Total Synthesis of (+)-Chaetocin and its Analogues: Their Histone Methyltransferase G9a Inhibitory Activity

Iwasa, Eriko; Hamashima, Yoshitaka; Fujishiro, Shinya; Higuchi, Eisuke; Ito, Akihiro; Yoshida, Minoru; Sodeoka, Mikiko

doi:10.1021/ja101280p

Cited by 218 publications

(118 citation statements)

References 24 publications

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“…16 From this screen, we identified gliotoxin, an ETP secondary metabolite produced by fungal pathogens, as a G9a inhibitor (Figure 1a). To examine the inhibitory activity of gliotoxin against G9a in detail, we performed in vitro HMT assay by western blotting.…”

Section: In Vitro Hmt Assaymentioning

confidence: 99%

“…This is also consistent with the previous report of the activity of synthetic analogs of chaetocin lacking disulfide. 16 Because G9a and Suv39h1, but not Set7/9, possess pre-and post-SET domains, which bind zinc atoms with their cysteine-rich regions, 2 ETPs might interact with these domains through their own sulfur-containing functional groups.…”

Section: In Vitro Hmt Assaymentioning

confidence: 99%

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Inhibition of histone H3K9 methyltransferases by gliotoxin and related epipolythiodioxopiperazines

et al. 2012

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Histone lysine methyltransferases (HMTs) regulate transcriptional activity by writing epigenetic marks. Methylation of histone H3K9, a hallmark of silent chromatin, 1 is mainly regulated by two subgroups of HMTs, G9a/G9a-like protein 2 and Suv39h. 3 G9a and G9a-like protein induce mono-and di-methylation of histone H3K9 (H3K9me1 and H3K9me2) in the euchromatin region, 4 whereas Suv39h contributes to tri-methylation of histone H3K9 (H3K9me3) in the heterochromatin. 3 As methylation at histone H3K9, increased DNA methylation and reduced levels of activating chromatin modifications (e.g., histone acetylation) have been detected at promoter regions of aberrantly silenced tumor suppressor genes in cancer cells, 5,6 HMTs responsible for histone H3K9 methylation may represent promising targets for drug discovery. Indeed, overexpression of G9a is associated with several types of cancers and downregulation of G9a by RNAi inhibits tumor cell proliferation. 7-9 Chaetocin and BIX-01294 are small molecules that inhibit histone H3K9 HMTs. Chaetocin, which inhibit both G9a and Suv39h activities, is a member of the epipolythiodioxopiperazine (ETP) class of fungal metabolites. 10 On the other hand, BIX-01294 is a synthetic compound that selectively inhibits G9a but not Suv39h1 11 Based on a co-crystallization analysis of G9a with BIX-01294, BIX-01294-related molecules have been developed; these novel compounds are both more potent inhibitors and more membrane permeable than the parent compound. 12-15 MATERIALS AND METHODS MaterialsAnti-mono-and di-methylated histone H3K9 antibodies were purchased from Merck Millipore (Billerica, MA, USA). pGEX4T-1-mG9a (706Àstop amino acid (a.a.)), pGEX4T-3-histone H3 (1À57 a.a.) and pGEX4T-3-mSuv39h1-H320R (74À412 a.a.) were previously described. 2 The pET-28a(+)-Set7/9 was kindly provided by Dr Kenichi Nishioka (National Institute of Genetics). Bacterial protein expression and purificationpGEX4T-1-mG9a (706Àstop a.a.), pGEX4T-3-mSuv39h1-H320R (74À412 a.a.), pET-28a(+)-Set7/9, or pGEX4T-3-histone H3 (1À57 a.a.) was introduced into Escherichia coli BL21 (DE3). Expression of each recombinant protein was induced by 0.1 mM isopropyl-b-D-galactopyranosid at 18 1C for 24 h. Purification of GST-and (His) 6 -fused proteins were carried out by either glutathioneaffinity (GE Healthcare UK Ltd, Little Chalfont, UK) or nickel (Ni 2+ ) affinity (GE Healthcare UK Ltd). In vitro HMT assayEach purified recombinant HMT was pretreated in the presence or absence of a given compound in HMT buffer (50 mM Tris-HCl (pH 8.5), 10 mM MgCl 2 , 20 mM KCl, 10 mM 2-mercaptoethanol and 250 mM sucrose) containing 3 mg ml À1 of BSA for 1 h. Next, 10 mg ml À1 of SAM and 20 mg ml À1 of GST-fused histone H3 (1À57 a.a.) were added into the reaction mixture and further incubated for 1 h at 37 1C. To detect histone methylation on western blots, samples were transferred onto Immobilon membranes (Merck Millipore), probed using appropriate primary antibodies, and visualized using horseradish peroxidase-linked secondary antibodies.In or...

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Section: In Vitro Hmt Assaymentioning

confidence: 99%

Section: In Vitro Hmt Assaymentioning

confidence: 99%

Inhibition of histone H3K9 methyltransferases by gliotoxin and related epipolythiodioxopiperazines

et al. 2012

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“…To target other classes of chromatin-modifying enzymes, compounds were chosen that have mostly been previously described: as HKMT inhibitors, we used chaetocin (16), a nonspecific compound originally described as selective for SU(VAR)3-9 and later shown to inhibit G9a (17) and thioredoxin reductase (18); BIX-01294 (19,20), a G9a/GLP-specific compound, and its active analog BRD-K62233722. DNMT inhibitors were 5-azadeoxycytidine (decitabine) (21), zebularine (22), and RG-0108 (23).…”

Section: Resultsmentioning

confidence: 99%

Chromatin-targeting small molecules cause class-specific transcriptional changes in pancreatic endocrine cells

Kubicek

Gilbert²,

Fomina-Yadlin³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Under the instruction of cell-fate-determining, DNA-binding transcription factors, chromatin-modifying enzymes mediate and maintain cell states throughout development in multicellular organisms. Currently, small molecules modulating the activity of several classes of chromatin-modifying enzymes are available, including clinically approved histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors. We describe the genome-wide expression changes induced by 29 compounds targeting HDACs, DNMTs, histone lysine methyltransferases (HKMTs), and protein arginine methyltransferases (PRMTs) in pancreatic α-and β-cell lines. HDAC inhibitors regulate several hundred transcripts irrespective of the cell type, with distinct clusters of dissimilar activity for hydroxamic acids and orthoamino anilides. In contrast, compounds targeting histone methyltransferases modulate the expression of restricted gene sets in distinct cell types. For example, we find that G9a/GLP methyltransferase inhibitors selectively up-regulate the cholesterol biosynthetic pathway in pancreatic but not liver cells. These data suggest that, despite their conservation across the entire genome and in different cell types, chromatin pathways can be targeted to modulate the expression of selected transcripts.histone modification | gene regulation | chemical epigenetics | beta cell biology | cholesterol pathway E pigenetic mechanisms mediated through chromatin control cell-state and cell-type decisions during development and in the adult (1, 2). Chromatin-modifying enzymes have been shown to function by interacting with master transcription factors, regulating the expression of key target genes and conferring epigenetic memory through the propagation of modifications to the chromatin template. These modifications include methylation of the DNA itself and a variety of posttranslational modifications to histones, the proteins most closely interacting with DNA. Depending on the type of modification, e.g., acetylation or methylation, and the exact position of the modified amino acid, these modifications can activate or repress transcription of the underlying DNA sequence (3).Small molecules targeting chromatin have mainly been developed for the treatment of cancer, justified by the identification of genetic aberrations leading to overexpression or activation of several chromatin-modifying enzymes (4-7). In contrast to clinically approved HDAC and DNMT inhibitors, fewer compounds targeting histone lysine methyltransferases (HKMTs) and protein arginine methyltransferases (PRMTs) are available. These small molecules are mainly used as chemical probe compounds in basic research, and toxicity is often limiting for testing in animal models. Interestingly, for most of the 50 HKMTs and 30 histone demethylases encoded in the human genome, no specific compounds are available. Even for HDACs, most compounds inhibit HDACs 1, 2, 3, and 6 (8), and HDAC8-specific compounds are only now emerging (9).Chromatin-modifying enzymes play important roles in normal development,...

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“…It was selective over non-H3K9 PKMTs, such as H3K27 PKMT dE(z) complex, H3K4 PKMT SET7/9, and H4K20 PKMT SETD8 [67] (IC 50 dE(z) complex.90 mM; SET7/9 and SETD8.180 mM). Interestingly, a total synthesis report found natural (þ)-and synthetic (2)-chaetocin to be equipotent against G9a (IC 50 ¼ 2.4 and 1.7 mM, respectively) [69]. In addition, it was found that chaetocin inhibited thioredoxin reductase [70].…”

Section: Pkmt Inhibitorsmentioning

confidence: 99%

Targeting protein lysine methylation and demethylation in cancers

He¹,

Korboukh²,

Jin³

et al. 2012

ABBS

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During the last decade, we saw an explosion of studies investigating the role of lysine methylation/demethylation of histones and non-histone proteins, such as p53, NF-kappaB, and E2F1. These 'Ying-Yang' post-translational modifications are important to fine-tuning the activity of these proteins. Lysine methylation and demethylation are catalyzed by protein lysine methyltransferases (PKMTs) and protein lysine demethylases (PKDMs). PKMTs, PKDMs, and their substrates have been shown to play important roles in cancers. Although the underlying mechanisms of tumorigenesis are still largely unknown, growing evidence is starting to link aberrant regulation of methylation to tumorigenesis. This review focuses on summarizing the recent progress in understanding of the function of protein lysine methylation, and in the discovery of small molecule inhibitors for PKMTs and PKDMs. We also discuss the potential and the caveats of targeting protein lysine methylation for the treatment of cancer.

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Total Synthesis of (+)-Chaetocin and its Analogues: Their Histone Methyltransferase G9a Inhibitory Activity

Cited by 218 publications

References 24 publications

Inhibition of histone H3K9 methyltransferases by gliotoxin and related epipolythiodioxopiperazines

Inhibition of histone H3K9 methyltransferases by gliotoxin and related epipolythiodioxopiperazines

Chromatin-targeting small molecules cause class-specific transcriptional changes in pancreatic endocrine cells

Targeting protein lysine methylation and demethylation in cancers

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