Fig. 1: Characterization of autophagy modulators that target fungal ATG4-mediated cleavage of ATG8.

a Schematics of HTS using the bioluminescence resonance energy transfer (BRET)-based B. cinerea synthetic ATG8 (BcATG8)-sensor in which citrine fluorescence protein and modified Renilla luciferase, SuperhRLUC (ShR), are fused to the N- and the C-terminus of BcATG8, respectively. BcATG4 cysteine protease-mediated cleavage of Citrine-BcATG8-ShR leads to generation of Citrine-BcATG8 and ShR byproducts. Citrine-BcATG8-ShR exhibits a higher BRET ratio in the presence of ShR substrate coelenterazine (CLZ) and the cleavage of the sensor leads to lower BRET ratio. Compounds with high and low BRET ratios are considered as inhibitors and activators, respectively. b BRET ratio of the ebselen (EB) treatment (left). The cleavage of Citrine-BcATG8-ShR by BcATG4 (right) confirms the inhibition of the BcATG4-mediated BcATG8 processing by EB compared to the DMSO control. The graph presents mean and SE. p < 0.0001 (****), two-tailed t-test. c Chemical scaffolds of EB and its analogs, EO, PT, PID, and PIO. d EO and PT inhibit the BcATG8 cleavage similar to EB. PID and PIO analogs of EB have weak effects on the cleavage. Inhibition of the cleavage is reversible under reducing conditions (+DTT). e IC₅₀ values of EB, EO, and PT for the BcATG8 cleavage are estimated by in vitro cleavage assay (left panels). The graph (right) shows mean and SE. f The inhibition of the M. oryzae ATG4 (MoATG4)-mediated processing of MoATG8 by EB, EO, and PT. g The equilibrium dissociation constant of EB is estimated by ITC. The ITC data of EB with BcATG4 (left) and MoATG4 (right) are fitted to the model of multiple binding sites. Three independent experiments were performed with similar results in b–g. A representative image is shown in each panel. Arrows and arrowheads represent the full-length BRET-sensors and the cleavage byproducts, respectively.