Screening Assays for Epigenetic Targets Using Native Histones as Substrates

Abstract

In the past years, a lot of attention has been given to the identification and characterization of selective and potent inhibitors of chromatin-modifying enzymes to better understand their specific role in transcriptional regulation. As aberrant histone methylation is involved in different pathological processes, the search for methyltransferase and demethylase inhibitors has emerged as a crucial issue in current medicinal chemistry research. High-throughput in vitro assays are important tools for the identification of new methyltransferase or demethylase inhibitors. These usually use oligopeptide substrates derived from histone sequences, although in many cases, they are not good substrates for these enzymes. Here, the authors report about the setup and establishment of in vitro assays that use native core histones as substrates, enabling an assay environment that better resembles native conditions. They have applied these substrates for the known formaldehyde dehydrogenase assay for the histone demethylase LSD1 and have established two new antibody-based assays. For LSD1, a heterogeneous assay format was set up, and a homogeneous assay was used for the characterization of the arginine methyltransferase PRMT1. Validation of the system was achieved with reference inhibitors in each case.

Nonpeptidic Propargylamines as Inhibitors of Lysine Specific Demethylase 1 (LSD1) with Cellular Activity

“…17 The data is summarized in table 1. Only the benzoyl derivatives 1 showed considerable demethylase inhibition in the higher micromolar range, but we could show with this that in principle small molecules substrate analogues are able to inhibit LSD1.…”

Pure enantiomers of benzoylamino-tranylcypromine: LSD1 inhibition, gene modulation in human leukemia cells and effects on clonogenic potential of murine promyelocytic blasts

“…Resolution was good in all cases and several tens of mg of both enantiomers were collected in a pure state (enantiomeric excess (ee) >98%). The stereochemical characterization procedure was based on the determination of the absolute configuration of the Boc intermediates by CD correlation using the enantiomers of Br-tPCPA and Br-cPCPA as reference samples [66]. Comparison of the CD spectra shown in Fig.…”

High-throughput screening to identify inhibitors of lysine demethylases

“…Formaldehyde dehydrogenase (FDH) is used to oxidize formaldehyde into formic acid through the coupled reduction of NAD + into NADH. NADH formation is monitored by absorbance at 340 nm [26] or more precisely by fluorescence intensity with excitation at 330 nm and emission at 460 nm (Figure 1A) [27,28]. Based on this reaction, Simeonov and colleagues developed a HTS platform [29], which was used to screen approximately 236,000 compounds for inhibitory activity against the catalytic domain of JMJD2E [27].…”