Inhibitor screening and enzymatic activity determination for autophagy target Atg4B using a gel electrophoresis-based assay

  • Eur J Med Chem. 2016 Nov 10;123:631-638. doi: 10.1016/j.ejmech.2016.07.073.
Matthias Cleenewerck  1 ,  Mandy O J Grootaert  2 ,  Rafaela Gladysz  1 ,  Yves Adriaenssens  1 ,  Ria Roelandt  3 ,  Jurgen Joossens  1 ,  Anne-Marie Lambeir  4 ,  Guido R Y De Meyer  2 ,  Wim Declercq  3 ,  Koen Augustyns  1 ,  Wim Martinet  2 ,  Pieter Van der Veken  5
Affiliations
  • 1. Laboratory of Medicinal Chemistry (UAMC), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Antwerp, Belgium.
  • 2. Laboratory of Physiopharmacology, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Antwerp, Belgium.
  • 3. VIB Inflammation Research Center, Technologiepark 927, B-9052, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, B-9052, Ghent, Belgium.
  • 4. Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Antwerp, Belgium.
  • 5. Laboratory of Medicinal Chemistry (UAMC), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Antwerp, Belgium. Electronic address: [email protected].
PMID: 27517808 DOI: 10.1016/j.ejmech.2016.07.073
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.

Keywords
Atg4B; Autophagy; Inhibitor; LC3B-GST; Screening.
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