A Fluorescence‐Lifetime‐Based Binding Assay for Class IIa Histone Deacetylases

Abstract

Class IIa histone deacetylases (HDACs) show extremely low enzymatic activity and no commonly accepted endogenous substrate is known today. Increasing evidence suggests that these enzymes exert their effect rather through molecular recognition of acetylated proteins and recruiting other proteins like HDAC3 to the desired target location. Accordingly, class IIa HDACs like bromodomains have been suggested to act as "Readers" of acetyl marks, whereas enzymatically active HDACs of class I or IIb are called "Erasers" to highlight their capability to remove acetyl groups from acetylated histones or other proteins. Small-molecule ligands of class IIa histone deacetylases (HDACs) have gained tremendous attention during the last decade and have been suggested as pharmaceutical targets in several indication areas such as cancer, Huntington's disease and muscular atrophy. Up to now, only enzyme activity assays with artificial chemically activated trifluoroacetylated substrates are in use for the identification and characterization of new active compounds against class IIa HDACs. Here, we describe the first binding assay for this class of HDAC enzymes that involves a simple mix-and-measure procedure and an extraordinarily robust fluorescence lifetime readout based on [1,3]dioxolo[4,5-f]benzodioxole-based ligand probes. The principle of the assay is generic and can also be transferred to class I HDAC8.

Determination of the binding mechanism of histone deacetylase inhibitors

“…Dansyl probes containing hydroxamic acid were synthesized according to Meyners, Baud, Fuchter, & Meyer‐Almes, a and Dioxolobenzodioxol (DBD)‐probes according to Refs. (Meyners, Baud, Fuchter, & Meyer‐Almes, ) and (Meyners, Mertens, Wessig, & Meyer‐Almes, ). HDAH pa was prepared as described elsewhere (Meyners, Baud, Fuchter, & Meyer‐Almes, ).…”

“…In the FLT‐based assay format, the fluorescent ligand is used at low nanomolar concentrations and the protein binding partner is used in excess concentration (Figure ). We exploited the special features of DBD dyes, pronounced dependency on microenvironment hydrophobicity and large stokes shift, to develop fluorescent DBD ligands for all classes of zinc‐containing histone deacetylases (Meyners, Baud, Fuchter, & Meyer‐Almes, 2014b) (Figure ). Two series of ligands with varying linker lengths between DBD head group and zinc binding group have been synthesized: Hydroxamates that are mainly suitable for class I, IIb HDACs, and bacterial HDAHs, and trifluoromethyl ketones to bind to class IIa HDACs.…”

“…Here, the use of a trifluoromethyl ketone-DBD-ligand (14c) (Meyners, Mertens, et al, 2017) is exemplified by a FP-based binding assay ( Figure 12). Caution: The binding kinetics for this HDAC-ligand is very slow (min-h)…”

Detection of dsDNA with [1,3]Dioxolo[4,5‐f]benzodioxol (DBD) Dyes

“…[4] Further applicationsi nl ifetime-based assays were developed:Aconformation change study in proteins [5] and two binding assays, one for acetylpolyamine amidohydrolases [6] and one for histone deacetylases. [7] Applying the DBD dye in various contexts resultedi nn umerous ways that the DBD chromophore can connecttoa nd interactwith biological molecules. Progressh as also been made in utilizingt he DBD chromophorei nt he field of Fçrsterr esonance energy transfer (FRET) [8] and the design of the fluorescencep roperties of the DBD chromophore became increasingly sophisticated.…”

“…DBD dyes stand out because of their large Stokes shifts, large fluorescent lifetimes, and their sensitivity to the polarity of the environment, which blazed the trail to various applications of biological interest such as probing lipophilic environments, the presence of K + ions or carbohydrate‐lectin interactions . Further applications in lifetime‐based assays were developed: A conformation change study in proteins and two binding assays, one for acetylpolyamine amidohydrolases and one for histone deacetylases . Applying the DBD dye in various contexts resulted in numerous ways that the DBD chromophore can connect to and interact with biological molecules.…”