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    • Dithiodipyridine also known as aldrithiol is a known zinc

    2021-09-28

    2,2′-Dithiodipyridine , also known as aldrithiol, is a known zinc ejector, targeting for instance the zinc finger in nucleocapsid protein of human immunodeficiency Aldicarb type 1. inhibited G9a activity with IC = 0.65 µM and GLP with IC = 2.6 µM. We also tested disulfide cysteamine dihydrochloride salt , but its IC value was found to be above 100 µM for GLP, whereas for G9a an IC value of 15 µM was obtained. Glutathione, in both the reduced () and oxidized () form, did not inhibit G9a and GLP. Also, no inhibition was observed for cystamine , the reduced form of cysteamine dihydrochloride. Azidocarbonamide showed very high inhibition activity against both G9a and GLP (IC = 0.50 and 1.7 µM, respectively). Naphthoquinone and ninhydrin , both known to eject zinc from p300, were also tested against G9a and GLP. Naphthoquinone was observed to be a potent inhibitor of G9a with an IC value of 2.0 µM, whereas it inhibited GLP with IC = 14 µM. Ninhydrin only poorly inhibited GLP (∼40% inhibition at 100 µM, ) and an IC value of 54 µM was observed for G9a. In addition, anthraquinone , a structurally related analogue of naphthoquinone, exhibited very poor inhibition activity (∼20% for both G9a and GLP) at 100 µM. Finally, inhibitory activity of cisplatin was evaluated. Cisplatin is a potent chemotherapeutic drug and is known to be highly reactive towards Cys or CysHis type Zn-fingers. Importantly, we found that cisplatin inhibits G9a and GLP with similar IC values of 1.4 and 1.7 µM respectively. The observations that several Se- and S-based electrophiles, including clinically used ebselen and disulfiram, possess excellent inhibition activity against G9a and GLP prompted us to examine their mode of action. We envisioned that they inhibit G9a and GLP by the release of structural zinc ions, as a result of covalent modification of cysteine residues. The release of zinc from G9a and GLP was therefore monitored using the Zn-selective indicator FluoZin™-3. In line with observed MALDI-TOF data, we found that zinc was released from both methyltransferases in the presence of those compounds that showed inhibitory activity (). Because of their current use in clinics, we were particularly interested in inhibition of G9a/GLP by ebselen, disulfiram and cisplatin. Therefore, zinc release from G9a and GLP in the presence of various concentrations of ebselen, disulfiram and cisplatin was monitored over time in the presence of FluoZin-3 (a–c). We observed that ebselen ejects Zn(II) very rapidly (within minutes) for both G9a and GLP; disulfiram and cisplatin also have the ability to eject zinc ions from G9a/GLP, but require somewhat longer times to achieve it (b–c). Based on a calibration curve with known Zn(II) concentrations, a dose-response curve for the amount of zinc released from each methyltransferase was plotted (GLP d–f, G9a ). Each methyltransferase contains in total four structural zinc ions, one of which is very close to the active site, and three at a more distant location (). In the presence of ebselen (>25 μM), all 4 zinc ions were released from G9a and GLP, whereas higher concentrations of disulfiram (>50 µM) were required for complete zinc ejection. On average only ∼2.5 Zn(II) ions are removed after one hour in the presence of 100 µM cisplatin, although it is possible that all four zinc ions can be released after prolonged time, and/or at even higher concentrations of cisplatin. We also tested whether UNC0638, a very potent histone competitive inhibitor for G9a and GLP, was able to eject structural Zn(II); as expected, no zinc release was observed (). Having shown that ebselen and disulfiram effectively inhibit G9a and GLP via zinc ejection mechanism, we next explored whether the folding of these two enzymes has been affected in the presence ebselen and disulfiram. In order to investigate potential changes to the secondary and/or tertiary structure of these methyltransferases, we employed circular dichroism (CD), a well-established tool in protein/peptide research. Upon the addition of 100 µM of ebselen or disulfiram to either 2 µM G9a or GLP, we observed significant distortions in the CD spectrum (), implying that both enzymes became partially unfolded.