In-Line Screening Of Histone Deacetylase Inhibitors By Capillary Electrophoresis

Histone deacetylases(HDACs)catalyze the deacetylation of histone lysine residues,the substrates of which are histone or non-histone compounds.HDACs became popular target in cancer treatment as they are important enzymes in epigenetic study and the process of cell modulation and they are overexpressed in cancer cells.Histone deacetylase inhibitors(HDACi)lead to death of tumor,inhibition of gene transcription and damage of DNA.Till now there are several HDACs inhibitors on the market.Now,the development of new efficient high-throughput screening methods for HDAC inhibitor activities assay became very useful method for drug discovery to accelerate the discovery of new drugs.With the development in decades,capillary electrophoresis(CE)has successfully applied for inhibitor screening.The advantages of CE are various test mode,high separation efficiency,low consumption of samples,and automation.Besides the conventional offline CE analysis,electrophoresis-mediated microanalysis and immobilized enzyme microreactor are two new in-line CE assay methods and are widely used in the determination of enzyme reactions.Electrophoretical mediated microanalysis(EMMA)is an in-line assay method for enzymatic reactions and separation of substrates and products in capillary.The enzyme and substrate are injected into the inlet of capillary sequentially.Then a merge voltage is applied to make enzyme and substrate mix.When the reaction is finished,a voltage is applied to separate and determine the product and un-reacted substrate.Capillary electrophoresis immobilized enzyme microreactor(CE-IMERs)is a new in-line enzymatic assay method,which combine capillary electrophoresis and immobilized enzyme technology.The target enzyme is immobilized at the inlet of capillary by different immobilization measure,then CE-IMERs are prepared for online research on enzymatic reaction kinetics and enzyme inhibitor kinetics.The advantages of CE-IMERs are low reagent consumption,simple operation and repeated use of target enzyme.CE-IMERs is a novel high-throughput inhibitors screening method.This study established a capillary electrophoresis in-line screening method for HDAC inhibitors and provided a new screening method for the development of new anti-tumor agents.In this paper,the conditions of EMMA method are optimized by the separation conditions and mixing conditions.This method was verified and data showed that results are stable and repeatable.The results of HDAC inhibitory activities were consistent with the results of the classical method for fluorescent assay and data from the literature.In addition,a new CE-IMERs method for HDAC inhibition assay was established.The sodium alginate sol containing HDACs was injected into the inlet of capillary,then a voltage was applied to replace sodium with calcium.The calcium alginate hydrogel was formed and HDACs was immobilized at the inlet of capillary.The reactor was evaluated for its stability and reproducibility between batches,and the results suggested that enzymatic activity and inhibition kinetics of immobilized HDACs are consistent w ith the literatures.Therefore,the advantage of this method includes low reagent consumption,simple test procedure,repeatable enzyme usage,and this method could be used in high throughput screening for the discovery of HDAC inhibitors.