Design, synthesis and biological evaluation of small molecules as modulators of histone methyltransferases and demethylases

In chromatin remodeling, lysine methylation patterns are determined by the counteracting activity of two different classes of enzymes: histone lysine methyltransferases (HKMTs) and demethylases (HDMs). The former catalyze methyl group addition using on the є-amino group of Lys residues, using S-adenosylmethionine as cofactor. The latter regenerate unmodified Lys residues and are divided into two superfamilies: LSDs (or Lysine Specific Demethylases) and JMJDs (or Jumonji C domain-containing Demethylases) according to their mechanism and substrate specificity. Enhancer of Zeste Homolog 2 (EZH2) is a HKMT implicated in di- and tri-methylation of the Lys 27 of histone H3 (H3K27me2/me3), two marks associated with transcriptional repression. EZH2 has been reported to be over-expressed in aggressive and metastatic tumors, like breast, lung, liver, nasopharyngeal, colon, prostate, and many other types of carcinomas, and seems to play a key role in epithelial-mesenchymal transitions, thus being a promising epi-target for cancer diseases. Pursuing our researches on design, synthesis and biological evaluation of small molecule modulators of epigenetic targets, we described some bis(monobromo)-benzylidene ketones active against Lys methyltransferases, especially on EZH2. To further deepen our studies on structure/activity relationships we also prepared derivatives of piperidone scaffolds bearing acyl- or alkyl phenyl moieties on piperidonic nitrogen. Our compounds were tested both in vitro and in vivo and showed interesting activities related to inhibition of EZH2. Specifically our compounds exerted perturbation on cell cycle distribution and apoptosis induction in U937 leukemia cells. Some of them were also tested on colon and glioblastoma cancer stem cells. LSD1 (or Lysine Specific Demethylase 1) demethylates H3K4me1/2 and H3K9me2/1, leading to epigenetic silencing or activation, respectively, depending on its molecular partner. JmjCs (or Jumonji C domain-containing demethylases) catalyze methyl groups removal on trimethyl lysine forms too, and are correlated with epigenetic activation/silencing according to their substrates. In prostate cancer LSD1 and JMJD2 are co-expressed and co-localized with the androgen receptor. We designed and synthesized hybrid LSD1/JmjC – “pan-KDM” - inhibitors 1-6, by coupling the skeleton of tranylcypromine 7, a known LSD1 inhibitor, with 4-carboxy-4'-carbomethoxy-2,2'-bipyridine 8 or 5- carboxy-8-hydroxyquinoline 9, two JmjC inhibitors. Hybrid compounds 1-6 are able to simultaneously target both KDM families, and have been validated as potential antitumor agents in cells. Among them, compounds 2 and 3 increase H3K4 and H3K9 methylation levels in cells and cause growth arrest and substantial apoptosis in LNCaP prostate and HCT116 colon cancer cells. When tested in non-cancer mesenchymal progenitor (MePR) cells, 2 and 3 induced little and no apoptosis, respectively, thus showing a cancer-selective inhibiting action