Coumarin-suberoylanilide hydroxamic acid as a fluorescent probe for determining binding affinities and off-rates of histone deacetylase inhibitors

Abstract

Histone Deacetylases (HDACs) are intimately involved in the epigenetic regulation, and thus are one of the key therapeutic targets for cancer, and two HDAC inhibitors, namely suberoylanilide hydroxamic acid (SAHA) and romidepsin have been recently approved for the cancer treatment. Since the screening and detailed characterization of HDAC inhibitors has been time consuming, we synthesized Coumarin-SAHA (c-SAHA) as a fluorescent probe for determining the binding affinities (K d ) and the dissociation off-rates (k off ) of the enzyme-inhibitor complexes. The determination of the above parameters relies on the changes in the fluorescence emission intensity (λ ex = 325 nm, λ em = 400 nm) of c-SAHA due to its competitive binding against other HDAC inhibitors, and such determination neither requires employment of polarization accessories nor is dependent on the fluorescence energy transfer from the enzyme's tryptophan residues to the probe. Our highly sensitive and robust analytical protocol presented herein is applicable to most of the HDAC isozymes, and it can be easily adopted in a high-throughput mode for screening the HDAC inhibitors as well as for quantitatively determining their K d and k off values.

Mechanism of N-Acylthiourea-mediated Activation of Human Histone Deacetylase 8 (HDAC8) at Molecular and Cellular Levels

“…All of the structural, kinetic, and thermodynamic data suggest that SAHA binds to the active site pocket of HDAC8 (28,29,32,39). Because TM-2-51 does not obliterate the binding of SAHA to the enzyme (Fig.…”

“…1)), and deacetylated lysine-coumarin adduct (BML-KI142) were purchased from Enzo Life Sciences. The recombinant form of HDAC8 was expressed and purified as described previously (28). Trypsin used in the enzyme assay was purchased from Sigma.…”

A Substrate-Independent TR-FRET Histone Deacetylase Inhibitor Assay

“…6 competitive binding assays using fluorescent Hdac inhibitors have previously been developed, including a fluorescence resonance energy transfer (Fret)-based competitive displacement assay using internal tryptophan fluorescence and a fluorescent Hdac inhibitor, 7 a fluorescent polarization assay, 8 a combined fluorescence polarization and lifetime assay, 9 and a fluorescence-intensity assay based on a coumarin-labeled Hdac inhibitor. 10 these approaches were limited to use of a bacterial enzyme 7,9 or were demonstrated for only one 10 or two 11 human Hdacs. thus, how broadly applicable these competitive binding assays are to the study of human enzymes is unknown.…”

Coumarin-Derived Fluorescent Chemosensors

“…Histone deacetylases are intimately involved in epigenetic regulation and, thus, are one of the key therapeutic targets for cancer. Coumarinsuberoylanilide hydroxamic acid 128 is a fluorescent probe for determining binding affinities and off-rates of histone deacetylase inhibitors (Singh et al, 2011). A quinonemethide-rearrangement reaction as the off-on optical switch has been successfully implemented into the design of the first long-wavelength latent fluorogenic substrate 129 which is a sensitive fluorimetric indicator for analyte determination in salicylate hydroxylase-coupled dehydrogenase assay (S.-T. Huang et al, 2010).…”