A new quinoline-based chemical probe inhibits the autophagy-related cysteine protease ATG4B

The cysteine protease ATG4B is a key component of the autophagy machinery, acting to proteolytically prime and recycle its substrate MAP1LC3B. The roles of ATG4B in cancer and other diseases appear to be context dependent but are still not well understood. To help further explore ATG4B functions and potential therapeutic applications, we employed a chemical biology approach to identify ATG4B inhibitors. Here, we describe the discovery of 4–28, a styrylquinoline identified by a combined computational modeling, in silico screening, high content cell-based screening and biochemical assay approach. A structure-activity relationship study led to the development of a more stable and potent compound LV-320. We demonstrated that LV-320 inhibits ATG4B enzymatic activity, blocks autophagic flux in cells, and is stable, non-toxic and active in vivo. These findings suggest that LV-320 will serve as a relevant chemical tool to study the various roles of ATG4B in cancer and other contexts.


SCREENING:
The crystal structures of ATG4B were obtained from the RCSB Protein Data Bank (PDB, http://www.rcsb.org/pdb/). ICM package (version 3.6) was used for protein preparation, pocket identification and molecular docking-based screening of small molecule databases. The structure data (sdf files) of small molecules were downloaded from ZINC (http://zinc.docking.org).
The protein preparation tools implemented in ICM 1 were used to prepare the proteins for docking. Water molecules, ligands and ions were removed. Counter mutations were modeled for PDBs with mutated amino acids (catalytic residues). Hydrogens were added and global optimization was performed to find the best hydrogen bonding network. In addition, the orientations of His, Pro, Asn, Gln, Cys were optimized. The protonation states of His were also optimized.
The PocketFinder 2 was applied to those crystal structures for identifying small moleculebinding pockets. This method uses only the protein structure for the prediction of cavities and clefts. The position and size of the ligand-binding pocket are determined based on a transformation of the Lennard-Jones potential by convolution with a Gaussian kernel of a certain size, a grid map of a binding potential and construction of equipotential surfaces along the maps.
A set of grid maps was pre-calculated for each pocket. The maps represent hydrogen bonding potential, van der Waals potential, hydrophobic potential and electrostatic potential.
The maps were generated in a rectangular box with 0.5 Å grid spacing centered at the predicted small molecule binding site.  Figure S1. Full-length blots are shown next to corresponding cropped images. and the right side with Q100. The compound lies completely within the predicted pocket (orange chicken wire presentation). The ATG4B protein is in gray skin model. Figure S5A. ATG4B active conformation is in red color and inactive conformation is in blue.

SUPPLEMENTAL INFORMATION
3-mercaptopropanoate (129 mg, 1.02 mmol) were added and the reaction mixture was stirred at reflux for 2.5 h under inert conditions. The mixture was diluted with EtOAc and the organic phase was washed with saturated NaHCO 3 and brine, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on a silica gel column (EtOAc:hexanes; 98:2 to 40:60) to afford 68 as yellow solid (17%).