title: Inhibitor screening and enzymatic activity determination for autophagy target Atg4B using a gel electrophoresis-based assay.
abstract: Atg4B is a cysteine hydrolase that plays a key role in autophagy. Although it has been proposed as an attractive drug target, inhibitor discovery has proven highly challenging. The absence of a standardized, easily implementable enzyme activity/inhibition assay for Atg4B most likely contributes to this situation. Therefore, three different assay types for Atg4B activity/inhibition quantification were first compared: (1) an approach using fluorogenic Atg4B-substrates, (2) an in-gel densitometric quantification assay and (3) a thermal shift protocol. The gel-based approach showed the most promising results and was validated for screening of potential Atg4B inhibitors. A set of 8 literature inhibitors was included. Remarkably, in our hands only 2 literature references were found to have measurable Atg4B affinity. Furthermore, a fragment library (n = 182) was tested for Atg4B inhibition. One library member showed inhibition at high micromolar concentration and was found fit for further, fragment-based inhibitor design.
authors: Laboratory of Medicinal Chemistry (UAMC), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Antwerp, Belgium.
authors: Matthias
authors: M
authors: Cleenewerck
authors: Laboratory of Physiopharmacology, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Antwerp, Belgium.
authors: Mandy O J
authors: MOJ
authors: Grootaert
authors: Laboratory of Medicinal Chemistry (UAMC), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Antwerp, Belgium.
authors: Rafaela
authors: R
authors: Gladysz
authors: Laboratory of Medicinal Chemistry (UAMC), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Antwerp, Belgium.
authors: Yves
authors: Y
authors: Adriaenssens
authors: VIB Inflammation Research Center, Technologiepark 927, B-9052, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, B-9052, Ghent, Belgium.
authors: Ria
authors: R
authors: Roelandt
authors: Laboratory of Medicinal Chemistry (UAMC), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Antwerp, Belgium.
authors: Jurgen
authors: J
authors: Joossens
authors: Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Antwerp, Belgium.
authors: Anne-Marie
authors: AM
authors: Lambeir
authors: Laboratory of Physiopharmacology, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Antwerp, Belgium.
authors: Guido R Y
authors: GRY
authors: De Meyer
authors: VIB Inflammation Research Center, Technologiepark 927, B-9052, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, B-9052, Ghent, Belgium.
authors: Wim
authors: W
authors: Declercq
authors: Laboratory of Medicinal Chemistry (UAMC), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Antwerp, Belgium.
authors: Koen
authors: K
authors: Augustyns
authors: Laboratory of Physiopharmacology, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Antwerp, Belgium.
authors: Wim
authors: W
authors: Martinet
authors: Laboratory of Medicinal Chemistry (UAMC), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Antwerp, Belgium. Electronic address: pieter.vanderveken@uantwerpen.be.
authors: Pieter
authors: P
authors: Van der Veken
mesh_terms: D001343:Autophagy / Q000187:drug effects*; D000071183:Autophagy-Related Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical; D004586:Electrophoresis; D057075:Enzyme Assays; D006801:Humans; D013696:Temperature
chemical_list: D000071183:Autophagy-Related Proteins; D015853:Cysteine Proteinase Inhibitors; C507524:ATG4B protein, human; D003546:Cysteine Endopeptidases
keywords: Atg4B; Autophagy; Inhibitor; LC3B-GST; Screening