Screening of histone deacetylase 1 inhibitors in natural products by capillary electrophoresis

Abstract

A method for the screening of histone deacetylase 1 (HDAC1) inhibitors in natural products by using capillary electrophoresis (CE) coupled with laser induced fluorescence (LIF) detection was developed. The method was developed by employing a 5-carboxyfluorescein labelled peptide with an acetylated lysine residue as the substrate of HDAC1 and a small chemical library composed of 38 purified natural products. The biochemical assay was performed by means of CE separation, i.e. the deacetylated product in the enzymatic reaction solution was separated from the substrate peptide; therefore, the enzyme activity can be accurately calculated through the measurement of the peak area of the deacetylated product. For inhibitor screening, the tested samples were spiked in the substrate solution and the resulting solution mixtures were then incubated with the enzyme solution for proceeding the deacetylation reaction for a short period of time at 30 °C. After quenching the reaction by putting the reaction vial in boiling water, the resulting solution was injected for CE separation with 100 mM Tris–H₃PO₄ buffer (pH 8.0) containing 30 mM NaCl and 0.05% (m/v) hexadimethrine bromide (HDB). The inhibitor could be readily identified as long as the peak area of the deacetylated product is reduced in comparison with that of the negative control in the absence of any inhibitor. Dynamically coating the capillary wall with positively charged HDB is very necessary to diminish the serious adsorption of the substrate and product peptides onto the capillary wall. A rapid, cost-effective method for HDAC1 inhibitor screening is proposed.