Inhibitor screening and enzymatic activity determination for autophagy target Atg4B using a gel electrophoresis-based assay
Inhibitor screening and enzymatic activity determination for autophagy target Atg4B using a gel electrophoresis-based assay
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DOI：
10.1016/j.ejmech.2016.07.073
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发表时间：
2016-11-10
影响因子：
6.7
通讯作者：
Van der Veken, Pieter
中科院分区：
文献类型：
--
作者：
Cleenewerck, Matthias;Grootaert, Mandy O. J.;Van der Veken, Pieter
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. (C) 2016 Elsevier Masson SAS. All rights reserved.
