In order to establish proper folding and

In order to establish proper folding and enzymatic activity, G9a and GLP methyltransferases each contain four structural zinc ions. Both ZM 306416 synthesis contain two distinguishable types of zinc fingers; three Zn(II) ions are chelated in a triangular cluster by 9 cysteines (b, top left), whereas one Zn(II) ion is chelated by 4 cysteines in a Cys-type zinc finger (b, top right). The latter zinc finger is adjacent to the SAM-binding site. Recent studies have highlighted that significant efforts have been made in developing strategies for targeting labile Zn-fingers with electrophilic small molecules, most notably by ebselen and disulfiram., Inhibition of important biological processes by the release of structural zinc ions has been shown for a variety of proteins, including nucleocapsid 7, p300, γ-butyrobetaine, and histone lysine demethylase JMJD2A. We hypothesized that it would be possible to inhibit G9a and GLP methyltransferases by small molecule-mediated ejection of structural zinc ions. It was envisioned that the ejection of Zn(II) from the Cys-Zn finger, which is located adjacent to the SAM-binding site (b, top right), would lead to a loss of the methyltransferase activity of G9a and GLP. We initiated our investigations by testing whether 20 known and potential zinc ejectors, including clinically used ebselen, disulfiram and cisplatin, have an ability to inhibit the G9a and GLP methyltransferase activity (). The chosen examples include: selenium-based compounds –, sulfur-based compounds –, and various other potential Zn(II) ejectors –. Initially, all compounds were tested at a concentration of 10 µM against both G9a and GLP. Therefore, methylation of a synthetic 15-mer peptide (residues 1–15) mimic of the -terminal histone 3 tail containing a lysine at position 9 (H3K9) was monitored using matrix assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. Representative inhibition data for GLP-catalyzed methylation of H3K9 can be found in . Molecules that did not show significant inhibition at 10 µM, were also tested at 100 µM. Inhibition data at 10 and 100 µM for all other compounds can be found in . For those compounds that showed >50% inhibition at a concentration of 100 µM, half maximum inhibitory concentrations (IC) were obtained using a MALDI-TOF based assay using 200 nM enzyme concentrations ( and ). In the absence of inhibitor, 15-mer H3K9 histone mimic underwent near quantitative trimethylation (/ = 1603.1 Da, a); this result is consistent with our recent studies on KMT-catalyzed methylation of lysine. For ebselen , which is known to inhibit various zinc finger containing proteins, such as metallothionein, histone lysine demethylase JMJD2A, and γ-butyrobetaine hydroxylase, submicromolar IC values were obtained (0.40 µM for G9a and 0.73 µM for GLP), demonstrating very effective inhibition. Notably, at 10 µM concentration of ebselen, only unmethylated peptide was observed in MALDI-TOF spectrum (b, / = 1560.9). Related seleno compounds – were also observed to be excellent inhibitors of G9a and GLP; IC values were found to be 0.45–4.4 µM for G9a, and 1.0–4.9 µM for GLP. Sodium selenate only showed ∼10% inhibition for G9a and GLP at 100 µM concentration (). Diphenyl diselenide inhibited G9a and GLP with IC = 0.55 μM and 0.86 μM, respectively. Having shown that selenium-based compounds act as good inhibitors of G9a and GLP, we examined related sulfur-based small molecules as potential inhibitors for these two methyltransferases. Dithiocarbamates disulfiram (c) and thiram inhibited the activity of G9a with submicromolar IC values of 0.60 and 0.55 µM, whereas for GLP the observed IC values are 1.6 and 1.1 µM, respectively. Inhibition by clinically safe disulfiram is particularly important, as it has been used for decades for treatment alcoholism by targeting acetaldehyde dehydrogenase (ADH), thereby causing instantaneous nausea upon consumption of alcohol. Besides ADH, disulfiram is also known to inhibit hepatitis C viral replication by targeting a labile zinc finger, and to induce apoptosis in a number of human cancer cell lines or inhibit cancer cell growth., , Sodium diethyldithiocarbamate , the reduced form of disulfiram, was found to only poorly inhibit GLP. This result is consistent with observations on inhibition of HIV-1 nucleocapsid protein.