Title
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
Author
Faculty/Department
Faculty of Pharmaceutical, Biomedical and Veterinary Sciences. Pharmacy
Publication type
article
Publication
Paris ,
Subject
Pharmacology. Therapy
Source (journal)
European journal of medicinal chemistry. - Paris, 1974, currens
Volume/pages
123(2016) , p. 631-638
ISSN
0223-5234
Carrier
E
Target language
English (eng)
Full text (Publishers DOI)
Affiliation
University of Antwerp
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.
Full text (open access)
https://repository.uantwerpen.be/docman/irua/738037/134853.pdf
E-info
https://repository.uantwerpen.be/docman/iruaauth/b36b52/134853.pdf
Handle