title: Identification of New ATG4B Inhibitors Based on a Novel High-Throughput Screening Platform.
abstract: Autophagy is an evolutionarily conserved homeostasis process through which aggregated proteins or damaged organelles are enveloped in a double-membrane structure called an autophagosome and then digested in a lysosome-dependent manner. Growing evidence suggests that malfunction of autophagy contributes to the pathogenesis of a variety of diseases, including cancer, viral infection, and neurodegeneration. However, autophagy is a complicated process, and understanding of the relevance of autophagy to disease is limited by lack of specific and potent autophagy modulators. ATG4B, a Cys-protease that cleaves ATG8 family proteins, such as LC3B, is a key protein in autophagosome formation and maturation process. A novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay measuring protease activity of ATG4B was developed, validated, and adapted into a high-throughput screening (HTS) format. HTS was then conducted with a Roche focus library of 57,000 compounds. After hit confirmation and a counterscreen to filter out fluorescence interference compounds, 267 hits were confirmed, constituting a hit rate of 0.49%. Furthermore, among 65 hits with an IC50< 50 µM, one compound mimics the LC3 peptide substrate (-TFG-). Chemistry modification based on this particular hit gave preliminary structure activity relationship (SAR) resulting in a compound with a 10-fold increase in potency. This compound forms a stable covalent bond with Cys74 of ATG4B in a 1:1 ratio as demonstrated by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Furthermore, this compound displayed cellular ATG4B inhibition activity. Overall, the novel TR-FRET ATG4B protease assay plus counterscreen assay provides a robust platform to identify ATG4B inhibitors, which would help to elucidate the mechanism of the autophagy pathway and offer opportunities for drug discovery.
authors.affiliation: 1 Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Shanghai, China.
authors.forename: Danqing
authors.initials: D
authors.lastname: Xu
authors.affiliation: 1 Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Shanghai, China.
authors.forename: Zhiheng
authors.initials: Z
authors.lastname: Xu
authors.affiliation: 1 Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Shanghai, China.
authors.forename: Li
authors.initials: L
authors.lastname: Han
authors.affiliation: 1 Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Shanghai, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Liu
authors.affiliation: 1 Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Shanghai, China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Zhou
authors.affiliation: 1 Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Shanghai, China.
authors.forename: Zongxing
authors.initials: Z
authors.lastname: Qiu
authors.affiliation: 1 Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Shanghai, China.
authors.forename: Xianfeng
authors.initials: X
authors.lastname: Lin
authors.affiliation: 1 Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Shanghai, China.
authors.forename: Guozhi
authors.initials: G
authors.lastname: Tang
authors.affiliation: 1 Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Shanghai, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Shen
authors.affiliation: 2 Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Basel, Switzerland.
authors.forename: Johannes
authors.initials: J
authors.lastname: Aebi
authors.affiliation: 2 Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Basel, Switzerland.
authors.forename: Claus
authors.initials: C
authors.lastname: Riemer
authors.affiliation: 2 Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Basel, Switzerland.
authors.forename: Bernd
authors.initials: B
authors.lastname: Kuhn
authors.affiliation: 2 Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Basel, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Stahl
authors.affiliation: 3 Roche Pharma Research and Early Development, Roche Innovation Center, New York, NY, USA.
authors.forename: David
authors.initials: D
authors.lastname: Mark
authors.affiliation: 1 Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Shanghai, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Qin
authors.affiliation: 1 Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Shanghai, China.
authors.forename: Haiyuan
authors.initials: H
authors.lastname: Ding
mesh_terms: D001343:Autophagy / Q000187:drug effects* / Q000235:genetics; D000071190:Autophagy-Related Protein 8 Family / Q000737:chemistry; D000071183:Autophagy-Related Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000737:chemistry; D062313:Databases, Pharmaceutical; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017930:Genes, Reporter; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry*; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D059748:Proteolysis / Q000187:drug effects; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; C498698:GABARAPL2 protein, human; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D011480:Protease Inhibitors; D008156:Luciferases; C507524:ATG4B protein, human; D003546:Cysteine Endopeptidases
keywords: ATG4B; HTS; TR-FRET; autophagy