Figure 2.

Screening and Evaluation of Drugs for ATG4 Inhibition. (A) The ability of the 22 hits obtained from the in silico drug screening to inhibit ATG4 was evaluated using LC3-PLA₂ biochemical assays (red dots) and ATG4 reporter yeast cells (green dots). EGTA (5 mM, red dot line) and NEM (10 mM, green dot line) were used as positive controls for biochemical and cellular ATG4 reporter assays, respectively. (B) Tioconazole (Tc, 2.5 μM) was confirmed with ATG4 reporter yeast cells and LC3-PLA₂ biochemical assay and counter assayed with caspase-1 reporter yeast cells for selectivity (left panel). Various concentrations of Tc were mixed with 0.5 nM caspase-3 and 100 μM Ac-DEVD-AFC to determine the effects of Tc on the caspase-3 activity (right panel). (C) The activities of 2-fold serially titrated Tc and its analog miconazole (Mc) on ATG4B were compared with an LC3-PLA₂ assay. (D) ATG4B (0.1 nM) or (E) ATG4A (5 nM) were mixed with 2-fold serially diluted Tc and 100 nM GABARAPL2-PLA₂ to determine the IC₅₀ of Tc for ATG4 members. Quantitative results are shown in each panel (n=4). (D) ATG4B (1 nM) or (E) ATG4A (50 nM) were mixed with 10-fold serially diluted Tc and 500 nM GABARAPL2-PLA₂ for 1 h. The cleavage of GABARAPL2-PLA₂ was validated with immunoblotting using an anti-Myc antibody. (F) The expression vector that encoded ATG4-cleavable luciferase was constructed with LC3 and a luciferase chimera gene, as shown in the schematic diagram (upper panel). HEK293T cells were transfected with scramble siRNA (siCtrl) or siRNA against ATG4B (siATG4B) for 48 h, followed by transfection with the ATG4 reporter vector for 24 h. The luciferase activity was measured with a luminescent reader (n=6), and the knockdown efficiency of ATG4B was determined using immunoblotting. (G) HEK293T cells were transfected overnight with the ATG4 reporter vector as described in (I) and treated with Tc for 8 h to measure the luciferase activity (lower panel). The cleavage of LC3 and luciferase chimera protein by ATG4 in cells treated with Tc or untreated cells was verified via immunoblotting (upper panel). (H) N-terminal Venus fused with ATG4B and C-terminal Venus fused with LC3 were co-transfected into HEK 293T cells. After overnight culture, the cells were fixed to observe Venus complementary under fluorescence microscopy (right panel). Bar: 100 μm. (I) The transfected cells were treated with Tc for 8 h and fixed for flow cytometry to quantify Venus fluorescence using GFP as a counter assay. The results are expressed as the mean ± SEM from at least 3 individual experiments. *p < 0.05; **p < 0.01; ***p < 0.001.