α1,6フコシルトランスフェラーゼ(FUT8)阻害剤の探索と機能解析

概要

α1,6-fucosyltransferase (FUT8) transfers fucose from GDP-fucose to N-acetylglucosamine at the base of N-linked sugar chains by α1,6 binding to form a core fucose structure. The core fucose suppresses immunoglobulin-dependent cytotoxic activity and is involved in immune regulation, as well as in various diseases such as fibrosis and cancer. In this study, we are developing an inhibitor of FUT8 that can be used in vivo for the purpose of functional analysis and activity control of core fucose.

　We have already discovered the pharmacophore of FUT8 inhibitor by high throughput screening (HTS) using about 30,000 compounds. We aimed to improve the inhibitor by evaluating the function of FUT8 inhibitors at cell level and performing a structure-activity relationship based on the pharmacophore of the FUT8 inhibitor. First, we aimed to create a compound with high FUT8 inhibitory activity by synthesizing this analog based on FUT8 inhibitor 1 that based on pharmacophore. The structure of compound 1 was divided into two units, and each unit was optimized. As a result of synthesizing about 50 kinds of compounds and measuring their activities, we have synthesized the FUT8 inhibitor 2, with an IC50 of about 10 µM in vitro. Next, the FUT8 inhibitory activity of the obtained compound 2 was measured, and the expression level of core fucose 72 hours after treated was confirmed by PANC-1, but the expression level of core fucose on the cell surface did not decrease. Therefore the physical property of compound 2 were investigated and as a result, compound 2 showed instability in water condition and completely decomposed after 12 hours in PBS at 37 ˚C. When the decomposition product was analyzed by MS, a peak of the compound in which the phenylpiperazine ring portion was considered to be eliminated was detected due to the formation of an enone by deprotonation of the hydroxy group of compound 2. The structure of the decomposition product of compound 2 was confirmed using MS and NMR. From the results of 13C-NMR, 1H-NMR and LC-MS (nega), the structure of the decomposition product was identified, and the hydroxy group of compound 2 was determined to be the cause of destabilization in aqueous solution.

　Therefore, we synthesized compound 3 which was designed so that the hydroxy group of compound 2 was protected and lipophilicity was increased to allow it to rapidly permeate the cell membrane and the hydroxy group was exposed by intracellular enzymes. Compound 3 showed improvement of stability in aqueous solution compared to compound 2. Compound 3 was treated to PANC-1 every 12 hours for 3 days, and the FUT8 inhibitory activity was evaluated by flow cytometry. As a result, compound 3 reduced the expression level of core fucose on the cell surface at a level comparable to 2-Fluro-peracetylfucose, which is generally used as an inhibitor of fucosyltransferase.

　Next, the mechanism of action of compound 2 was investigated. The reaction product of FUT8 and compound 2 was measured using HPLC-MS and MS-MS, and the multivalent peak and binding site where FUT8 and compound 2 were bound were confirmed.