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

Journal Contribution - Journal Article

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.
Journal: European journal of medicinal chemistry
ISSN: 0223-5234
Volume: 123
Pages: 631 - 638
Publication year:2016
Keywords:A1 Journal article
BOF-publication weight:1
CSS-citation score:1
Authors from:Government