Kifunensine

Tunicamycin (1 µg/mL; Sigma) and kifunensine (10 µM; cat. 10009437, Cayman chemical company, Michigan, USA) were used to inhibit N-glycosylation or the formation of complex glycosylation, respectively. Cells were treated overnight (18 h) for western blot and enzyme kinetics and 6 h for immunocytochemistry.

For crystallization, hingeless Fc was appended to RBD (RBD-noHg-Fc), and the protein was expressed in Expi293F cells in the presence of 5 µM α-mannosidase inhibitor, kifunensine (Cayman Chemical Co.). After purification using the rProtein A Sepharose, RBD-noHg-Fc was treated with His-tagged IdeS protease to cleave between the RBD and the Fc, followed by subjecting to the Ni-NTA agarose and the rProtein A Sepharose sequentially to remove His-IdeS and Fc, respectively, by recovering the unbound fractions. The sample was further purified by size-exclusion chromatography (SEC) on a Superdex 200 Increase 10/300 GL column (Cytiva) equilibrated with 20 mM Tris, 150 mM NaCl, pH 7.5. 3N39 mutant sACE2-His was also expressed in Expi293F cells in the presence of kifunensine. After Ni-NTA purification, the sample was purified by anion exchange chromatography on a MonoQ 5/50 GL column equilibrated with 20 mM Tris, pH 8.0. To obtain sACE2-RBD complex, the purified sACE2 and RBD were mixed at a molar ratio of 1: 1.3 and subjected to SEC. Fractions containing the complex sample were concentrated to 9.2 mg/ml prior to crystallization. The diffraction quality crystal was grown under the condition of 1.6 M ammonium sulfate, 0.25 M lithium sulfate, and 0.05 M CAPS pH 10.5. The crystal was cryoprotected with the same buffer containing 25% ethylene glycol and used for data collection.

Pseudotyped viruses were produced by transfection of HEK-293 cells. DNA comprising Env plasmid and pSG3ΔEnv or pNL4-3.Luc.R^-.E^- at a mass ratio of 1 3.5 was mixed with transfection reagent polyethylene imine (PEI 25K, Sigma-Aldrich). Kifunensine (Cayman Chemical Co.) was added to HEK-293 cells 30 min prior to transfection [6] (link). Virus was alternatively produced in 293 GnTI- cells as described previously [85] (link). Virus containing supernatant was harvested 72 hours post transfection and 0.2 µm filtered to remove cellular debris. Viral supernatants were aliquoted and stored at −80°C.

Curcumin or curcumin derivative was dissolved in dimethyl sulfoxide (DMSO; Wako) to obtain stock solution (10 mM), and this stock solution was then stored at −20 °C. Each of the various compounds tested were added as DMSO solution to medium. For 24 h treatment, cells were treated with 10 μM curcumin, 10 μM curcumin derivative, or 3 μM GT863. For 48 h treatment, cells were treated with 0.5–3 μM GT863 for 24 h, following which an additional 0.5–3 μM GT863 was added without changing the medium until 48 h. To confirm γ-secretase activity, cells were treated with 1 μM N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (Wako) for 24 h. To inhibit N-glycosylation, cells were treated with 1 μg/ml kifunensine (Cayman Chemical Company, Ann Arbor, MI), 1 μg/ml swainsonine (Cayman), or 1 mM N-butyl-deoxynojirimycin (NB-DNJ, Wako) for 48 h.