Novel Assays and Inhibitors of the Key Autophagy Protease

The process of macroautophagy has been implicated in a number of human diseases, most notably in cancer where it may serve as a survival and resistance mechanism for tumour cells growing in otherwise hostile environments. It has been hypothesized that an inhibitor of autophagy may serve as a useful adjunctive therapy for cancer but no selective inhibitors are available to test this concept. Atg4B converts LC3 and LC3-PE to LC3-I during macroautophagy and thus may be an appropriate macromolecular target. To enable discovery of inhibitors we have developed three enzyme assays to monitor Atg4B activity: Assay 1) a mass spectrometric assay (MS) monitoring proteolysis of LC3B allows measurement of enzyme kinetics and evaluation of putative inhibitors but is low throughput; Assay 2) Atg4-B-mediated cleavage of novel fluorogenic peptide substrates is amenable to higher throughput but Vmax for these substrates was quite slow requiring high enzyme concentration for practical rates of cleavage; Assay 3) utilizes a novel fluorescent LC3B construct, YFP-MAP1LC3B-CyFP where cleavage is monitored by fluorescence changes resulting from the loss of FRET after cleavage. MS and fluorescence shows that this construct is a substrate for Atg4B and is cleaved after the same glycine residue and with Km similar to that observed for native LC3B. This assay has been optimized for high throughput screening and initial screen has yielded several interesting hit structures. In addition cellular assays for autophagy were developed using SKBR3-GFP-LC3 cells to monitor autophagosome content, and MDA-MB-231 cells to measure autophagic flux using Western blot analysis of endogenous p62. Based on the optimized fluorogenic peptide substrates several irreversible inhibitors of Atg4B were designed and shown to have efficacy both in enzyme and cellular assays. These halo-methyl ketone analogs inhibit by alkylation of the active site cysteine thiol and may be useful tools to explore the role of authophagy in health and diseases such as cancer.

The authors acknowledge grant support from NSERC and CIHR GPG102167 as well as funding for RY’s chair from the BC-LEEF and Merck Canada.