An High-Throughput In Vivo Screening System to Select H3K4-Specific Histone Demethylase Inhibitors

Cecilia Mannironi,Enrico Cundari,Angela Alagia,Antonio Coluccia,Marco Proietto,Valeria Famiglini,Rodolfo Negri,Giuseppe La Regina,Teresa Rinaldi,Francesca Bufalieri,Romano Silvestri,Svetlana Danovska,Arthur J Lustig

doi:10.1371/journal.pone.0086002

Abstract

BackgroundHistone demethylases (HDMs) have a prominent role in epigenetic regulation and are emerging as potential therapeutic cancer targets. The search for small molecules able to inhibit HDMs in vivo is very active but at the present few compounds were found to be specific for defined classes of these enzymes.Methodology/Principal FindingsIn order to discover inhibitors specific for H3K4 histone demethylation we set up a screening system which tests the effects of candidate small molecule inhibitors on a S.cerevisiae strain which requires Jhd2 demethylase activity to efficiently grow in the presence of rapamycin. In order to validate the system we screened a library of 45 structurally different compounds designed as competitive inhibitors of α -ketoglutarate (α-KG) cofactor of the enzyme, and found that one of them inhibited Jhd2 activity in vitro and in vivo. The same compound effectively inhibits human Jumonji AT-Rich Interactive Domain (JARID) 1B and 1D in vitro and increases H3K4 tri-methylation in HeLa cell nuclear extracts (NEs). When added in vivo to HeLa cells, the compound leads to an increase of tri-methyl-H3K4 (H3K4me3) but does not affect H3K9 tri-methylation. We describe the cytostatic and toxic effects of the compound on HeLa cells at concentrations compatible with its inhibitory activity.Conclusions/SignificanceOur screening system is proved to be very useful in testing putative H3K4-specific HDM inhibitors for the capacity of acting in vivo without significantly altering the activity of other important 2-oxoglutarate oxygenases.

Highlights

Chromatin structure governs several aspects of cell metabolism
In order to screen for specific H3K4 demethylase inhibitors, we developed an experimental system based on S.cerevisiae, taking advantage of the fact that this yeast contains only one H3K4-specific JHDM, i.e. Jhd2
This strategy seemed really ideal in searching for inhibitors of histone demethylases specific for H3K4 that, existing in multiple forms in mammalians, have a unique orthologue in S.cerevisiae

Summary

Introduction

Chromatin structure governs several aspects of cell metabolism. Histone N-terminal tails are subjected to several covalent modifications which form a sophisticated combinatory code which is read and interpreted by a plethora of regulatory protein complexes [1,2]. In S.cerevisiae, three lysine methyl transferases, Set, Set and Dot, catalyse histone mono-, di- or tri-methylation at K4, K36 and K79, respectively These epigenetic marks, which are absolutely conserved among eukaryotes, have been associated with actively transcribed loci [4], their roles in controlling transcription efficiency may be distinct and strongly contextdependent [5]. JARID1B is up-regulated in 90% of human breast cancers and recently it has been shown to promote breast tumor cell cycle progression through epigenetic repression of microRNA let-7e [25]. Both JARID1A and JARID1B appear to contribute to retinoblastoma-mediated gene silencing during cellular senescence [26]. The stringent requirement on Jhd demethylase activity of a particular strain to grow in the presence of rapamycin allowed to detect the possible inhibitory activity of 45 compounds, selected by a computer-driven drug design approach, by determining their cytostatic effects on yeast cells

Materials and Methods

Results

Discussion