DAXX safeguards heterochromatin formation in embryonic stem cells.

Genomes comprise a large fraction of repetitive sequences folded into constitutive heterochromatin, which protect genome integrity and cell identity. De novo formation of heterochromatin during preimplantation development is an essential step for preserving the ground-state of pluripotency and the self-renewal capacity of embryonic stem cells (ESCs). However, the molecular mechanisms responsible for the remodeling of constitutive heterochromatin are largely unknown. Here, we identify that DAXX, an H3.3 chaperone essential for the maintenance of mouse ESCs in the ground state, accumulates in pericentromeric regions independently of DNA methylation. DAXX recruits PML and SETDB1 to promote the formation of heterochromatin, forming foci that are hallmarks of ground-state ESCs. In the absence of DAXX or PML, the three-dimensional (3D) architecture and physical properties of pericentric and peripheral heterochromatin are disrupted, resulting in de-repression of major satellite DNA, transposable elements and genes associated with the nuclear lamina. Using epigenome editing tools, we observe that H3.3, and specifically H3.3K9 modification, directly contribute to maintaining pericentromeric chromatin conformation. Altogether, our data reveal that DAXX is crucial for the maintenance and 3D organization of the heterochromatin compartment and protects ESC viability.

A genetic screen identifies BEND3 as a regulator of bivalent gene expression and global DNA methylation.

Epigenetic mechanisms are essential to establish and safeguard cellular identities in mammals. They dynamically regulate the expression of genes, transposable elements and higher-order chromatin structures. Consequently, these chromatin marks are indispensable for mammalian development and alterations often lead to disease, such as cancer. Bivalent promoters are especially important during differentiation and development. Here we used a genetic screen to identify new regulators of a bivalent repressed gene. We identify BEND3 as a regulator of hundreds of bivalent promoters, some of which it represses, and some of which it activates. We show that BEND3 is recruited to a CpG-containg consensus site that is present in multiple copies in many bivalent promoters. Besides having direct effect on the promoters it binds, the loss of BEND3 leads to genome-wide gains of DNA methylation, which are especially marked at regions normally protected by the TET enzymes. DNA hydroxymethylation is reduced in Bend3 mutant cells, possibly as consequence of altered gene expression leading to diminished alpha-ketoglutarate production, thus lowering TET activity. Our results clarify the direct and indirect roles of an important chromatin regulator, BEND3, and, more broadly, they shed light on the regulation of bivalent promoters.

Advances in Chemical Probing Concepts for Chemical Biology Applications.

Chemical probes allow us to identify, validate and confirm novel targets for therapeutic applications, enable the development of drug candidates, and open the way to new therapeutic strategies, vaccines and diagnostic tools.

EFMC: Trends in Medicinal Chemistry and Chemical Biology.

Ground-breaking research in disease biology and continuous efforts in method development have uncovered a range of potential new drug targets. Increasingly, the drug discovery process is informed by technologies involving chemical probes as tools. Applications for chemical probes comprise target identification and assessment, as well as the qualification of small molecules as chemical starting points and drug candidates. Progress in probe chemistry has opened the way to novel assay formats and pharmaceutical compound classes. The European Federation of Medicinal Chemistry and Chemical Biology (EFMC) has launched the Chemical Biology Initiative to advance science in the field of medicinal chemistry and chemical biology, while representing all members of this extended scientific community. This review provides an overview of the many important developments in the field of chemical biology that have happened at the lively interface of academic and industrial research.

Inhibitors of DNA Methylation.

DNA methylation is involved in numerous biological processes and is deregulated in human diseases. The modulation of the activity of the enzymes and proteins in charge of DNA methylation, for example, DNA methyltransferases (DNMTs), can represent a powerful strategy to alter DNA methylation patterns and restore biological processes that are aberrant in diseases. In this chapter, we present examples of inhibitors of DNMTs (DNMTi). We review their fields of application either as therapeutic molecules, for example, in cancers, cardiovascular, neurological, and infectious diseases or as bioengineering tools. Finally, novel strategies to target DNA methylation and overcome the limits of single DNMT inhibitors will be described. These strategies consist in either targeting the methyl group reader proteins rather than targeting directly DNMTs or to combine within the same molecule a DNMT inhibitor with an additional active moiety, e.g., HDAC inhibitor, to improve efficacy and lower secondary effect of such drug.

Synthesis and Biological Activity of a Cytostatic Inhibitor of MLLr Leukemia Targeting the DOT1L Protein.

Histone methyltransferase DOT1L catalyzes mono-, di- and trimethylation of histone 3 at lysine residue 79 (H3K79) and hypermethylation of H3K79 has been linked to the development of acute leukemias characterized by the MLL (mixed-lineage leukemia) rearrangements (MLLr cells). The inhibition of H3K79 methylation inhibits MLLr cells proliferation, and an inhibitor specific for DOT1L, pinometostat, was in clinical trials (Phase Ib/II). However, the compound showed poor pharmacological properties. Thus, there is a need to find new potent inhibitors of DOT1L for the treatment of rearranged leukemias. Here we present the design, synthesis, and biological evaluation of a small molecule that inhibits in the nM level the enzymatic activity of hDOT1L, H3K79 methylation in MLLr cells with comparable potency to pinometostat, associated with improved metabolic stability and a characteristic cytostatic effect.

The DNMT3A R882H mutant displays altered flanking sequence preferences.

The DNMT3A R882H mutation is frequently observed in acute myeloid leukemia (AML). It is located in the subunit and DNA binding interface of DNMT3A and has been reported to cause a reduction in activity and dominant negative effects. We investigated the mechanistic consequences of the R882H mutation on DNMT3A showing a roughly 40% reduction in overall DNA methylation activity. Biochemical assays demonstrated that R882H does not change DNA binding affinity, protein stability or subnuclear distribution of DNMT3A. Strikingly, DNA methylation experiments revealed pronounced changes in the flanking sequence preference of the DNMT3A-R882H mutant. Based on these results, different DNA substrates with selected flanking sequences were designed to be favored or disfavored by R882H. Kinetic analyses showed that the R882H favored substrate was methylated by R882H with 45% increased rate when compared with wildtype DNMT3A, while methylation of the disfavored substrate was reduced 7-fold. Our data expand the model of the potential carcinogenic effect of the R882H mutation by showing CpG site specific activity changes. This result suggests that R882 is involved in the indirect readout of flanking sequence preferences of DNMT3A and it may explain the particular enrichment of the R882H mutation in cancer patients by revealing mutation specific effects.

Assembly of the Entire Carbon Backbone of a Stereoisomer of the Antitumor Marine Natural Product Hemicalide.

A strategy for the assembly of the entire carbon backbone of a stereoisomer of the antitumor marine natural product hemicalide has been investigated. The devised convergent approach relies on Horner-Wadsworth-Emmons and Julia-Kocienski olefination reactions for the construction of the C6=C7 and C34=C35 double bonds, respectively, an aldol reaction to create the C27-C28 bond, and a Suzuki-Miyaura cross-coupling as the endgame to form the C15-C16 bond.

Natural Products and Chemical Biology Tools: Alternatives to Target Epigenetic Mechanisms in Cancers.

DNA methylation and histone acetylation are widely studied epigenetic modifications. They are involved in numerous pathologies such as cancer, neurological disease, inflammation, obesity, etc. Since the discovery of the epigenome, numerous compounds have been developed to reverse DNA methylation and histone acetylation aberrant profile in diseases. Among them several were inspired by Nature and have a great interest as therapeutic molecules. In the quest of finding new ways to target epigenetic mechanisms, the use of chemical tools is a powerful strategy to better understand epigenetic mechanisms in biological systems. In this review we will present natural products reported as DNMT or HDAC inhibitors for anticancer treatments. We will then discuss the use of chemical tools that have been used in order to explore the epigenome.

Disruptor of telomeric silencing 1-like (DOT1L): disclosing a new class of non-nucleoside inhibitors by means of ligand-based and structure-based approaches.

Chemical inhibition of chromatin-mediated signaling involved proteins is an established strategy to drive expression networks and alter disease progression. Protein methyltransferases are among the most studied proteins in epigenetics and, in particular, disruptor of telomeric silencing 1-like (DOT1L) lysine methyltransferase plays a key role in MLL-rearranged acute leukemia Selective inhibition of DOT1L is an established attractive strategy to breakdown aberrant H3K79 methylation and thus overexpression of leukemia genes, and leukemogenesis. Although numerous DOT1L inhibitors have been several structural data published no pronounced computational efforts have been yet reported. In these studies a first tentative of multi-stage and LB/SB combined approach is reported in order to maximize the use of available data. Using co-crystallized ligand/DOT1L complexes, predictive 3-D QSAR and COMBINE models were built through a python implementation of previously reported methodologies. The models, validated by either modeled or experimental external test sets, proved to have good predictive abilities. The application of these models to an internal library led to the selection of two unreported compounds that were found able to inhibit DOT1L at micromolar level. To the best of our knowledge this is the first report of quantitative LB and SB DOT1L inhibitors models and their application to disclose new potential epigenetic modulators.

New insights on the mechanism of quinoline-based DNA Methyltransferase inhibitors.

Among the epigenetic marks, DNA methylation is one of the most studied. It is highly deregulated in numerous diseases, including cancer. Indeed, it has been shown that hypermethylation of tumor suppressor genes promoters is a common feature of cancer cells. Because DNA methylation is reversible, the DNA methyltransferases (DNMTs), responsible for this epigenetic mark, are considered promising therapeutic targets. Several molecules have been identified as DNMT inhibitors and, among the non-nucleoside inhibitors, 4-aminoquinoline-based inhibitors, such as SGI-1027 and its analogs, showed potent inhibitory activity. Here we characterized the in vitro mechanism of action of SGI-1027 and two analogs. Enzymatic competition studies with the DNA substrate and the methyl donor cofactor, S-adenosyl-l-methionine (AdoMet), displayed AdoMet non-competitive and DNA competitive behavior. In addition, deviations from the Michaelis-Menten model in DNA competition experiments suggested an interaction with DNA. Thus their ability to interact with DNA was established; although SGI-1027 was a weak DNA ligand, analog 5, the most potent inhibitor, strongly interacted with DNA. Finally, as 5 interacted with DNMT only when the DNA duplex was present, we hypothesize that this class of chemical compounds inhibit DNMTs by interacting with the DNA substrate.

DNA Methyltransferase Inhibitors: Development and Applications.

As described in previous chapters of this book, DNA methylation is involved in numerous biological processes, and modulation of the activity of DNA methyltransferases (DNMTs) is a powerful strategy to modulate, restore, or reduce DNA methylation. In this chapter, we will present examples of inhibitors of DNMTs (DNMTi) and review the fields of applications of DNMTi mainly as therapeutic molecules, for example, in cancers, cardiovascular or neurological diseases, but also as bioengineering tools. Finally, the limits of currently available inhibitors will be discussed and the perspectives to discover improved DNMTi will be presented.

Design and synthesis of new non nucleoside inhibitors of DNMT3A.

DNA methylation, an epigenetic modification regulating gene expression, is a promising target in cancer. In an effort to identify new non nucleosidic inhibitors of DNA methyltransferases, the enzymes responsible for DNA methylation, we carried out a high-throughput screening of 66,000 chemical compounds based on an enzymatic assay against catalytic DNMT3A. A family of propiophenone derivatives was identified. After chemical optimization and structure activity relationship studies, a new inhibitor (33) was obtained with an EC50 of 2.1 µM against DNMT3A. The mechanism of inhibition of the compound was investigated as it forms a reactive Michael acceptor group in situ. Thereby, the Michael acceptor 20 was identified. This compound was further characterized for its biological activity in cancer cells.

Development of a universal radioactive DNA methyltransferase inhibition test for high-throughput screening and mechanistic studies.

DNA methylation is an important epigenetic mark in eukaryotes, and aberrant pattern of this modification is involved in numerous diseases such as cancers. Interestingly, DNA methylation is reversible and thus is considered a promising therapeutic target. Therefore, there is a need for identifying new small inhibitors of C5 DNA methyltransferases (DNMTs). Despite the development of numerous in vitro DNMT assays, there is a lack of reliable tests suitable for high-throughput screening, which can also give insights into inhibitor mechanisms of action. We developed a new test based on scintillation proximity assay meeting these requirements. After optimizing our assay on human DNMT1 and calibrating it with two known inhibitors, we carried out S-Adenosyl-l-Methionine and DNA competition studies on three inhibitors and were able to determine each mechanism of action. Finally, we showed that our test was applicable to 3 other methyltransferases sources: human DNMT3A, bacterial M.SssI and cellular extracts as well.

DNA methylation associated with polycomb repression in retinoic acid receptor ß silencing.

Retinoic acid receptor ß 2 (RARß2) is a tumor suppressor gene whose loss of expression is recurrent in prostate cancers. Here we studied the epigenetic mechanisms leading to its stable silencing. First, we characterized all RARß isoforms in 6 human tumor cell lines (prostate DU145, LNCaP, PC3, lung A549, breast Hs578T, and colon HCT116) by RT-PCR and Western blot. We excluded loss of heterozygosity (2D-FISH) and loss of RARa expression, an upstream regulator, as origin of RARß2 silencing. All data concluded to an epigenetic silencing. In agreement, a DNA methylation inhibitor restored its expression. Second RARß2 loss of expression was found associated with different epigenetic profiles in LNCaP and DU145 cells. According to bisulfite sequencing and ChIP analysis, we observed heavy methylation (97%) of the RARß2 promoter with repressive histone mark H3K9me3 in LNCaP. While DNA methylation and polycomb repression are described to be mutually exclusive at CpG-rich promoters, we observed that in DU145, moderate DNA methylation (36%) and H3K9me3 mark were present concomitantly with H3K27me3, a signature of polycomb repression. In summary, we provide new insights on how the RARß2 promoter is silenced, reveal the existence of two distinct repressive chromatin profiles at the same locus, and support a polycomb-mediated epigenetic repression process in prostate cancer.

Non-canonical functions of UHRF1 maintain DNA methylation homeostasis in cancer cells.

DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It is yet unclear if UHRF1 functions solely by stimulating DNA methylation maintenance by DNMT1, or if it has important additional functions. Using degron alleles, we show that UHRF1 depletion causes a much greater loss of DNA methylation than DNMT1 depletion. This is not caused by passive demethylation as UHRF1-depleted cells proliferate more slowly than DNMT1-depleted cells. Instead, bioinformatics, proteomics and genetics experiments establish that UHRF1, besides activating DNMT1, interacts with DNMT3A and DNMT3B and promotes their activity. In addition, we show that UHRF1 antagonizes active DNA demethylation by TET2. Therefore, UHRF1 has non-canonical roles that contribute importantly to DNA methylation homeostasis; these findings have practical implications for epigenetics in health and disease.

Correction: Zwergel et al. Novel Quinoline Compounds Active in Cancer Cells through Coupled DNA Methyltransferase Inhibition and Degradation. Cancers 2020, 12, 447.

In the original publication [...].

To target or not to target? The role of DNA and histone methylation in bacterial infections.

Epigenetics describes chemical modifications of the genome that do not alter DNA sequence but participate in the regulation of gene expression and cellular processes such as proliferation, division, and differentiation of eukaryotic cell. Disruption of the epigenome pattern in a human cell is associated with different diseases, including infectious diseases. During infection pathogens induce epigenetic modifications in the host cell. This can occur by controlling expression of genes involved in immune response. That enables bacterial survival and replication within the host and evasion of the immune response. Methylation is an example of epigenetic modification that occurs on DNA and histones. Reasoning that DNA and histone methylation of human host cells plays a crucial role during pathogenesis, these modifications are promising targets for the development of alternative treatment strategies in infectious diseases. Here, we discuss the role of DNA and histone methyltransferases in human host cell upon bacterial infections. We further hypothesize that compounds targeting methyltransferases are tools to study epigenetics in the context of host-pathogen interactions and can open new avenues for the treatment of bacterial infections.

Plasmodium falciparum Eukaryotic Translation Initiation Factor 3 is Stabilized by Quinazoline-Quinoline Bisubstrate Inhibitors.

Malaria drug resistance is hampering the fight against the deadliest parasitic disease affecting over 200 million people worldwide. We recently developed quinoline-quinazoline-based inhibitors (as compound 70) as promising new antimalarials. Here, we aimed to investigate their mode of action by using thermal proteome profiling (TPP). The eukaryotic translation initiation factor 3 (EIF3i) subunit I was identified as the main target protein stabilized by compound 70 in Plasmodium falciparum. This protein has never been characterized in malaria parasites. P. falciparum parasite lines were generated expressing either a HA tag or an inducible knockdown of the PfEIF3i gene to further characterize the target protein. PfEIF3i was stabilized in the presence of compound 70 in a cellular thermal shift Western blot assay, pointing that PfEIF3i indeed interacts with quinoline-quinazoline-based inhibitors. In addition, PfEIF3i-inducible knockdown blocks intra-erythrocytic development in the trophozoite stage, indicating that it has a vital function. We show that PfEIF3i is mostly expressed in late intra-erythrocytic stages and localizes in the cytoplasm. Previous mass spectrometry reports show that PfEIF3i is expressed in all parasite life cycle stages. Further studies will explore the potential of PfEIF3i as a target for the design of new antimalarial drugs active all along the life cycle of the parasite.

Design and synthesis of naturally-inspired SARS-CoV-2 inhibitors.

A naturally inspired chemical library of 25 molecules was synthesised guided by 3-D dimensionality and natural product likeness factors to explore a new chemical space. The synthesised chemical library, consisting of fused-bridged dodecahydro-2a,6-epoxyazepino[3,4,5-c,d]indole skeletons, followed lead likeness factors in terms of molecular weight, C-sp3 fraction and Clog P. Screening of the 25 compounds against lung cells infected with SARS-CoV-2 led to the identification of 2 hits. Although the chemical library showed cytotoxicity, the two hits (3b, 9e) showed the highest antiviral activity (EC50 values of 3.7 and 1.4 µM, respectively) with an acceptable cytotoxicity difference. Computational analysis based on docking and molecular dynamics simulations against main protein targets in SARS-CoV-2 (main protease Mpro, nucleocapsid phosphoprotein, non-structural protein nsp10-nsp16 complex and RBD/ACE2 complex) were performed. The computational analysis proposed the possible binding targets to be either Mpro or the nsp10-nsp16 complex. Biological assays were performed to confirm this proposition. A cell-based assay for Mpro protease activity using a reverse-nanoluciferase (Rev-Nluc) reporter confirmed that 3b targets Mpro. These results open the way towards further hit-to-lead optimisations.