Continuous enzyme activity assay for high-throughput classification of histone deacetylase 8 inhibitors

Abstract

Aim: Human histone deacetylase 8 (KDAC8) is a well-recognized pharmaceutical target in Cornelia de Lange syndrome and different types of cancer, particularly childhood neuroblastoma. Several classes of chemotypes have been identified, which interfere with the enzyme activity of KDAC8. These compounds have been identified under equilibrium or near equilibrium conditions for inhibitor binding to the target enzyme. This study aims for the classification of KDAC8 inhibitors according to the mode of action and identification of most promising lead compounds for drug development. Methods: A continuous enzyme activity assay is used to monitor inhibition kinetics. Results: A high-throughput continuous KDAC8 activity assay is developed that provides additional mechanistic information about enzyme inhibition enabling the classification of KDAC8 inhibitors according to their mode of action. Fast reversible inhibitors act as a molecular chaperone and are capable to rescue the enzyme activity of misfolded KDAC8, while covalent inactivators and slow dissociating inhibitors do not preserve KDAC8 activity. Conclusions: The application of continuous KDAC8 activity assay reveals additional information about the mode of interaction with inhibitors, which can be used to classify KDAC8 inhibitors according to their mode of action. The approach is compatible with the high-throughput screening of compound libraries. Fast reversible inhibitors of KDAC8 act as molecular chaperones and recover enzyme activity from misfolded protein conformations. In contrast, slow-binding inhibitors and covalent inactivators of KDAC8 are not capable to recover enzyme activity.

In-Cell Testing of Zinc-Dependent Histone Deacetylase Inhibitors in the Presence of Class-Selective Fluorogenic Substrates: Potential and Limitations of the Method

“…For in vitro testing of HDACi selectivity and potency, recombinant HDACs and their fluorogenic substrates, namely lysine derivatives or various peptides (some of which were described for the first time two decades ago), are most often used [ 16 , 17 ]. Currently, the most popular and commercially available fluorogenic HDAC substrates have become Boc-Lys(Ac)-AMC (substrate selectivity for HDAC1/2/3 class I and HDAC6 class IIb) and Boc-Lys(Tfa)-AMC (substrate selectivity for HDAC class IIa and to a lesser extent for HDAC8/10/11) [ 18 , 19 ]. In both cases, enzymatic removal of the acyl group from the substrate results in the formation of a product, Boc-Lys-AMC (Prod Lys ), which, after incubation with trypsin, is detected by the release of brightly fluorescent 7-amino-4-methylcoumarin (AMC) [ 17 ].…”