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Updated: Aug 25, 2025
Assays for Validating Histone Acetyltransferase Inhibitors
Published on: August 6, 2020
Methionine 274 Is Not the Determining Factor for Selective Inhibition of Histone Deacetylase 8 (HDAC8) by L-Shaped
Niklas Jänsch1, Kim Leoni Lang1, Franz-Josef Meyer-Almes1
1Department of Chemical Engineering and Biotechnology, University of Applied Sciences Darmstadt, Haardtring 100, 64295 Darmstadt, Germany.
Abstract:
HDAC8 is an important target in several indication areas including childhood neuroblastoma. Several isozyme selective inhibitors of HDAC8 with L-shaped structures have been developed. A theoretical study has suggested that methionine 274 (M274) would act as a "switch" that controls a transient binding pocket, which is induced upon binding of L-shaped inhibitors. This hypothesis was experimentally examined in this study. The thermostability and functionality of HDAC8 wildtype and mutant variants with exchanged M274 were analyzed using biophysical methods. Furthermore, the binding kinetics of L-shaped and linear reference inhibitors of these HDAC8 variants were determined in order to elucidate the mode of interaction. Exchange of M274 has considerable impact on enzyme activity, but is not the decisive factor for selective recognition of HDAC8 by L-shaped inhibitors.
Insights
Methionine 274 in Histone Deacetylase 8 (HDAC8) does not solely control the binding pocket for L-shaped inhibitors. While M274 exchange impacts enzyme activity, it is not the key factor for selective inhibitor recognition.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Structural biology
Background:
- Histone Deacetylase 8 (HDAC8) is a therapeutic target for neuroblastoma.
- L-shaped inhibitors selectively target HDAC8.
- A theoretical study proposed Methionine 274 (M274) acts as a switch for a transient binding pocket induced by L-shaped inhibitors.
Purpose of the Study:
- To experimentally validate the role of M274 in HDAC8 inhibition by L-shaped compounds.
- To investigate the impact of M274 exchange on HDAC8 thermostability, functionality, and inhibitor binding kinetics.
Main Methods:
- Site-directed mutagenesis to create M274 variants of HDAC8.
- Biophysical techniques (thermostability assays, enzyme activity assays) to characterize enzyme variants.
- Binding kinetics analysis using L-shaped and linear inhibitors.
Main Results:
- Exchange of M274 significantly affected HDAC8 enzyme activity.
- M274 was not the sole determinant for selective binding of L-shaped inhibitors to HDAC8.
- Binding kinetics revealed insights into inhibitor-enzyme interactions.
Conclusions:
- The role of M274 in HDAC8 inhibition is complex and not limited to controlling the binding pocket for L-shaped inhibitors.
- Further studies are needed to fully elucidate the mechanism of selective HDAC8 inhibition.
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