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.
Subject(s)
Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Epigenesis, Genetic , High-Throughput Screening Assays/methods , Histones/metabolism , Aldehyde Oxidoreductases/metabolism , Antibodies/metabolism , Dose-Response Relationship, Drug , Histone Demethylases/antagonists & inhibitors , Histone Demethylases/immunology , Histone Demethylases/metabolism , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Protein-Arginine N-Methyltransferases/metabolism , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/metabolism , Reproducibility of ResultsABSTRACT
We present a combination of database screening, synthesis and in vitro testing to identify novel histone acetyltransferase (HAT) inhibitors. The National Cancer Institute compound collection (NCI) and several commercial databases were filtered by similarity-based virtual screening to find new HAT inhibitors. Employing the recombinant HAT p300/CBP-associated factor (PCAF) and two different histone substrates for screening, pyridoisothiazolones were identified as inhibitors of human PCAF. Due to the limited solubility of the initial hits, we synthesized and tested them on PCAF. The compounds inhibit the proliferation of cancer cells. In summary, valuable chemical tools and potential lead candidates for new anticancer agents directed against HATs as new targets have been identified.
Subject(s)
Antineoplastic Agents/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Histone Acetyltransferases/antagonists & inhibitors , Pyridines/chemistry , Thiazoles/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Computer Simulation , Enzyme Inhibitors/chemistry , Humans , Models, Molecular , Molecular Structure , Thiazoles/chemistry , Thiazoles/pharmacologyABSTRACT
Arginine methylation is an epigenetic modification that receives increasing interest as it plays an important role in several diseases. This is especially true for hormone-dependent cancer, seeing that histone methylation by arginine methyltransferase I (PRMT1) is involved in the activation of sexual hormone receptors. Therefore, PRMT inhibitors are potential drugs and interesting tools for cell biology. A dapsone derivative called allantodapsone previously identified by our group served as a lead structure for inhibitor synthesis. Acylated derivatives of p-aminobenzenesulfonamides and the antilepra drug dapsone were identified as new inhibitors of PRMT1 by in vitro testing. The bis-chloroacetyl amide of dapsone selectively inhibited human PRMT1 in the low micromolar region and was selective for PRMT1 as compared to the arginine methyltransferase CARM1 and the lysine methyltransferase Set7/9. It showed anticancer activity on MCF7a and LNCaP cells and blocked androgen dependent transcription specifically in a reporter gene system. Likewise, a transcriptional block was also demonstrated in LNCaP cells using quantitative RT-PCR on the mRNA of androgen dependent genes.
Subject(s)
Antineoplastic Agents/chemical synthesis , Dapsone/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Methyltransferases/antagonists & inhibitors , Sulfonamides/chemical synthesis , Acylation/drug effects , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Dapsone/chemistry , Dapsone/pharmacology , Enzyme Inhibitors/chemistry , Humans , Inhibitory Concentration 50 , Intracellular Signaling Peptides and Proteins , Models, Molecular , Molecular Structure , Protein-Arginine N-Methyltransferases , Receptors, Androgen/genetics , Sulfonamides/chemistry , Sulfonamides/pharmacologyABSTRACT
Methyltransferases and demethylases are emerging targets for epigenetic therapy. Wigle et al. (2010) present a new assay for the enzyme inhibition of methyltransferases and demethylases based on selective enzymatic cleavage of unmethylated versus methylated peptides and their subsequent electrophoretic separation.
