Fanny L. Cherblanc

Chaetocin is a nonspecific inhibitor of histone lysine methyltransferases.

To the Editor—Histone lysine methyltransferases (HKMTs) are important epigenetic enzymes1 and exciting new druggable targets for drug discovery2. In a Brief Communication published in Nature Chemical Biology, Greiner et al.3 identified the fungal metabolite chaetocin (1, Fig. 1a) as the first specific inhibitor of histone lysine methyltransferase SU(VAR)3-9 in Drosophila melanogaster and concluded that (i) chaetocin is a ‘specific’ inhibitor of the H3K9 HKMT SU(VAR)3-9, on the basis of a comparison of the half-maximum inhibitory concentration (IC50) values for a range of HKMTs; (ii) it is competitive with the cofactor S-adenosyl methionine (SAM); and (iii) the bridging disulfide bond of the epipolythiodioxopiperazine unit (or units) is not required for HKMT inhibitory activity. On the contrary, we have found that chaetocin inhibits the protein in a time-dependent and nonspecific manner, most likely through chemical modification of the enzyme by the disulfide group. We conclude that chaetocin, or related natural products, are not fit for application as selective chemical probes of HKMT function. Chaetocin is a member of a large family of fungal toxins, which all bear the epipolythiodioxopiperazine (ETP) ‘warhead’4. Chaetocin has further been shown to affect a number of structurally and functionally unrelated proteins, with compound activity dependent on the disulfide functionality5,6. We therefore questioned whether the molecule could serve as a specific enzyme inhibitor. We tested the dependence of chaetocin’s HKMT activity on its ETP warhead. Chaetocin (1)7 was indeed found to have in vitro inhibitory activity (IC50 = 0.11 mM; Fig. 1b) on purified human SUV39H1, in agreement with the data of Greiner et al.3 (IC50 = 0.8 mM). However, a close structural analog, in which the disulfide had been converted to the chemically unreactive bisthioether (2), was inactive. This result mirrors a recent report that a chaetocin analog lacking the disulfide was inactive against the related HKMT G9a8. Additionally, we found a structurally simple ETP model compound (3) to have considerable inhibitory activity (IC50 = 3.2 mM), suggesting that the disulfide functionality was the principal feature required for SUV39H1 inhibition. We found inhibition by chaetocin to be time dependent, with potency increasing with longer preincubation times (Fig. 1c), and enzyme concentration dependent (Fig. 1d). We further conducted SAM-competition experiments to define the mechanism of action. In opposition to the published results3, we found no dependence on [SAM]/Km (Fig. 1e,f), demonstrating that inhibition by chaetocin is not prevented by SAM. Taken together, these data suggest that the disulfide bridge of the ETP unit is central to chaetocin’s HKMT inhibitory activity by a nonspecific mechanism. Indeed, it has recently been reported that other ETP natural products can also inhibit HKMTs9, Chaetocin is a nonspecific inhibitor of histone lysine methyltransferases

On the Histone Lysine Methyltransferase Activity of Fungal Metabolite Chaetocin

Histone lysine methyltransferases (HKMTs) are an important class of targets for epigenetic therapy. 1 (chaetocin), an epidithiodiketopiperazine (ETP) natural product, has been reported to be a specific inhibitor of the SU(VAR)3-9 class of HKMTs. We have studied the inhibition of the HKMT G9a by 1 and functionally related analogues. Our results reveal that only the structurally unique ETP core is required for inhibition, and such inhibition is time-dependent and irreversible (in the absence of DTT), ultimately resulting in protein denaturation. Mass spectrometric data provide a molecular basis for this effect, demonstrating covalent adduct formation between 1 and the protein. This provides a potential rationale for the selectivity observed in the inhibition of a variety of HKMTs by 1 in vitro and has implications for the activity of ETPs against these important epigenetic targets.

On the determination of the stereochemistry of semisynthetic natural product analogues using chiroptical spectroscopy: desulfurization of epidithiodioxopiperazine fungal metabolites.

Isolation and semisynthetic modification of the fungal metabolite chaetocin gave access to a desulfurized analogue of this natural product. Detailed chiroptical studies, comparing experimentally obtained optical rotation values, electronic circular dichroism spectra, and vibrational circular dichroism spectra to computationally simulated ones, reveal the desulfurization of chaetocin to unambiguously proceed with retention of configuration. Consideration of the plausible mechanisms for this process highlighted inconsistencies in the stereochemical assignment of related molecules in the literature. This in turn allowed the stereochemical reassignment of the natural product analogue dethiodehydrogliotoxin.

Current limitations and future opportunities for epigenetic therapies

This article reviews progress in epigenetic therapies that hope to improve the treatment of cancer. Tumors show widespread, aberrant epigenetic changes, leading to changes in the expression of genes involved in all the hallmarks of cancer. These epigenetic changes can potentially be reversed using small-molecule inhibitors of enzymes involved in maintenance of the epigenetic state. DNA-demethylating agents and histone deacetylase inhibitors have shown anti-tumor activity against certain hematological malignancies; however, their activity in solid tumors remains more uncertain. Major challenges remain in delivery of epigenetic therapy, maintenance of a pharmacodynamic response and achievement of a therapeutic index. We believe histone lysine methyl transferases are a highly promising epigenetic target, which has yet to be clinically exploited. Crystallographic studies on histone lysine methyl transferases provide insights into their mechanism and specificity crucial for the design and development of small-molecule inhibitors.

Dual EZH2 and EHMT2 histone methyltransferase inhibition increases biological efficacy in breast cancer cells

BackgroundMany cancers show aberrant silencing of gene expression and overexpression of histone methyltransferases. The histone methyltransferases (HKMT) EZH2 and EHMT2 maintain the repressive chromatin histone methylation marks H3K27me and H3K9me, respectively, which are associated with transcriptional silencing. Although selective HKMT inhibitors reduce levels of individual repressive marks, removal of H3K27me3 by specific EZH2 inhibitors, for instance, may not be sufficient for inducing the expression of genes with multiple repressive marks.ResultsWe report that gene expression and inhibition of triple negative breast cancer cell growth (MDA-MB-231) are markedly increased when targeting both EZH2 and EHMT2, either by siRNA knockdown or pharmacological inhibition, rather than either enzyme independently. Indeed, expression of certain genes is only induced upon dual inhibition. We sought to identify compounds which showed evidence of dual EZH2 and EHMT2 inhibition. Using a cell-based assay, based on the substrate competitive EHMT2 inhibitor BIX01294, we have identified proof-of-concept compounds that induce re-expression of a subset of genes consistent with dual HKMT inhibition. Chromatin immunoprecipitation verified a decrease in silencing marks and an increase in permissive marks at the promoter and transcription start site of re-expressed genes, while Western analysis showed reduction in global levels of H3K27me3 and H3K9me3. The compounds inhibit growth in a panel of breast cancer and lymphoma cell lines with low to sub-micromolar IC50s. Biochemically, the compounds are substrate competitive inhibitors against both EZH2 and EHMT1/2.ConclusionsWe have demonstrated that dual inhibition of EZH2 and EHMT2 is more effective at eliciting biological responses of gene transcription and cancer cell growth inhibition compared to inhibition of single HKMTs, and we report the first dual EZH2-EHMT1/2 substrate competitive inhibitors that are functional in cells.

Mechanistic and chiroptical studies on the desulfurization of epidithiodioxopiperazines reveal universal retention of configuration at the bridgehead carbon atoms.

The stereochemistry of the desulfurization products of chiral natural and synthetic 3,6-epidithiodiketopiperazines (ETPs) is specified inconsistently in the literature. Qualitative mechanisms have been put forward to explain apparently divergent stereochemical pathways, but the quantitative feasibility of such mechanistic pathways has not been assessed. We report a computational study revealing that desulfurization of ETPs should occur universally with retention of configuration. While the majority of stereochemically assigned and reassigned cases fit this model, until now desulfurization of the synthetic gliotoxin analogue shown has remained assigned as proceeding via inversion of configuration. Through detailed chiroptical studies comparing experimentally obtained optical rotation values, electronic circular dichroism spectra, and vibrational circular dichroism spectra to their computationally simulated counterparts as well as chemical derivatization studies, we have unambiguously demonstrated that contrary to its current assignment in the literature, the desulfurization of this synthetic ETP also proceeds with retention of configuration.

Chiroptical studies on brevianamide B: Vibrational and Electronic Circular Dichroism confronted

Chiroptical spectroscopies, such as electronic circular dichroism (ECD) and vibrational circular dichroism (VCD), are highly sensitive techniques to probe molecular conformation, configuration, solvation, and aggregation. Here we report the application of these techniques to study the fungal metabolite brevianamide B. Comparison of the experimental ECD and VCD spectra with the density functional theory simulated counterparts establishes that VCD is the more reliable technique to assign absolute configuration due to the larger functional and dispersion dependence of computed ECD spectra. Despite a low amount of available material and a relatively unusual example of using VCD carbonyl multiplets, the absolute configuration could be reliably predicted, strengthening the case for application of VCD in the study of complex natural products. Spectral and crystallographic evidence for or against the formation of a dimeric aggregate is discussed; in solution, the VCD spectra strongly suggest only monomeric species are present.

Abstract A162: Targeting chromatin: Small molecules reactivating H3K27me3 silenced genes.

There is a need for the development of compounds allowing more targeted and efficient reversal of aberrant epigenetic silencing in cancer. Recently, the discovery of histone lysine methyltransferases (HKMT) being involved in tumorgenesis as well as tumor stem cell like maintenance has sparked interest in this particular class of enzymes. We have set-up a cell based assay (MDA-MB-231) in order to identify small molecule inhibitors which are able to re-express endogenous genes where EZH2 has been shown to be involved in the silencing process. In order to identify compounds which inhibit a specific chromatin remodeler class (silencing of chromatin), we chose two DNA unmethylated EZH2 target genes (KRT17, FBXO32), which we would be expect to be re-expressed and one DNA methylated EZH2 target gene (RUNX3) which we would expect to be unaffected in case of an HKMT inhibitor. SiRNA to EZH2 increases expression of KRT17 and FBXO32, but not RUNX3. We have assayed compounds chemically related to a known HKMT inhibitor and identified compounds that up-regulate KRT17 as well as FBXO32 but fail to act on RUNX3. These compounds inhibit tumor cell growth in the low microMolar range. Chromatin immunoprecipitation (ChIP) experiments verified a decrease in silencing marks (H3K27me3, H3K9me3) and importantly an increase in active chromatin marks (e.g. H3K4me2, H3K4me3) at the promoter region of KRT17 and FBXO32. Of note, the H3K27me3 demethylase JMJD3 also showed increased binding at the promoter region which correlated with the presence of H3K4me2/3. In conclusion, we have identified compounds that induce re-expression of genes, reverse H3K27me3 mediated gene silencing and induce inhibition of tumor cell growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A162.

Abstract B165: Structural and biological studies on analogues of the natural product histone methyltransferase inhibitor chaetocin.

Epigenetic mechanisms play a critical role in the regulation of gene expression, and how such regulation becomes aberrant in cancer. Along with DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), histone methyltransferases (HKMTs) have recently appeared as new promising drug targets for epigenetic cancer therapies. While several HDAC and DNMT inhibitors have been clinically validated, to date only few HKMT inhibitors have been reported. Chaetocin, a fungal metabolite belonging to the 3,6-epidithio-diketopiperazine (ETP) class was first described as a specific inhibitor of the histone methyltransferase SU(VAR)3–9.(1) Like other fungal metabolites of the ETP class however, it exhibits a broad range of antibacterial and cytostatic activity, including a remarkable cytotoxicity against HeLa cells. Since the broad cytotoxicity of the ETPs is due to the presence of the disulfide bridge of the 3,6-epidithio-diketopiperazine (causing protein crosslinking, reactive oxygen species generation, or zinc chelation), we reasoned that access to semi-synthetic derivatives devoid of such functionality, would provide valuable insight into the chemical biology of this natural product, particularly its reported HKMT activity. We developed conditions to extract and purify the natural product culture (Chaetomium virescens var. thielavioideum), followed several semi-synthetic approaches to obtain chaetocin analogues. Comparison of experimental and computationally simulated chiroptical spectroscopy allowed unambiguous structural characterization of our products. Evaluation of our compounds in a number of in vitro HKMT assays revealed a total loss in inhibitory potency upon modification of the disulfide bridge. These studies thus demonstrate the ETP functionality of chaetocin to be responsible for its reported HKMT activity. In conclusion, we have synthetically prepared distinct and novel chaetocin analogues from the natural product and explored their HKMT activity, implicating the ETP functionality as the active pharmacophore. Reference : 1. Greiner, D.; Bonaldi, T.; Eskeland, R.; Roemer, E.; Imhof, A. Nat. Chem. Biol. 2005, 1, 143. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B165.