Inhibitor screening and enzymatic activity determination for autophagy target Atg4B using a gel electrophoresis-based assay
(2016) European Journal of Medicinal Chemistry — Vol. 123, n° 123, p. 631-638 (2016)
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Grootaert, Mandy 
- 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.
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Cleenewerck, M., Grootaert, M., Gladysz, R., Adriaenssens, Y., Roelandt, R., Joossens, J., Lambeir, A.-M., De Meyer, G. R. Y., Declercq, W., Augustyns, K., Martinet, W., & Van der Veken, P. (2016). Inhibitor screening and enzymatic activity determination for autophagy target Atg4B using a gel electrophoresis-based assay. European Journal of Medicinal Chemistry, 123(123), 631-638. https://doi.org/10.1016/j.ejmech.2016.07.073 (Original work published 2016)