Abstract
Deferiprone (DFP) is a hydroxypyridinone-derived iron chelator currently in clinical use for iron chelation therapy. DFP has also been known to elicit antiproliferative activities, yet the mechanism of this effect has remained elusive. We herein report that DFP chelates the Fe2+ ion at the active sites of selected iron-dependent histone lysine demethylases (KDMs), resulting in pan inhibition of a subfamily of KDMs. Specifically, DFP inhibits the demethylase activities of six KDMs - 2A, 2B, 5C, 6A, 7A and 7B - with low micromolar IC50s while considerably less active or inactive against eleven KDMs - 1A, 3A, 3B, 4A-E, 5A, 5B and 6B. The KDM that is most sensitive to DFP, KDM6A, has an IC50 that is between 7- and 70-fold lower than the iron binding equivalence concentrations at which DFP inhibits ribonucleotide reductase (RNR) activities and/or reduces the labile intracellular zinc ion pool. In breast cancer cell lines, DFP potently inhibits the demethylation of H3K4me3 and H3K27me3, two chromatin posttranslational marks that are subject to removal by several KDM subfamilies which are inhibited by DFP in cell-free assay. These data strongly suggest that DFP derives its anti-proliferative activity largely from the inhibition of a sub-set of KDMs. The docked poses adopted by DFP at the KDM active sites enabled identification of new DFP-based KDM inhibitors which are more cytotoxic to cancer cell lines. We also found that a cohort of these agents inhibited HP1-mediated gene silencing and one lead compound potently inhibited breast tumor growth in murine xenograft models. Overall, this study identified a new chemical scaffold capable of inhibiting KDM enzymes, globally changing histone modification profiles, and with specific anti-tumor activities.
Highlights
On the account that high levels of iron are essential for tumor cell growth, the antiproliferative effect of DFP has been largely attributed to its iron chelation activity which results in the depletion of free intracellular iron and removal of iron from the active sites of key iron-dependent enzymes
We used molecular docking to interrogate the interaction of DFP with KDM6A, a representative KDM which regulates gene expression programs associated with breast cancer (BCa) proliferation and invasion[23,24]
This docking analysis built on our previous studies where we used the molecular docking program AutoDock 4.225 to successfully identify from this library 3-hydroxypyridin-2-thione as a non-hydroxamate zinc binding group that is compatible with histone deacetylase (HDAC) inhibition[15,16]
Summary
On the account that high levels of iron are essential for tumor cell growth, the antiproliferative effect of DFP has been largely attributed to its iron chelation activity which results in the depletion of free intracellular iron and removal of iron from the active sites of key iron-dependent enzymes. For those HDAC isoforms that have been subject to structural characterization, the architecture of the enzymes’ active sites is nearly identical, consisting of Zn2+ ion bound to the base of the active site pocket that is in turn exposed to the enzyme surface through a short channel lined with hydrophobic residues Another class of epigenetic modifiers whose active sites architecture resemble HDACs’ is 2-oxoglutarate- and Fe2+-dependent histone lysine demethylases (KDMs) that remove specific histone methylation posttranslational marks[18,19,20,21,22]. This study furnished several novel DFP-based KDM inhibitors which alter the velocity of HP1-mediated heterochromatin gene repression These compounds displayed tumor-selective cytotoxicity against the breast cancer (BCa) cell lines tested, with potency enhancement as high as 65-fold relative to DFP. Our findings demonstrate that DFP can serve as a novel template for the discovery of tumor-selective KDM inhibitors
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