhibitory activity against other glycosidases, β-glucosidase, α- and β-galactosidases, and α-mannosidase. The results indicated that these inhibitors were specific for α-glucosidase. Although in the aforementioned assay the α-glucosidase inhibitory activity was evaluated for yeast α-glucosidase, it was more appropriate to evaluate the enzyme inhibitory activity of GII derived from human origin to further develop them into host-targeting antivirals. Subsequently, a cell-based GII assay system was constructed using HeLa cells and our newly developed fluorogenic substrate. The inhibitory activity of the AR200 series inhibitors was then evaluated using the constructed system. Therefore, most of the inhibitors in the sub-µM–50 µM range caused a decrease in fluorescence based on GII inhibition without showing any cytotoxicity in HeLa cells. Finally, photoaffinity labeling experiments were performed for the target identification of the AR200 compound series using AR200 compound series -based photoaffinity labeling probes to pull-down GII proteins in the HeLa cells. These pulled-down proteins were then analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the gels were silver stained and subjected to Western blotting. As a result, the probe could pull-down GII protein. Given these results, almost AR200 series compounds were indicated the GII inhibition at human cells and the inhibitors will be specifically bound to human GII. Continued evaluation of this AR200 compound series is needed in the future to elucidate the mechanistic details of bioactivity expression and to determine the beneficial applications of these compounds as host-targeting antivirals. In particular, it is essential to clarify the relationship between the accumulation of N-glycan caused by GII inhibition and the compounds’ antiviral activity.