An high-throughput in vivo screening system to select H3K4-specific histone demethylase inhibitors.
BACKGROUND
Histone 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.


RESULTS
In 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
Our 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.
Istituto di Biologia e Patologia Molecolari Consiglio Nazionale delle Ricerche, Rome, Italy.
Cecilia
C
Mannironi
Istituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Biologia e Biotecnologie "C. Darwin", Sapienza Università di Roma, Rome, Italy.
Marco
M
Proietto
Istituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Biologia e Biotecnologie "C. Darwin", Sapienza Università di Roma, Rome, Italy.
Francesca
F
Bufalieri
Istituto di Biologia e Patologia Molecolari Consiglio Nazionale delle Ricerche, Rome, Italy.
Enrico
E
Cundari
Istituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Biologia e Biotecnologie "C. Darwin", Sapienza Università di Roma, Rome, Italy.
Angela
A
Alagia
Istituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Biologia e Biotecnologie "C. Darwin", Sapienza Università di Roma, Rome, Italy.
Svetlana
S
Danovska
Istituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Biologia e Biotecnologie "C. Darwin", Sapienza Università di Roma, Rome, Italy.
Teresa
T
Rinaldi
Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Rome, Italy.
Valeria
V
Famiglini
Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Rome, Italy.
Antonio
A
Coluccia
Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Rome, Italy.
Giuseppe
G
La Regina
Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Rome, Italy.
Romano
R
Silvestri
Istituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Biologia e Biotecnologie "C. Darwin", Sapienza Università di Roma, Rome, Italy.
Rodolfo
R
Negri
D003067:Coenzymes / Q000378:metabolism; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006657:Histones / Q000378:metabolism*; D006801:Humans; D056484:Jumonji Domain-Containing Histone Demethylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D008745:Methylation; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D020123:Sirolimus / Q000494:pharmacology; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*
D003067:Coenzymes; D006657:Histones; D007656:Ketoglutaric Acids; D054852:Small Molecule Libraries; D056484:Jumonji Domain-Containing Histone Demethylases; D020123:Sirolimus