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Title
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Inhibitor screening and enzymatic activity determination for autophagy target Atg4B using a gel electrophoresis-based assay
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Author
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Abstract
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| 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. |
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Language
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English
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Source (journal)
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European journal of medicinal chemistry. - Paris, 1974, currens
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Publication
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Paris
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2016
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ISSN
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0223-5234
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DOI
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10.1016/J.EJMECH.2016.07.073
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Volume/pages
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123
(2016)
, p. 631-638
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ISI
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000385319000049
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Full text (Publisher's DOI)
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Full text (open access)
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Full text (publisher's version - intranet only)
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